YEASTBOOK

CELL SIGNALING & DEVELOPMENT

Architecture and Biosynthesis of the Saccharomycescerevisiae Cell WallPeter Orlean1

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

ABSTRACT The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating,sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of b1,3- and b1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. Thesecomponents become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking varyduring growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells,covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphos-phatidylinositol membrane anchors, b1,3- and b1,6-linked glucans, and chitin; the reactions that cross-link wall components; and thepossible functions of enzymatic and nonenzymatic cell wall proteins.

TABLE OF CONTENTS

Abstract 775

Introduction 777

Wall Composition and Architecture 777Polysaccharides 778

Chitin: 778b-Glucans: 778Cross-links between polysaccharides: 779

Cell wall mannoproteins 779GPI proteins: 780Mild alkali-releasable proteins: 780Disulfide-linked proteins: 780

Strategies to identify CWP 780

Cell wall phenotypes 781

Precursors and Carrier Lipids 781Sugar nucleotides 781

Dolichol and dolichol phosphate sugars 781Dolichol phosphate synthesis: 781

Continued

Copyright © 2012 by the Genetics Society of Americadoi: 10.1534/genetics.112.144485Manuscript received May 17, 2012; accepted for publication August 6, 2012Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.144485/-/DC1.1Address for correspondence: Department of Microbiology, University of Illinois at Urbana-Champaign, B-213 Chemical and Life Sciences Laboratory, 601 South Goodwin Ave.,Urbana, IL 61801. E-mail: p-orlean@illinois.edu

Genetics, Vol. 192, 775–818 November 2012 775

CONTENTS, continued

Dol-P-Man and Dol-P-Glc synthesis: 781

Biosynthesis of Wall Components Along the Secretory Pathway 781N-Glycosylation 782

Assembly and transfer of the Dol-PP-linked precursor oligosaccharide: 782Steps on the cytoplasmic face of the ER membrane: 782Transmembrane translocation of Dol-PP-oligosaccharides: 782Lumenal steps in Dol-PP-oligosaccharide assembly: 783Oligosaccharide transfer to protein: 783

N-glycan processing in the ER and glycoprotein quality control: 783Mannan elaboration in the Golgi: 784

Formation of core-type N-glycan and mannan outer chains: 784Mannan side branching and mannose phosphate addition: 784

O-Mannosylation 785Protein O-mannosyltransferases in the ER: 785Extension and phosphorylation of O-linked manno-oligosaccharide chains: 785Importance and functions of O-mannosyl glycans: 785

GPI anchoring 785GPI structure and proteins that receive GPIs: 785

GPI structure: 785Identification of GPI proteins: 786

Assembly of the GPI precursor and its attachment to protein in the ER: 786Steps on the cytoplasmic face of ER membrane: 786Lumenal steps in GPI assembly: 787GPI transfer to protein: 788

Remodeling of protein-bound GPIs: 788

Sugar nucleotide transport 789GDP-Man transport: 789Other sugar nucleotide transport activities: 789

Biosynthesis of Wall Components at the Plasma Membrane 789Chitin 789

Septum formation: 789Chitin synthase biochemistry: 790S. cerevisiae’s chitin synthases and auxiliary proteins: 791

Chitin synthase I: 791Chitin synthase II and proteins impacting its localization and activity: 791Chitin synthase III and proteins impacting its localization and activity: 792

Chitin synthesis in response to cell wall stress: 793Chitin synthase III in mating and ascospore wall formation: 794

b1,3-Glucan 794Fks family of b1,3-glucan synthases: 794Roles of the Fks proteins in b1,3-glucan synthesis: 794Rho1 GTPase, a regulatory subunit of b1,3-glucan synthase: 795

b1,6-Glucan 795In vitro synthesis of b1,6-glucan 795

Proteins involved in b1,6-glucan assembly 796ER proteins: 796

Homologs of the UGGT/calnexin protein quality control machinery: 796Fungus-specific ER chaperones required for b1,6-glucansynthesis: 796

More widely distributed secretory pathway proteins: 797Kre6 and Skn1: 797Kre9 and Knh1: 797

Plasma membrane protein Kre1: 797How might b1,6-glucan be made?: 797

Continued

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CONTENTS, continued

Remodeling and Cross-Linking Activities at the Cell Surface 797Order of incorporation of components into the cell wall 797

Incorporation of GPI proteins into the wall 798

Incorporation of PIR proteins into the wall 798

Cross-linkage of chitin to b1,6- and b1,3-glucan 799

Cell Wall-Active and Nonenzymatic Surface Proteins and Their Functions 799Known and predicted enzymes 799

Chitinases: 799b1,3-glucanases: 799

Exg1, Exg2, and Ssg/Spr1 exo-b1,3-glucanases: 799Bgl2, Scw4, Scw10, and Scw11 endo-b1,3-glucanases: 800Eng1/Dse4 and Eng2/Acf2 endo-b1,3-glucanases: 800

Gas1 family b1,3-glucanosyltransferases: 800Yapsin aspartyl proteases: 800

Nonenzymatic CWPs 801Structural GPI proteins: 801

Sps2 family: 801Tip1 family: 801Sed1 and Spi1: 801Ccw12: 801Other nonenzymatic GPI proteins: 802Flocculins and agglutinins: 802

Non-GPI-CWP: 802PIR proteins: 802Scw3 (Sun4): 803Srl1: 803

What Is Next? 803

THE wall gives Saccharomyces cerevisiae its morphologiesduring budding growth, pseudohypha formation, mat-

ing, and sporulation; it preserves the cell’s osmotic integrity;and it provides a scaffold to present agglutinins and floccu-lins to other yeast cells. The wall consists of mannoproteins,b-glucans, and a small amount of chitin, which becomecross-linked in various ways. Wall composition and organi-zation vary during growth and development. During thebudding cycle, deposition of chitin is tightly controlled,and expression of certain hydrolases involved in cell separa-tion is daughter cell-specific. The wall can be weakened, andthe cell consequently stressed, by treatment with polysaccha-ride binding agents such as Calcofluor White (CFW), CongoRed, sodium dodecyl sulfate (SDS), aminoglycoside antibio-tics, and b-glucanase preparations or by mutational loss ofcapacity to make a wall component. Such stresses commonlyactivate the cell wall integrity (CWI) pathway (Levin 2011)and result in compensatory synthesis of wall material.

Up to a quarter of the genes in S. cerevisiae have some rolein maintenance of a normal wall. From the results of a surveyof deletion strains for cell wall phenotypes, De Groot et al.(2001) estimated that �1200 genes, not counting essentialones, impact the wall. Most of the effects, however, are in-

direct, and the number of genes that encode enzymes directlyinvolved in biosynthesis or remodeling of the wall, or non-enzymatic wall proteins, is now �180 (see Supporting Infor-mation, Table S1). This review covers these proteins, withemphasis on the wall of vegetative cells during the buddingcycle and in response to stress. Wall synthetic activities will becovered in the context of their cellular localization, startingwith precursors in the cytoplasm, proceeding along the secre-tory pathway from the endoplasmic reticulum (ER) to theplasma membrane, and culminating with the events outsidethe plasma membrane that generate covalent cross-links be-tween wall components. Additional information about indi-vidual proteins and the phenotypes of strains lacking them ispresented in File S1, File S2, File S3, File S4, File S5, File S6,File S7, File S8, and File S9. Earlier work on the yeast cellwall has been reviewed by Ballou (1982), Fleet (1991),Orlean (1997), Kapteyn et al. (1999a), Cabib et al. (2001),Klis et al. (2002, 2006), and Lesage and Bussey (2006).

Wall Composition and Architecture

The wall accounts for 15–30% of the dry weight of a vege-tative S. cerevisiae cell (Aguilar-Uscanga and François 2003;

S. cerevisiae Cell Wall 777

Yin et al. 2007). It is 110–200 nm wide, as estimated fromtransmission electron micrographs and by using an atomicforce microscope to detect surface accessibility of “molecularrulers” consisting of versions of the plasma membrane sensorWsc1 with different lengths (Dupres et al. 2010; Yamaguchiet al. 2011). The wall’s major components are b1,3- andb1,6-linked glucans, mannoproteins, and chitin, which canbe covalently joined to form higher-order complexes. Theb1,6-glucan, although quantitatively a minor componentof the wall, has a central role in cross-linking wall compo-nents (Kollar et al. 1997). Some mannoproteins have or arepredicted to have enzymatic activity as hydrolases or cross-linkers; others may have structural roles or mediate “socialactivity” by serving as mating agglutinins or flocculins.Among the latter, Flo1 and Flo11 promote formation of ex-tensive mats of cells, or biofilms (Reynolds and Fink 2001;Beauvais et al. 2009; Bojsen et al. 2012).

Electron micrographs of thin sections through the wall ofvegetative cells reveal two layers. The outer one is electron-dense, has a brush-like surface (Osumi et al. 1998) (Babaet al. 1989; Osumi et al. 1998); Kapteyn et al. 1999a; Hagenet al. 2004; Yamaguchi et al. 2011), and can be removedby proteolysis (Kopecka et al. 1974; Zlotnik et al. 1984); ittherefore consists mostly of mannoproteins. The inner layer,more electron transparent, is microfibrillar and b-glucanase-digestible, indicating that its major components are glucans.The relative thicknesses of the two layers and their apparentorganization can be altered in cell wall mutants.

Relative amounts and localization of individual wallcomponents vary depending on cell cycle or developmentalstage, growth phase, nutritional conditions, and wall stressesimposed by hypo-osmolarity, mutational loss of wall bio-synthetic activities or wall proteins, or drug treatment.Variations in wall composition and organization impact theextent to which the wall is a barrier to export of soluble,secreted proteins to the medium. Some proteins can beretained by the wall outside the plasma membrane in theperiplasmic space; in the case of Suc2, this is due to the abilityof the protein to form large multimers (Orlean 1987). Thebarrier function of the wall is dependent on growth phase andcultural conditions, with the walls of growing cells beingmore porous (De Nobel and Barnett 1991). Native glycopro-teins such as Cts1, as well as many heterologously expressedsoluble glycoproteins with masses up to 400 kDa, can passthrough the wall of logarithmically growing cells to the me-dium, whereas walls of stationary-phase cells are less porous(De Nobel et al. 1990; Kuranda and Robbins 1991). Therelatively high porosity of walls of logarithmic-phase cellscould reflect a lower degree of cross-linking, but the dissolu-tion of septal material that occurs when dividing cells sepa-rate could also release wall proteins to the medium (seeOrder of incorporation of components into the cell wall). Per-spectives on wall organization are provided by Kapteyn et al.(1999a), Klis et al. (2002, 2006), Latgé (2007), Pitarch et al.(2008), and Gonzalez et al. (2010a). The major wall compo-nents and strategies for isolating them are as follows.

Polysaccharides

Wall polysaccharides are typically separated into threefractions defined on the basis of their solubility in alkaliand acid (Fleet 1991). These fractions contain differing rel-ative amounts of b1,3- and b1,6-linked glucans and mannan(Magnelli et al. 2002) and also differ in whether and to whatextent the glucans are cross-linked to chitin, which deter-mines their solubility in alkali. Determination of Man-to-Glcratios in total acid hydrolysates of walls has been useful inassessing the impact of mutations on wall composition (Ramet al. 1994; Dallies et al. 1998). Digestion of isolated wallsand wall fractions with linkage-specific glycosidases hasbeen used to quantify wall components and determine thefine structure of b1,6-glucan (Boone et al. 1990; Magnelliet al. 2002; Aimanianda et al. 2009), as well as to generateoligosaccharides for structural analysis and characterizationof linkages between polymers (Kollar et al. 1995, 1997).

Chitin: This polymer of b1,4-linked GlcNAc contributes only1–2% of the dry weight of the wall of unstressed wild-typecells. Chitin is normally deposited in a ring in the neck be-tween a mother cell and its emerging bud, in the primarydivision septum, and in the lateral walls of newly separateddaughter cells. Chitin can be visualized in situ by stainingwith CFW, which reveals that most of it is present in divisionsepta and bud scars. Chitin in lateral walls and in divisionsepta can also be detected by immunoelectron microscopy(Shaw et al. 1991). Chitin levels are typically determinedafter extraction of walls with acid and alkali or hot SDS,followed by acid or enzymatic hydrolysis and quantificationof GlcNAc (Kang et al. 1984; Orlean et al. 1985; Dallies et al.1998; Magnelli et al. 2002). The average length of chitin inb-glucanase-digested septa is �110 GlcNAc residues (Kanget al. 1984). However, chitin occurs in three different andpolydisperse forms in the wall: in addition to free chitin,some is bound to b1,3-glucan and present mainly in theneck between mother and daughter cell, whereas a lesseramount, found in lateral walls, is bound to b1,6-glucan,which is in turn linked to mannan and b1,3-glucan (Cabiband Duran 2005; Cabib 2009). Chitin levels increase in re-sponse to mating pheromones (Schekman and Brawley1979; Orlean et al. 1985; see Sugar nucleotides) and delo-calized chitin in lateral walls can increase to as much as 20%of the wall in S. cerevisiae mutants mounting the cell wallstress response (Kapteyn et al. 1997, 1999a; Popolo et al.1997; Dallies et al. 1998; Ram et al. 1998; Osmond et al.1999; Valdivieso et al. 2000; Magnelli et al. 2002; see Chitinsynthesis in response to cell wall stress).

b-Glucans: b-linked glucans compose 30–60% of the dryweight of the wall and can be separated into three fractionsthat contain both b1,3 and b1,6 linkages. The major frac-tion, which makes up �35% of the dry weight of the wall, isan acid- and alkali-insoluble b1,3-glucan with a degree ofpolymerization of �1500 and b1,3-linked glucan side chainsinitiated at branching b1,6-linked glucoses that represent

778 P. Orlean

�3% of the whole polymer (Fleet 1991). The nonreducingends of b1,3-glucan chains in this fraction can be linked tochitin, rendering the b-glucan insoluble (Kollar et al. 1995;see below). A second b-glucan fraction, representing �20%of the dry weight of the wall, is similar in size and compo-sition to the alkali-insoluble b1,3-glucan, but soluble in al-kali because it is not cross-linked to chitin (Hartland et al.1994). A third fraction, making up �5% of the dry weight ofthe wall, can be released from alkali-insoluble glucan byextraction with acid or digestion with endo-b1,3-glucanase(Manners et al. 1973; Boone et al. 1990). This fraction isa b1,6-glucan with a degree of polymerization of 140, inwhich 14% of the b1,6-linked residues bear a side-branchingb1,3 Glc (Manners et al. 1973). A procedure involving serialdigestion with purified hydrolases has also been used toseparate and quantify b1,3- and b1,6-glucan, mannan, andchitin (Magnelli et al. 2002). The b1,6-glucan was releasedfrom the high-molecular-weight material remaining aftertreatment of walls with a mixture of b1,3-glucanase and chi-tinase by digestion with recombinant endo-b1,6-glucanase.The b1,6-glucan was therefore recovered as a mixture ofoligosaccharides whose major component was Glcb1,6Glc,and which also contained Glcb1,6Glcb1,6Glc and smalleramounts of Glcb1,3Glcb1,6Glc and Glcb1,6Glcb1,6Glc witha b1,3-Glc branching from its middle Glc (Magnelli et al.2002). The degree of b1,3 branching inferred from the oli-gosaccharide profile was similar to that reported by Mannerset al. (1973). This b1,6-glucan analysis would also includethe b1,6-glucan present in the alkali-soluble cell wall frac-tion, which is not included in procedures involving alkaliextraction. In another approach, b1,6-glucan was isolatedfollowing extraction of intact cells with hot SDS and mer-captoethanol, treatment with hot alkali under reducing con-ditions, and b1,3-glucanase digestion of the alkali-insolublematerial (Aimanianda et al. 2009). The b1,3-glucanase re-leasable material was a b1,6-glucan of 190–200 glucoseswith, on average, a b1,3-Glc or a b1,3-Glcb1,3-Glc side

branch on every fifth b1,6-linked glucose (Aimaniandaet al. 2009).

Cross-links between polysaccharides: Three types of link-ages between wall polysaccharides have been described(Figure 1). The first is a b1,4-linkage between the reducingend of a chitin chain and the nonreducing end of a b1,3-linked glucan (Kollar et al. 1995), and up to half of the chitinchains in the wall may be linked to b-glucan in this way.Because there is about one chitin-b-glucan linkage per 8000hexoses, these rare cross-links have a major impact on thesolubility of b-glucan (Kollar et al. 1995). The second linkageis between the reducing end of chitin and the nonreducingend of a b1,3-Glc that branches off b1,6-glucan (Kollar et al.1997; see Remodeling and Cross-Linking Activities at the CellSurface). The configuration of this linkage is either b1,2- orb1,4-. The two types of chitin-b-glucan linkage are found indifferent parts of the wall. In the third linkage, the reducingends of b1,6-glucan chains can be attached to b1,3-glucan,but the configuration is unknown (Kollar et al. 1997).

Cell wall mannoproteins

Yeast cell wall proteins can bear asparagine- (N-)linkedglycans, O-linked manno-oligosaccharides, and often a gly-cosylphosphatidylinositol (GPI) as well. The N-linked gly-cans can be extended with an outer chain of 50 or morea1,6-linked Man that is extensively decorated with shorta1,2-Man side branches terminated in a1,3-Man. Phospho-diester-linked mannoses can also be attached to a1,2-linkedresidues. Many glycoproteins also bear O-mannosyl glycans,which are often present in Ser/Thr-rich stretches.

Proteins relevant to the wall can be placed into one ofthree groups. The first contains those with the potential toparticipate in wall construction as hydrolases or trans-glycosidases. The second contains nonenzymatic aggluti-nins, flocculins, or b1,3-glucan cross-connectors (Klis et al.2006, 2010; Dranginis et al. 2007; Goossens and Willaert

Figure 1 Wall components and cross-links be-tween them. (A) Reducing end of chitin linked toa side-branching b1,3-Glc on b1,6-glucan. (B) Re-ducing end of chitin linked to a nonreducing endof b1,3-glucan. (C) Reducing end of b1,3-glucanchain linked to a side-branching b1,6-Glc on b1,3-glucan. (D) Reducing end of GPI glycan (possiblythe a1,4-Man) to internal Glc in b1,6-glucan (link-age to nonreducing end of b1,6-glucan is also pos-sible). (E) Ester linkages between b1,3-Glc andg-carboxyl groups of glutamates in PIR protein in-ternal repeats. (F) Disulfide link between CWP.Chemical treatments used to release CWP areindicated.

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2010). Most, if not all the proteins in these two groups areglycosylated. Proteins that are covalently attached to cell wallglycan are referred to as CWP (Yin et al. 2005) and fall intothe subgroups below. The third group consists of single-passplasma membrane proteins with short C-terminal cytoplasmicdomains and long Ser/Thr-rich extracellular regions. Theseinclude Wsc1, Wsc2, and Wsc3, which also have N-terminalcysteine-rich domains, as well as Mtl1 and Mid2. These aremechanosensors that detect cell wall stress and activate theCWI pathway (Rodicio and Heinisch 2010; Levin 2011). CWPand cell wall-active enzymes are discussed in Cell Wall-Activeand Nonenzymatic Surface Proteins and Their Functions.

GPI proteins: These receive a GPI that initially anchors themin the outer face of the plasma membrane, but many thenbecome cross-linked to b1,6-glucan via a remnant of the GPI(Gonzalez et al. 2009). Results to date suggest that the GPIis cleaved between its GlcN residue and Man, whereuponthe mannose’s reducing end is glycosidically linked to a non-reducing end of b1,6-glucan or to a Glc in a b1,6-Glc chain(Kollar et al. 1997; Fujii et al. 1999). The b1,6-glucan towhich the GPI-CWP is attached is in turn linked to b1,3-glucan and chitin (Kapteyn et al. 1996; Van der Vaartet al. 1996; Kollar et al. 1997; Fujii et al. 1999; Figure 1).Some wall-bound GPI proteins may retain enzymatic activ-ity, whereas others may have a structural role (Yin et al.2005). GPI-CWP are released by treatment with hydrogenfluoride (HF)/pyridine, which cleaves the phosphodiesterof the GPI that links Man and the phosphoethanolamine(Etn-P) moiety that is linked to protein (Yin et al. 2005).Proteins released in this way have a C-terminal GPI signal-anchor sequence, and this, and signals for wall anchorage ofGPI-CWP, are discussed in Lumenal steps in GPI assembly andin Incorporation of GPI proteins into the cell wall. At least oneGPI-CWP, Cwp1, can additionally be linked to the wall viaan alkali-labile linkage (Kapteyn et al. 2001).

Mild alkali-releasable proteins: These include four proteinswith internal repeats (PIR proteins), which have multiplecopies of the internal repeat sequence SQ[I/V][S/T/G]DGQ[I/V]Q[A][S/T/A] (Toh-E et al. 1993) [simplified to DGQ[hydrophobic amino acid]Q by Klis et al. (2010)] and arereleased by mild alkali or b1,3-glucanase (Mrša et al. 1997).PIR proteins have no GPI attachment sequence and are notlinked to b1,6-glucan; rather, they are ester-linked to b1,3-glucan via side chains of amino acids in the repeat sequences(Ecker et al. 2006; see Incorporation of PIR proteins into thecell wall). Because PIR proteins can form several linkages tob1,3-glucan, they could interconnect glucans. Single PIRrepeats are also present in certain GPI-CWP (see Incorpora-tion of PIR proteins into the cell wall), and additional proteinslacking PIR sequences can be also extracted with alkali orb1,3-glucanase (Yin et al. 2005; see Cell Wall-Active andNonenzymatic Surface Proteins and Their Functions).

Disulfide-linked proteins: Various proteins can be releasedfrom the walls of living cells with sulfhydryl reagents,

indicating that they are directly attached via disulfides orretained behind a network of disulfide-linked proteins(Orlean et al. 1986; Cappellaro et al. 1998; Moukadiriet al. 1999; Moukadiri and Zueco 2001; Insenser et al.2010). Disulfide-linked mannoproteins create a barrier thatprotects wall polysaccharides from externally added glyco-sylhydrolases, making mercaptoethanol and protease pre-treatment necessary for spheroplasting with lytic enzymes(Zlotnik et al. 1984). Furthermore, the ability of the cyste-ine-rich domain of Wsc1 to form disulfide cross-links is im-portant for this mechanosensor in forming clusters and infunctioning in CWI signaling (Heinisch et al. 2010; Dupreset al. 2011).

Strategies to identify CWP

Biochemistry and bioinformatics have been used to identifyCWP. Because proteins can be associated with the wall indifferent ways, different treatments are necessary to releasethem. Separation and identification of individual CWP canbe complicated by their heavy and heterogeneous glycosyl-ation. CWP can be released from the wall by treatment withb1,3- and b1,6-glucanases (Van der Vaart et al. 1995; Mršaet al. 1997; Shimoi et al. 1998). In one approach, labeling ofintact cells with a membrane-impermeable biotinylation re-agent, followed successively by SDS and mercaptoethanolextraction and then mild alkali or b1,3-glucanase treatment,led to identification of nine “soluble cell wall” (Scw) and 11“covalently linked cell wall” (Ccw) proteins (Mrša et al.1997). In another approach, isolated walls, extracted withSDS, mercaptoethanol, NaCl, and EDTA, were then treatedwith HF/pyridine or mild alkali, and the CWP released wereidentified by mass spectrometry. Additional CWP were iden-tified following proteolytic digestion of walls, the two pro-cedures yielding 19 CWP, including GPI and PIR proteinsand alkali-releasable proteins without PIR sequences (Yinet al. 2005, 2007). These studies led to the estimate thata dividing haploid cell contains �2 · 106 covalently at-tached CWPs and the suggestion that CWP form a denselypacked surface layer (Yin et al. 2007). A strategy that alsopermitted identification of noncovalently associated surfaceproteins used treatment of intact cells with dithiothreitolfollowed by two-dimensional electrophoretic separation, ordirect proteolytic digestion and isolation of peptides, andthen mass spectrometric protein fingerprinting (Insenseret al. 2010). The 99 proteins so identified included CWPand glycosylhydrolases, as well as proteins associated withintracellular functions. The presence in the wall of proteinsconsidered cytosolic raises the possibility that they reach thewall via a nonconventional export pathway (Nombela et al.2006; Insenser et al. 2010). However, mercaptoethanol canmake the plasma membrane permeable to cytosolic proteins(Klis et al. 2007).

Bioinformatics has been used identify proteins likely toreceive a GPI anchor; hence, members of the major class ofCWP. In silico surveys for GPI attachment sequences revealthat the S. cerevisiae proteome contains 60–70 potential GPI

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proteins, which often contain Ser/Thr-rich stretches (Caroet al. 1997; Hamada et al. 1998a; De Groot et al. 2003;Eisenhaber et al. 2004).

Cell wall phenotypes

Cell wall phenotypes that are typically scored are sensitivityto hypo-osmotic stress, which can be tested on half-strengthyeast extract peptone medium (Valdivia and Schekman2003); sensitivity or resistance to CFW and Congo Red; sen-sitivity to aminoglycosides, b1,3-glucan synthase inhibitors,caffeine, SDS, and K1 killer toxin; and sensitivity to b1,3-glucanase preparations (Ram et al. 1994; Hampsey 1997;Lussier et al. 1997b; De Groot et al. 2001).

Precursors and Carrier Lipids

Sugar nucleotides

Glycosyltransferases involved in wall biogenesis use UDP-Glc,UDP-GlcNAc, and GDP-Man or dolichol phosphate (Dol-P)Man or Dol-P-Glc as donors. UDP-Glc is formed from UTP andGlc-1-P by the essential UDPGlc pyrophosphorylase Ugp1(Daran et al. 1995). Impairment of UDP-Glc synthesis ulti-mately impacts formation of cell wall b-glucans, althoughcells with no more than 5% of the activities of the phospho-glucomutases and Ugp1 that generate UDP-Glc are unaf-fected in growth and viability (Daran et al. 1997). GDP-Man is formed from Fru-6-P by the successive actions ofphosphomannose isomerase (Pmi40), phosphomannomutase(Sec53), and GDP-Man pyrophosphorylase (Psa1/Srb1/Vig9),which are all encoded by essential genes, and loss of any ofthese enzyme activities leads to severe glycosylation and se-cretion defects (Hashimoto et al. 1997; Orlean 1997; Yodaet al. 2000). Elevated expression of GDP-Man pyrophosphor-ylase, which presumably increases GDP-Man levels, correctsthe N-glycosylation defects in alg1 and alg2 mutants and themannosylation and GPI synthetic defects in dpm1 cells (Janiket al. 2003). GDP-Man transport into the Golgi lumen is dis-cussed in Sugar nucleotide transport.

The pathway for UDP-GlcNAc formation (Milewski et al.2006) involves conversion of Fru-6-P to GlcN-6-P by gluta-mine:Fru-6-P amidotransferase Gfa1 (Watzele and Tanner1989), N-acetylation of GlcN-6-P by Gna1 (Mio et al.1999), conversion of GlcNAc-6-P to GlcNAc-1-P by theGlcNAc phosphate mutase Agm1/Pcm1 (Hofmann et al.1994), and formation of UDP-GlcNAc by the pyrophosphor-ylase Uap1/Qri1 (Mio et al. 1998). Deficiencies in theseenzymes lead to formation of short chains of undivided cells,swelling, and eventual lysis, a phenomenon known as glu-cosamineless death (Ballou et al. 1977; Mio et al. 1998,1999). Glucosamine supply is highly regulated and impactschitin levels, which increase in response to mating phero-mones and cell wall stress (File S1).

Dolichol and dolichol phosphate sugars

Dolichol phosphate synthesis: Yeast dolichols contain 14–18 isoprene units (Jung and Tanner 1973). Biosynthesis of

dolichol (Schenk et al. 2001a; Grabinska and Palamarczyk2002) starts with extension of trans farnesyl-PP by succes-sive addition of cis-isoprene units by the homologous cis-prenyltransferases Rer2 and Srt1 (Sato et al. 1999; Schenket al. 2001b). Rer2 is dominant and makes dolichols with10–14 isoprene units, whereas dolichols made by Srt1 incells lacking Rer2 contain 19–22 isoprenes. rer2D strainshave severe defects in growth and in N- and O-glycosylation(Sato et al. 1999). The next two steps are likely the removalof the two phosphates from dehydrodolichyl diphosphate byunknown enzymes. The a-isoprene unit of the polyprenol isthen reduced, and Dfg10 is responsible for much of thisactivity (Cantagrel et al. 2010; File S1). Dolichol is likelynext phosphorylated by the CTP-dependent Dol kinaseSec59 (Heller et al. 1992).

Dol-PP generated on the lumenal side of the ER mem-brane after transfer of the N-linked oligosaccharide toprotein is dephosphorylated to Dol-P and Pi on that side ofthe membrane by the phosphatase Cwh8/Cax4 (Van Berkelet al. 1999; Fernandez et al. 2001). CWH8-disruptants havean N-glycosylation defect and a growth defect that is par-tially suppressed by high-level expression of RER2, SEC59,and the lipid phosphatase gene LPP1. Cwh8 likely has a role inrecycling of Dol-PP for use in new rounds of N-glycosylationon the cytoplasmic face of the ER membrane.

Dol-P-Man and Dol-P-Glc synthesis: Dol-P-Man and Dol-P-Glc are the donors in the lumenal glycosyltransfers thatoccur in protein O-mannosylation and the assembly path-ways for the Dol-PP-linked precursor in N-glycosylation andthe GPI anchor precursor glycolipid. Dol-P-Man is formedupon transfer of Man from GDP-Man to Dol-P by the Dol-P-Man synthase Dpm1 (Orlean et al. 1988; Orlean 1990).Temperature-sensitive dpm1 mutants have cell wall defects,consistent with a general block of glycosylation and GPIanchoring, and these phenotypes are suppressed by high-level expression of RER2, which presumably elevates Dol-Plevels (Orlowski et al. 2007).

Dol-P-Glc is formed from UDP-Glc and Dol-P. Deletion ofthe synthase gene, ALG5, is not lethal, and the disruptantsshow no obvious growth defects (Te Heesen et al. 1994).Because Dol-P-Man and Dol-P-Glc are used in lumenal reac-tions, and because spontaneous transmembrane transloca-tion of these glycolipids is not favored energetically, theirtranslocation may be protein-mediated. Assays for Dol-P-Man flipping have been reported (Haselbeck and Tanner1982; Sanyal and Menon 2010), but a protein involvedhas yet to be identified. One possibility is that the Dol-P-Man and Dol-P-Glc-utilizing transferases are their own flip-pases (Burda and Aebi 1999).

Biosynthesis of Wall Components Along theSecretory Pathway

Cell surface proteins can be modified with N-glycans,O-linked manno-oligosaccharides, and a GPI anchor as they

S. cerevisiae Cell Wall 781

transit the secretory pathway. Initial attachment of thesestructures occurs in the ER lumen, and the glycans aremodified in the Golgi before the glycoproteins are depositedin the plasma membrane or secreted from the cell, where-upon many become cross-linked to wall polysaccharides.

N-Glycosylation

N-glycosylation involves preassembly of a branched 14-sugar oligosaccharide on the carrier Dol-PP in the ERmembrane and then transfer of the oligosaccharide toselected asparagines in the ER lumen (Burda and Aebi1999; Helenius and Aebi 2004; Lehle et al. 2006; Larkinand Imperiali 2011). The first 7 sugars are transferredfrom sugar nucleotides on the cytosolic side of the ER mem-brane, and the remainder from Dol-P on the lumenal side(Figure 2).

Assembly and transfer of the Dol-PP-linked precursoroligosaccharide: Steps on the cytoplasmic face of the ERmembrane: These steps are (i) transfer of GlcNAc-1-P fromUDP-GlcNAc to Dol-P by Alg7, the target of the N-glycosylationinhibitor tunicamycin (Barnes et al. 1984), (ii) transfer of b1,4-

GlcNAc from UDP-GlcNAc by heterodimeric Alg13/Alg14(Bickel et al. 2005; Chantret et al. 2005; Gao et al. 2005),(iii) transfer of a b1,4-linked Man by Alg1 (Couto et al.1984), (iv) successive transfer of an a1,3 and an a1,6Man by Alg2 (O’Reilly et al. 2006; Kämpf et al. 2009), and(v) transfer of two a1,2-linked Man by Alg11 (Cipollo et al.2001; O’Reilly et al. 2006; Absmanner et al. 2010). Theseproteins act in higher-order complexes (Gao et al. 2004;Noffz et al. 2009; File S2).

Transmembrane translocation of Dol-PP-oligosaccharides:Dol-PP-GlcNAc2Man5 formed on the cytoplasmic face of theER membrane is somehow translocated into the lumen(Burda and Aebi 1999; Helenius and Aebi 2002), and Rft1is a candidate for the flippase (Helenius et al. 2002). Strainsdeficient in Rft1 accumulate Dol-PP-GlcNAc2Man5, but retainAlg3 Man-T activity and are unaffected in O-mannosylationor in GPI assembly, ruling out deficiences in Dol-P-Man sup-ply to the lumen. Furthermore, high level expression ofRFT1 partially suppresses the growth defect of alg11D andleads to increased levels of lumenal Dol-PP-GlcNAc2Man6-7and an increase in glycosylation of the reporter carboxypepti-dase Y, consistent with enhanced flipping of the suboptimal

Figure 2 Assembly of the Dol-PP-linked precursor oligosaccharide in N-glycosylation, its transfer to protein, and subsequent glycan processing. Residuesadded at the cytoplasmic face of the ER membrane originate from sugar nucleotides, whereas Dol-P sugars generated at the cytoplasmic face of themembrane are the donors in lumenal transfers. Symbols are adaptations of those used by the Consortium of Glycobiology Editors in Essentials inGlycobiology (Varki and Sharon 2009).

782 P. Orlean

substrate Dol-PP-GlcNAc2Man3 (Helenius et al. 2002). How-ever, although the above evidence is consistent with Rft1being the flippase, depletion of Rft1 did not lead to loss offlipping activity measured in vitro (Frank et al. 2008; Rushet al. 2009; File S2).

Lumenal steps in Dol-PP-oligosaccharide assembly: Dol-PP-GlcNAc2Man5 is extended by four Man and three Glc on thelumenal side of the ER membrane using Dol-P-Man and Dol-P-Glc as donors. Alg3 adds the sixth, a1,3-Man to the a1,6Man of Dol-PP-GlcNAc2Man5 (Aebi et al. 1996; Sharma et al.2001), Alg9 then transfers an a1,2-linked Man to the Manadded by Alg3 (Burda et al. 1999; Cipollo and Trimble2002), and Alg12 next adds the eighth, a1,6-Man to theMan added by Alg9 (Burda et al. 1999). Alg9 acts again toadd the ninth Man, a1,2-linked Man to the Man added byAlg12 (Frank and Aebi 2005). Two a1,3-linked Glc are suc-cessively added by Alg6 and Alg8 to extend the arm of theheptasaccharide ending in the a1,2-linked Man transferredby Alg11, and finally, Alg10 adds an a1,2-Glc (Stagljar et al.1994; Reiss et al. 1996; Burda and Aebi 1998). The six Dol-P-sugar-utilizing transferases are members of a family ofmultispanning membrane proteins that includes Man-T in-volved in GPI biosynthesis (Oriol et al. 2002).

Oligosaccharide transfer to protein: GlcNAc2Man9Glc3 istransferred from Dol-PP to asparagines by the oligosacchar-yltransferase complex (OST) (Knauer and Lehle 1999a; Yanand Lennarz 2005a; Kelleher and Gilmore 2006; Lehle et al.2006; Weerapana and Imperiali 2006; Lennarz 2007; Larkinand Imperiali 2011). Acceptor asparagines occur in thesequon Asn-X-Ser/Thr, where X can be any amino acid exceptPro. Mass spectrometric analyses of wall-derived peptidesrevealed that 85% of sequons were completely occupied, withpreferential usage Asn-X-Thr over Asn-X-Ser sites (Schulzand Aebi 2009). Analyses of protein-linked N-glycans inmutants defective in the elaboration of the Dol-PP-linkedprecursor indicate that structures smaller than GlcNAc2-Man9Glc3 can be transferred in vivo.

Yeast OST consists of Stt3, Ost1, Ost2, Wbp1, Swp1,Ost4, Ost5, and either of the paralogues Ost3 or Ost6. Thefirst five are encoded by essential genes. Two OST com-plexes can be formed, containing either Ost3 or Ost6(Schwarz et al. 2005; Spirig et al. 2005; Yan and Lennarz2005b). The Ost3-containing complex is about four times asabundant as the Ost6-containing one (Spirig et al. 2005).Genetic interaction studies and coimmunoprecipitation andchemical cross-linking experiments suggest the existence ofthree OST subcomplexes: (i) Swp1-Wbp1-Ost2, (ii) Stt3-Ost4-Ost3, and (iii) Ost1-Ost5 (Karaoglu et al. 1997; Reisset al. 1997; Spirig et al. 1997; Knauer and Lehle 1999b; Kimet al. 2003; Li et al. 2003; Kelleher and Gilmore 2006; FileS2). OST complexes themselves may function as dimers(Chavan et al. 2006).

Stt3 is the catalytic subunit of OST. It can be chemicallycross-linked to peptides derivatized with photoactivatablegroups (Yan and Lennarz 2002; Nilsson et al. 2003), andits bacterial and protist homologs transfer glycans to protein

substrates (Wacker et al. 2002; Kelleher and Gilmore 2006;Kelleher et al. 2007; Nasab et al. 2008; Hese et al. 2009).Ost3 and Ost6 have a lumenal thioreductase fold witha CXXC motif common to proteins involved in disulfide bondshuffling during oxidative protein folding (Kelleher andGilmore 2006; Schulz et al. 2009), and the proteins likelyform transient disulfide bonds with nascent proteins andpromote efficient glycosylation of Asn-X-Ser/Thr sites bydelaying oxidative protein folding (Schulz and Aebi 2009;Schulz et al. 2009). The Swp1p, Wbp1p, and Ost2p subcom-plex may confer the preference of OST for GlcNAc2Man9Glc3(Pathak et al. 1995; Kelleher and Gilmore 2006), Ost4 isrequired for recruitment of Ost3 and Ost6 to OST and alsointeracts with Stt3 (Karaoglu et al. 1997; Spirig et al. 1997;Knauer and Lehle 1999b; Kim et al. 2000, 2003; Spirig et al.2005), and Ost1 may funnel nascent polypeptides to Stt3(Lennarz 2007). OST may be subject to regulation by theCWI pathway via an interaction between Pkc1 or compo-nents of the PKC pathway with Stt3 (Park and Lennarz2000; Chavan et al. 2003a; File S2).

N-glycan processing in the ER and glycoprotein qualitycontrol: Protein-linked GlcNAc2Man9Glc3 is processed toglycans that are recognized by mechanisms that monitorcorrect protein folding and permit export from the ER orensure degradation if the protein misfolds (Herscovics1999; Aebi et al. 2010). Processing proceeds by removal ofthe a1,2-linked Glc by glucosidase I, Gls1/Cwh41 (Romeroet al. 1997), and then of the two a1,3-linked Glc by solubleglucosidase II, a heterodimer of catalytic Gls2/Rot2 andGtb1 (Trombetta et al. 1996; Wilkinson et al. 2006; Quinnet al. 2009; Figure 2). ER mannosidase I, Mns1, removesan a1,2 Man to generate GlcNAc2Man8 (Jakob et al. 1998;Herscovics 1999), and, if correctly folded, proteins bearingthis glycan are exported from the ER. Un- or misfolded pro-teins are bound by protein disulfide isomerase Pdi1, some ofwhich is in complex with Mns1 homolog Htm1, which trimsthe glycan to a GlcNAc2Man7 (Clerc et al. 2009; Gauss et al.2011; File S2). Misfolded proteins with GlcNAc2Man7 aretargeted to the cytosol for destruction by the ER-associatedprotein degradation (ERAD) system (Helenius and Aebi2004). They are bound by the lectin Yos9 (Buschhorn et al.2004; Bhamidipati et al. 2005; Kim et al. 2005; Szathmaryet al. 2005) and in turn directed to the HRD-ubiquitin ligasecomplex of Hrd1 and Hrd3 for retrotranslocation to the cy-toplasm (Bays et al. 2001; Deak and Wolf 2001; Gauss et al.2006), where they are deglycosylated by peptide N-glycanasePng1 (Suzuki et al. 2000; Hirayama et al. 2010).

In mammals and Schizosaccharomyces pombe, followingglucosidase II action, UDP-Glc:glycoprotein glucosyltransfer-ase (UGGT) adds back an a1,3-Glc, allowing the monoglu-cosylated N-glycans to interact with the lumenal lectindomains of calnexin or calreticulin (Parodi 1999; Carameloand Parodi 2007; Aebi et al. 2010). This interaction retainspartially folded or misfolded proteins in the ER and buysthem time to fold properly and be deglucosylated. Properly

S. cerevisiae Cell Wall 783

folded proteins are no longer recognized by UGGT andexported to the Golgi, whereas persistent misfolders are re-moved by ERAD. In S. cerevisiae, however, this quality con-trol mechanism does not operate because UGGT activity isnot detectable, and although the S. cerevisiae ER proteinKre5 is a sequence homolog of S. pombe UGGT, expressionof the S. pombe UGGT cannot rescue the growth defect ofkre5 mutants. However, kre5, as well as glucosidase I and IImutants and mutants in the calnexin homolog Cne1, are de-fective in b1,6-glucan synthesis, indicating roles for S. cerevisiaehomologs of players in the UGGT/calnexin quality controlsystem in b1,6-glucan synthesis (Jiang et al. 1996; Shahinianet al. 1998; Simons et al. 1998; see b1,6-Glucan).

Mannan elaboration in the Golgi: N-linked glycans onproteins are extended with a Man10-14 core-type structure orwith mannan outer chains containing up to 150–200 Man.Both structures can be modified with mannose phosphate(Figure 3) (Ballou 1990; Orlean 1997; Jigami 2008). Themannoses all originate from GDP-Man and are transferredby members of several families of redundant Golgi Man-T.

Formation of core-type N-glycan and mannan outer chains:Formation of core structures and mannan is initiated inthe cis-Golgi by Och1, which transfers an a1,6-Man to thea1,3-Man of the N-glycan that had been added by Alg2(Nakayama et al. 1997). OCH1 deletion is lethal in somestrain backgrounds, and och1D strains have severe growthdefects, highlighting the importance of mannan.

Synthesis of the poly-a1,6-mannan backbone is carriedout in the cis-Golgi by two protein complexes: Man-Pol I,see containing homologs Mnn9 and Van1, and Man-Pol II,

containing Mnn9, Anp1, Hoc1, and related Mnn10 andMnn11 (Hashimoto and Yoda 1997; Jungmann and Munro1998; Jungmann et al. 1999; File S2). M-Pol I acts first, withits Mnn9 subunit adding the first a1,6-Man to the Och1-derived Man, upon which 10–15 a1,6-Man are added inVan1-requiring reactions (Stolz and Munro 2002; Rodionovet al. 2009). This a1,6 backbone is further elongated with40–60 a1,6-Man by M-Pol II, whose Mnn10 and Mnn11subunits are responsible for the majority of the a1,6-Man-T activity (Jungmann et al. 1999). Hoc1’s role is unclear.

Core-type N-glycans are formed when an a1,2-Man is addedto the Och1-derived Man, blocking elongation of an a1,6 man-nan chain. The protein(s) involved have not been identified,but presumably either they, or M-Pol I, can tell from the contextof an N-glycan which type of structure it is to bear (Lewis andBallou 1991; Stolz and Munro 2002; Rodionov et al. 2009).Core-type structures are completed when that a1,2-Man, aswell as the two other terminal a1,2-Man on the Man8GlcNAc2structure, receives a1,3 mannoses from Mnn1.

Mannan side branching and mannose phosphate addition:Branching of the a1,6 mannan backbone is initiated by theMnn2 a1,2-Man-T, and Mnn5 adds a second a1,2-Man(Rayner and Munro 1998). Mnn2 and Mnn5 make up oneof two Mnn1 subfamilies (Lussier et al. 1999). Five membersof the Ktr1 protein subfamily, Kre2/Mnt1, Yur1, Ktr1, Ktr2,and Ktr3, also contribute to N-linked outer chain synthesis,acting collectively in the addition of the second and subse-quent a1,2-mannoses to mannan side branches (Lussieret al. 1996, 1997a, 1999).

Core-type glycans and mannan can be modified with Man-Pon a1,2-linked mannoses. Mnn6/Ktr6, a Ktr1 subfamily

Figure 3 Formation of mannan outer chains and core-type N-glycans in the Golgi. Protein-bound Man8-GlcNAc2 structures are first acted on by theOch1 a1,6-Man-T in the cis-Golgi. The initiating a1,6-Man is then elongated with �10 a1,6-linked Man by mannan polymerase (M-Pol)-I, and this chainis then extended with up to �50 a1,6-linked Man by M-Pol-II. Kre2/Mnt1, Ktr1, Ktr2, Ktr3, and Yur1 collectively add a1,2-linked mannoses. Core-typeglycans are formed when an a1,2-linked Man is added to the Och1-derived a1,6-Man. Symbols are as used in Figure 2.

784 P. Orlean

member, is mostly responsible for transferring Man-1-Pfrom GDP-Man, generating GMP (Wang et al. 1997; Jigamiand Odani 1999; File S2). Mnn4 is also involved in Man-Paddition but does not resemble glycosyltransferases andmay be regulatory (Odani et al. 1996). Levels of mannanphosphorylation are highest in the late log and stationaryphases, when MNN4 expression is elevated (Odani et al.1997). Terminal a1,2 mannoses and Man-1-Ps can be cap-ped with a1,3-Man, added by Mnn1 (Ballou 1990; Yip et al.1994).

O-Mannosylation

Many yeast proteins are modified on extracytoplasmic Ser orThr residues with linear manno-oligosaccharides. The firstMan is attached in a-linkage in the lumen of the ER, and upto four further Man are added by Man-T that act mostly inthe Golgi.

Protein O-mannosyltransferases in the ER: The first Man istransferred from Dol-P-Man (Strahl-Bolsinger et al. 1999;Lehle et al. 2006; Lommel and Strahl 2009). Consistent withthe requirement for Dol-P-Man, O-mannosylation of the modelprotein Cts1 is blocked in a dpm1-Ts mutant (Orlean 1990).There are six protein O-mannosyltransferases (PMTs) in yeast.Prototypical Pmt1 is an ER protein with seven membrane-spanning domains with conserved residues important forcatalysis and for interactions with acceptor peptides locatedin the first lumenal loop (Strahl-Bolsinger and Scheinost1999; Girrbach et al. 2000; Lommel et al. 2011).

Pmts function as hetero- or homodimers, and the pairs thatare formed are determined by membership of a subunit inone of three Pmt subfamilies. Pmt1 family members Pmt1 andPmt5 can form heterodimers with members of the Pmt2 fam-ily (which also contains Pmt3 and Pmt6), for example, Pmt1-Pmt2 and Pmt5-Pmt3 dimers, which are the most prevalentcomplexes (Girrbach and Strahl 2003). Pmt4, the lone repre-sentative of the third family, forms homodimers.

Analyses of O-mannosylation of individual proteins in pmtDstrains reveal that the different Pmt complexes have specificityfor different protein substrates (File S3). Substrates for Pmt4need to be attached to the membrane by a transmembranedomain or a GPI anchor and have an adjacent, lumenal Ser/Thr-rich domain, whereas Pmt1/Pmt2 substrates can be solu-ble or membrane-associated (Hutzler et al. 2007).

Because PMTs modify Ser and Thr, N-linked glycosylationsites are also potential targets, and this is the case with Cwp5.This protein contains a single sequon, NAT, that is normallyO-mannosylated by Pmt4, but which receives an N-linkedglycan in pmt4D cells (Ecker et al. 2003). O-mannosylation,therefore, normally precedes the action of OST on Cwp5 andmay control N-glycosylation of this protein, and perhapsothers as well.

Extension and phosphorylation of O-linked manno-oligosaccharide chains: The Ser- or Thr-linked Man is ex-tended with up to four a-linked Man by GDP-Man-dependent

Man-T of the Ktr1 and Mnn1 families (Lussier et al. 1999;Figure 4; File S3). Transfer of the first two a1,2-Man iscarried out by the Ktr1 subfamily members Ktr1, Ktr3, andKre2 and extension of the trisaccharide chain with oneor two a1,3-linked Man by Mnn1 family members Mnn1,Mnt2, and Mnt3 (Lussier et al. 1997a; Romero et al. 1999).The second a1,2-Man of an O-linked glycan can be modifiedwith Man-1-P by Mnn6 with the involvement of the regula-tor Mnn4 (Nakayama et al. 1998).

Importance and functions of O-mannosyl glycans: Noindividual PMT deletion is lethal, but strains lacking certaincombinations of three Pmts, such as pmt1D pmt2D pmt4D orpmt2D pmt3D pmt4D, are inviable, even with osmotic sup-port, indicating that yeast must carry out some minimumlevel of O-mannosylation to be viable or that one or moreessential proteins need to be O-mannosylated (Gentzsch andTanner 1996; Lommel et al. 2004). Moreover, strains lackingother combinations of Pmts, such as the pmt2D pmt3D andpmt2D pmt4D double nulls or the pmt1D pmt2D pmt3D tri-ple null, are osmotically fragile, indicating impaired wallassembly (Gentzsch and Tanner 1996). Analyses of pmtmutants show that O-mannosylation can be important forfunction of individual O-mannosylated proteins (File S3).

The phenotypes of pmtmutants are mimicked by treatmentwith the rhodanine-3-acetic acid derivative OGT1458, whichinhibits PMT activity in vitro (Orchard et al. 2004; Arroyo et al.2011). OGT1458 was used to analyze genome-wide transcrip-tional changes in response to inhibition of O-mannosylation.Consistent with the importance of O-mannosylation in wallconstruction and protein stability, consequences of defectiveO-mannosylation were activation of the CWI pathway andthe unfolded protein response (Arroyo et al. 2011). Further-more, certain genes involved in N-linked mannan outer chainassembly were upregulated. This, together with the findingthat PMT gene transcription is elevated when N-glycosylationis inhibited by tunicamycin (Travers et al. 2000), suggeststhat the N- and O-linked glycans of cell wall mannoproteinscan compensate for one another to some extent (Arroyoet al. 2011).

GPI anchoring

GPI structure and proteins that receive GPIs: GPI structure:S. cerevisiae GPI anchors have the core structure protein CO-NH2-CH2-CH2-PO4-6-Mana1,2Mana1,6Mana1,4GlcNa1,6-myoinositol phospholipid. In addition, the third, a1,2-Man,bears a fourth a1,2-Man that is added during precursor as-sembly, and this Man may receive another a1,2- or a1,3-linked Man in the Golgi (Fankhauser et al. 1993). Thea1,4- and a1,6-linked Man are also modified with Etn-P attheir 29- and 69-OHs, respectively, and the 2-OH of inositol istransiently modified with palmitate (Orlean and Menon2007; Pittet and Conzelmann 2007) (Figure 5). The lipidmoiety, initially diacylglycerol, is remodeled to a diacylgly-cerol with C26, acyl chains, or, in many cases, to a ceramide(Conzelmann et al. 1992; Fankhauser et al. 1993).

S. cerevisiae Cell Wall 785

Identification of GPI proteins: Biochemical demonstrationsof a GPI on a yeast protein are rare, and the criterion ofrelease of a protein by treatment with Ptd-Ins-specificphospholipase C (PI-PLC) is unreliable because althoughprotein-bound GPIs are mostly sensitive to PI-PLC, thistreatment does not always render the protein aqueoussoluble in the commonly used Triton X-114 fractionationprocedure (Conzelmann et al. 1990). Many GPI proteinsbecome covalently linked to wall polysaccharide, and re-lease from walls by treatment with HF/pyridine is a cluethat the protein had received a GPI (see GPI proteins; Yinet al. 2005). The presence of a GPI is usually inferred fromthe results of in silico analyses of a protein’s sequence.

Features of a likely GPI protein are a hydrophobicN-terminal secretion signal and a C-terminal GPI signal-anchor sequence that includes the amino acid residue, v, towhich the GPI will be amide-linked. Amino acids N-terminalto v are designated v(2), and those C-terminal, are desig-nated v(+). Proceeding from the C-terminal amino acid ofthe unprocessed protein, the signal anchor signal consists of(i) a variable stretch of hydrophobic amino acids capableof spanning the membrane; (ii) a spacer region of moder-ately polar amino acids in positions v+3 to v+9 or more;(iii) the v+2 residue, restricted mostly to G, A, or S; (iv) thev residue itself, generally G, A, S, N, D, or C; and (v)a stretch of some 10 amino acids that may form a flexiblelinker region and whose relative polarity may influenceplasma membrane or wall localization of the protein(Nuoffer et al. 1991, 1993; see Incorporation of GPI proteinsinto the cell wall). Some C-terminal sequences may containalternative candidates for the v and v+2 amino acids. Ev-

idence that a predicted GPI attachment sequence is func-tional can be obtained by fusing the sequence to the Cterminus of a reporter protein and testing whether the re-porter becomes expressed at the plasma membrane or in thewall (Hamada et al. 1998a).

Assembly of the GPI precursor and its attachment toprotein in the ER: At least 21 proteins are involved in GPIprecursor synthesis and attachment to protein (Figure 5).Eighteen are encoded by essential genes, and mutants lack-ing any of the other noncatalytic proteins or GPI side-branching enzymes have severe growth defects. Additionalinformation about GPI synthetic proteins and phenotypesassociated with deficiencies in them is given in File S4.

Steps on the cytoplasmic face of ER membrane: GPI assemblystarts with transfer of GlcNAc from UDP-GlcNAc to PI. Acomplex of at least six proteins (GPI-GnT) is involved, ofwhich Gpi3 is catalytic because it can be labeled with a photo-activatable UDP-GlcNAc analog (Kostova et al. 2000). GlcNActransfer occurs at the cytoplasmic face of the ER membrane(Vidugiriene and Menon 1993; Watanabe et al. 1996; Tiedeet al. 2000). Essential Gpi2, Gpi15, and Gpi19 (Leidich et al.1995; Yan et al. 2001; Newman et al. 2005), and nonessentialGpi1 and Eri1 (Leidich and Orlean 1996; Sobering et al.2004), are also required for GlcNAc-PI synthesis. ERI1 andGPI1 null mutants are temperature-sensitive. The mammalianorthologs of these proteins form a complex (Watanabe et al.1998; Tiede et al. 2000; Eisenhaber et al. 2003; Murakamiet al. 2005), and the yeast proteins likely also do, for Eri1 andGpi19 associate with Gpi2 (Sobering et al. 2004; Newmanet al. 2005). Roles of the noncatalytic subunits are unclear.

Figure 4 Biosynthesis of O-linked glycans. (A) Addition ofa-Man by protein O-mannosyltransferases in the ER lu-men. Pmt4 homodimers act on membrane proteins orGPI proteins. Representative Pmt heterodimers are shown.(B) Extension of O-linked manno-oligosaccharides in theGolgi. Ktr1 family members have a collective role in addinga1,2-linked mannoses, and Mnt1 family members adda1,3-linked mannoses. The dominant Man-T active ateach step are shown in boldface type. Man-P may beadded to saccharides with two a1,2-linked Man.

786 P. Orlean

Ras2, in its GTP-bound form, can also join GPI-GnT(Sobering et al. 2004). Membranes from ras2D cells have8- to 10-fold higher in vitro GPI-GnT activity than wild-typemembranes, whereas membranes from cells expressingconstitutively active Ras2-Val19 have almost undetectableactivity. These findings indicate that Ras2-GTP is a negativeregulator of GPI-GnT, and, depending on the degree towhich the GTPase is activated, this could permit abouta 200-fold range of GlcNAc-PI synthetic activity.

Once formed, GlcNAc-PI is de-N-acetylated at the cyto-plasmic face of the ER membrane by Gpi12 (Vidugiriene andMenon 1993; Watanabe et al. 1999). GlcN-PI is the precursorlikely to be translocated to the lumenal side of the ER mem-brane. Its flipping has been reconstituted in rat liver micro-somes, but the protein involved is unknown (Vishwakarmaand Menon 2005).

Lumenal steps in GPI assembly: The inositol ring in GlcN-PIis next acylated on its 2-OH, making the glycolipid resistantto cleavage by PI-specific phospholipase C. The reaction usesacyl CoA as donor (Costello and Orlean 1992), and the acylchain transferred in vivo is likely palmitate. Gwt1, the acyl-transferase, was identified in a screen for resistance to 1-[4-butylbenzyl] isoquinoline, which inhibits surface expressionof GPI proteins (Tsukahara et al. 2003; Umemura et al.2003). Disruption of GWT1 is lethal or leads to slow growthand temperature sensitivity, depending on the strain back-ground (Tsukahara et al. 2003). The inositol acyl chain mayprevent GPIs from being translocated back to the cytoplas-mic side of the ER membrane (Sagane et al. 2011), be im-portant for later steps in GPI assembly or transfer to protein,or block the action of PI-specific phospholipases.

GlcN-(acyl)PI is next extended with four Man by GPI-Man-T I-IV, and the first three Man are concurrentlymodified with Etn-P by Etn-P-T I, II, and III. Dol-P-Mandonates the mannoses because the dpm1 mutant accumu-

lates GlcN-(acyl)PI (Orlean 1990). The first, a1,4-linkedMan (Man-1, Figure 5) is added by Gpi14 (Maeda et al.2001), and two additional proteins are involved at this step.One, Arv1, was originally implicated in ceramide and sterolmetabolism. ARV1 disruptants are impaired in ER-to-Golgitransport of GPI proteins and accumulate GlcN-(acyl)PIin vitro (but not in vivo), although they are not defective inin vitro GPI-Man-T-I or Dpm1 activity or in N-glycosylation,and it was proposed that Arv1 has a role in delivering GlcN-(acyl)PI to Gpi14 (Kajiwara et al. 2008). The second protein,Pbn1, was implicated at the GPI-Man-T-I step becauseexpression of both GPI14 and PBN1 is necessary to comple-ment mammalian cell lines defective in Pbn1’s mammalianhomolog Pig-X, and co-expression of PIG-X and the gene forGpi14’s mammalian homolog, PIG-M, partially rescues thelethality of gpi14D (Ashida et al. 2005; Kim et al. 2007).Furthermore, Pbn1 depletion leads to accumulation of someof the ER form of the GPI protein Gas1, a phenotype of GPIprecursor assembly mutants (Subramanian et al. 2006;File S4).

Addition of a1,6-linked Man-2 requires catalytic Gpi18(Fabre et al. 2005; Kang et al. 2005) and Pga1 (Sato et al.2007), which form a complex (Sato et al. 2007). Gpi18-deficient cells accumulate both a Man1-GPI with Etn-P esteri-fied to its Man and an unmodified Man1-GPI, suggesting thatGPI-Man-T-II can use either as acceptor (Fabre et al. 2005;Scarcelli et al. 2012).

Gpi10 and Smp3 successively add a1,2-linked Man-3 andMan-4 (Canivenc-Gansel et al. 1998; Sütterlin et al. 1998;Grimme et al. 2001). Smp3-dependent addition of Man-4 isessential because addition of this residue precedes additionof the Etn-P that subsequently becomes linked to protein(Grimme et al. 2001).

As the GPI glycan is extended, Etn-P moieties are addedto the 2-OH of Man-1 and to the 6-OH of Man-2 and Man-3

Figure 5 Biosynthesis of the GPI precursor and its transfer to protein in the ER membrane. GlcNAc addition to PI and de-N-acetylation of GlcNAc-PI toGlcN-PI occur at the cytoplasmic face of the ER membrane, and further additions to the GPI occur on the lumenal side of the ER membrane. Gpi18 andMcd4 need not act in a defined order. Man3- and Man4-GPIs either bearing Etn-P on Man-2 but not Man-1 or without any Etn-Ps (not shown) have alsobeen detected in radiolabeling experiments with certain late-stage GPI assembly mutants.

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(Orlean 2009). The Etn-Ps likely originate from Ptd-Etn(Menon and Stevens 1992; Imhof et al. 2000; File S4).The Etn-P-T-I, II, and III transferases are Mcd4, Gpi7, andGpi13, respectively, which are 830- to 1100-amino-acid pro-teins predicted to have 10–14 transmembrane domains anda large lumenal loop containing sequences characteristicof the alkaline phosphatase superfamily that are importantfor function (Benachour et al. 1999; Gaynor et al. 1999;Galperin and Jedrzejas 2001; File S4). GPI-Etn-P-T-II andIII also require small, hydrophobic Gpi11 for activity. mcd4mutants accumulate unmodified Man1 and Man2-GPI(Wiedman et al. 2007; Scarcelli et al. 2012), suggesting thatboth structures can serve as Etn-P acceptors. From this, andbecause Gpi18-depleted cells accumulate Etn-P-modifiedMan1-GPI (Fabre et al. 2005), it seems that both Mcd4and Gpi18 can use Man1-GPI as acceptor and then modifythe GPI that the other has acted on (Figure 5; File S4). Etn-Ptransfer to Man-1 and GPI-dependent processing of Gas1 areinhibited by the terpenoid lactone YW3548 (Sütterlin et al.1997, 1998). The Etn-P on Man-1 may enhance the ability ofGpi10 to add Man-3, promote export of GPI proteins fromthe ER, and be necessary for remodeling of the lipid moietyto ceramide (Zhu et al. 2006).

Gpi7 is the catalytic subunit of GPI-Etn-P-T-II, and GPI7nulls, which are viable but temperature-sensitive, accumulatea Man4-GPI with Etn-P on Man-1 and Man-3 (Benachouret al. 1999). Essential Gpi11 was implicated at this stepbecause Gpi11-deficient cells have similar GPI precursor ac-cumulation profiles to gpi7D (Taron et al. 2000). The Etn-Pon Man-2 enhances transfer of GPIs to protein, ER-to-Golgitransport of GPI proteins, GPI lipid remodeling to ceramide,transfer of GPI proteins to the wall, and targeting of certainGPI-anchored proteins in daughter cells (Benachour et al.1999; Toh-E and Oguchi 1999; Richard et al. 2002; Fujitaet al. 2004).

Gpi13 is the catalytic subunit of GPI-Etn-P-T-III. The ma-jor GPI accumulated upon Gpi13 depletion is a Man4-GPIwith a single Etn-P on Man-1 (Flury et al. 2000; Taron et al.2000). Gpi11 is likely involved in the GPI-Etn-P-T-III reac-tion because a gpi11-Ts mutant also accumulates a Man4-GPI with its Etn-P on Man-1 (K. Willis and P. Orlean, un-published results), and human Gpi11 interacts with andstabilizes human Gpi13 (Hong et al. 2000).

GPI transfer to protein: Man4-GPIs bearing three Etn-Psare transferred to proteins with a C-terminal GPI signal-anchor sequence in a transamidation reaction in which theamino group of the Etn-P on Man-3 acts as nucleophile. Fiveessential membrane proteins are involved: Gaa1, Gab1,Gpi8, Gpi16, and Gpi17 (Hamburger et al. 1995; Benghezalet al. 1996; Fraering et al. 2001; Ohishi et al. 2000, 2001;Hong et al. 2003; Grimme et al. 2004). Gpi18 is catalyticbecause it resembles cysteine proteases and mutation ofpredicted active site residues eliminates its function (Meyeret al. 2000). The five transamidase subunits form a complexitself consisting of two subcomplexes: one containing Gaa1,Gpi8, and Gpi16, and the other, Gab1 and Gpi17 (Fraering

et al. 2001; Grimme et al. 2004; Zhu et al. 2005). Roles forthe noncatalytic subunits include recognition of the peptideand glycolipid substrates (Signorell and Menon 2009), and,in the case of Gab1 and Gpi8, possible interactions with theactin cytoskeleton (Grimme et al. 2004; File S4)

Remodeling of protein-bound GPIs: Following GPI transferto protein, both the anchor’s lipid and glycan remodeled(Figure 6; Fujita and Kinoshita 2010). The earliest event,which occurs in the ER, is removal of the inositol acyl moietyby lipase-related Bst1 (Tanaka et al. 2004; Fujita et al.2006a). Next, the sn-2 acyl chain of the diacylglycerol isremoved by the ER membrane protein Per1 to generatea lyso-GPI (Fujita et al. 2006b), whereupon a C26:0 acylchain is transferred to the sn-2 position by Gup1 in the ERmembrane (Bosson et al. 2006). Modifications of the GPIlipid by Bst1, Per1, and Gup1 are necessary for efficienttransport of GPI proteins from the ER to the Golgi (File S4).

Many GPIs are next remodeled by replacement of theirdiacylglycerol with ceramide by Cwh43 (Martin-Yken et al.2001; Ghugtyal et al. 2007; Umemura et al. 2007). Ceramideremodeling requires prior action of Bst1, and, because per1Dand gup1D strains show defects in remodeling, the exchangereaction likely takes place after the first three lipid modificationsteps. The mechanism could involve a phospholipase-like re-action that replaces diphosphatidic acid with ceramide phos-phate or diacylglycerol with ceramide (Ghugtyal et al. 2007;Fujita and Kinoshita 2010). Ceramide remodeling is notobligatory because certain GPI proteins, such as Gas1, reachthe plasma membrane with a diacylglycerol-based anchor(Fankhauser et al. 1993). Moreover, ceramide remodelingdoes not seem to be required for incorporation of GPI pro-teins into the wall (Ghugtyal et al. 2007).

Further GPI processing events may be the removal of theEtn-P moieties from Man-2 and Man-1. This is inferred fromthe fact that mammalian PGAP5, which removes the side-branching Etn-P from Man-2 (Fujita et al. 2009), has twohomologs in yeast: ER-localized Ted1 and Cdc1. Export ofGas1 is retarded in ted1D cells, and genetic interactionsconnect TED1 and CDC1 with processing and export ofGPI proteins (Haass et al. 2007). Because Etn-P side chainsare important for ceramide remodeling, they are likely re-moved after Cwh43 has acted.

Finally, a fifth, a1,2- or a1,3-linked Man can be added toMan-4 of protein-bound GPIs (Fankhauser et al. 1993).This modification is made to 20–30% of GPI proteins andoccurs in the Golgi, but none of the many Golgi Man-T seemsto be involved (Sipos et al. 1995; Pittet and Conzelmann2007). On reaching the plasma membrane, the GPIs onmany proteins become cross-linked to b1,6-glucan (see In-corporation of GPI proteins into the cell wall), and these GPI-CWP play structural or enzymatic roles in the wall (see CellWall-Active and Nonenzymatic Surface Proteins and TheirFunctions).

No individual GPI protein is essential in unstressedwild-type cells, so the lethality of mutations blocking GPI

788 P. Orlean

anchoring may be due to the collective effects of retardingER exit and plasma membrane or wall anchorage of multipleproteins. Consistent with this, temperature-sensitive GPIanchoring mutants grown at semipermissive temperaturehave aberrant morphologies and shed wall proteins into themedium (Leidich and Orlean 1996; Vossen et al. 1997).

Sugar nucleotide transport

GDP-Man transport: Cytoplasmically generated GDP-Manused by Golgi Man-T is transported into the Golgi lumen byVrg4/Vig4. GMP, generated from GDP formed in Man-Treactions by GDPase activity, serves as antiporter. Vrg4/Vig4 is essential, and vrg4 mutants are defective in manno-sylation of N- and O-linked glycans and mannosyl inositol-phosphoceramides (Dean et al. 1997; Abe et al. 1999).

Two homologous Golgi proteins, Gda1 and Ynd1, haveGDP-hydrolyzing activity. Gda1 has the highest activity to-ward GDP (Abeijon et al. 1989), and, consistent with GMP’srole as antiporter, rates of in vitro GDP-Man import intoGolgi vesicles from gdaD cells are fivefold lower than thoseof vesicles from wild-type cells (Berninsone et al. 1994).Ynd1 is a broader specificity apyrase (Gao et al. 1999) thathas a partially overlapping function with Gda1, and bothYnd1 and Gda1 are necessary for full elongation of N- andO-linked glycans (Gao et al. 1999; File S5).

Other sugar nucleotide transport activities: Transportactivities for UDP-Glc, UDP-GlcNAc, and UDP-Gal also occurin S. cerevisiae (Roy et al. 1998, 2000; Castro et al. 1999), andthere are eight more candidate transporters (Dean et al.1997; Esther et al. 2008) whose functions are unclear. UDP-Glc transport activity is present in the ER (Castro et al. 1999),and one possible need for it might be for a glucosylation re-action at an early stage of b1,6-glucan assembly (see b1,6-Glucan). Yea4 is an ER-localized UDP-GlcNAc transporter

whose deletion impacts chitin synthesis (Roy et al. 2000; FileS6). Hut1 is a candidate UDP-Gal transporter (Kainuma et al.2001), although galactose has not been detected on S. cere-visiae glycans. Both Hut1 and Yea4 may have broader speci-ficity and transport UDP-Glc (Esther et al. 2008).

Biosynthesis of Wall Components at the PlasmaMembrane

Chitin

S. cerevisiae has three chitin synthase activities—CS I, CS II,and CS III—which require the catalytic proteins Chs1, Chs2,and Chs3, respectively. The Chs proteins are active in theplasma membrane although they originate from the roughER. The pathways for trafficking and activation of Chs2 andChs3 involve different sets of auxiliary proteins that ensurethe correct spatial and temporal localization of chitin syn-thesis during septation.

Septum formation: Factors determining the site at whicha bud will be formed, and the proteins that recruit andorganize the participants in septum formation, includingseptins and an actin–myosin contractile ring, are reviewedby Cabib et al. (2001), Cabib (2004), Roncero and Sanchez(2010), and Bi and Park (2012). Two chitin-containingstructures are made during bud emergence and septum for-mation (Figure 7). The first is a ring deposited in the wallaround the base of the emerging bud. This chitin is formedby Chs3 (Shaw et al. 1991), and, after cell separation,remains on the mother cell as a component of the bud scar.Upon completion of mitosis, the primary septum is formedby centripetal synthesis of chitin by Chs2 in the neck regionbetween mother cell and bud (Shaw et al. 1991). Uponclosure, the septum separates the plasma membranes of

Figure 6 Remodeling of protein-bound GPIs. The inositol palmitoyl group and the sn-2 acyl chain are removed by Bst1 and Per1, respectively, and Gup1transfers a C26:0 acyl chain to the sn-2 position. Cwh43 can replace diphosphatidic acid with ceramide phosphate (shown here) or diacylglycerol withceramide. Etn-P on Man-1 and Man-2 may be removed by Ted1 and Cdc1. Steps through Etn-P removal occur in the ER. An a1,2- or an a1,3-linkedMan is added to Man-4 in the Golgi by as yet unknown Man-T. At the plasma membrane, the GPI can be cleaved, possibly between GlcN and Man, andthe reducing end of the GPI remnant transferred to b1,6-glucan. Symbols are as used in Figure 1 and Figure 5.

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the two cells, accomplishing cytokinesis. In budding wild-type cells, the primary septum is thickened on both sides bydeposition of a secondary septum that normally containschitin, b1,3-glucan, b1,6-glucan, and covalently cross-linkedmannoprotein (Rolli et al. 2009), resulting in a three-layeredstructure (Shaw et al. 1991).

Chs2 and Chs3 have important roles in septation andcytokinesis although in the absence of Chs2 or Chs3, or in-deed of all three chitin synthases, cytokinesis can still takeplace. In chs2D mutants, the primary septum is missing, anda thick, amorphous septum is formed that contains chitinmade by Chs3 (Shaw et al. 1991; Cabib and Schmidt2003). chs3D mutants form a three-layered septum, butthe neck region between mother cell and bud is elongated(Shaw et al. 1991). chs2D chs3D and chs1D chs2D chs3Dstrains grow very slowly on osmotically supported medium(Sanz et al. 2004; Schmidt 2004; File S6). The triplemutants, however, acquired a suppressor mutation thateliminated the need for osmotic support and conferreda growth rate as fast as that of a chs2D mutant althoughover a third of suppressed and unsuppressed cells in a cul-ture were dead (Schmidt 2004).

For mother and daughter cells to separate, septalmaterial must be degraded, a process that results fromsecretion of chitinase Cts1 (Kuranda and Robbins 1991),endo-b1,3-glucanases Eng1/Dse4 and Scw11 (Cappellaroet al. 1998; Colman-Lerner et al. 2001; Baladron et al.2002; see Known and predicted enzymes), and possibly addi-tional activities from the daughter cell’s side of the septum.Daughter cell-specific expression of these enzymes is underthe control of the transcription factor Ace2 (Colman-Lerneret al. 2001).

Chitin synthase biochemistry: Chs1, Chs2, and Chs3 useUDP-GlcNAc as donor and are members of GT family 2 ofprocessive inverting glycosyltransferases, which includeshyaluronate and cellulose synthases. Yeast’s chitin synthasesare predicted to have three to five transmembrane helicestoward their C termini, and Chs3 likely has two more trans-membrane domains nearer its N terminus (Jimenez et al.2010; Merzendorfer 2011). Amino acid residues importantfor catalysis lie in a large cytoplasmic domain containingthe signature sequences QXXEY, EDRXL, and QXRRW(Nagahashi et al. 1995; Saxena et al. 1995; Cos et al. 1998;Yabe et al. 1998; Ruiz-Herrera et al. 2002; Merzendorfer2011). An additional motif, (S/T)WG(X)T(R/K), predictedto be extracellularly oriented (Merzendorfer 2011), lies nearthe protein’s C terminus (Cos et al. 1998; Merzendorfer 2011).

The molecular mechanism of chitin synthesis is not yetclear. By analogy with bacterial NodC, which synthesizeschito-oligosaccharides, and with nonfungal chitin synthases,chain extension would be at the nonreducing end (Kamstet al. 1999; Imai et al. 2003). This topic, and the issue ofhow the synthases overcome the steric challenge that eachsugar in a b1,4-linked polymer is rotated by �180� relativeto its neighbor, are discussed further in File S6.

Chitin made in vitro by CS I or CS III contains, on aver-age, 115–170 GlcNAc residues (Kang et al. 1984; Orlean1987). Chitin synthases presumably make chitin chains witha range of lengths, and the range would be predicted to shiftto shorter chains as UDP-GlcNAc concentration drops belowKm, resulting in lowered rates of chain extension. Indeed,purified Chs1 and membranes from cells overexpressingChs2 make chito-oligosaccharides at low substrate concen-trations (Kang et al. 1984; Yabe et al. 1998). Chitin madein vivo is polydisperse (Cabib and Duran 2005), and in-creased chitin chain lengths are seen in fks1D and gas1Dmutants and CFW-treated cells, which mount the chitinstress response, whereas shorter chains were made ina strain expressing a Chs4 variant with lower in vitro CSIII activity (Grabinska et al. 2007). However, GlcN treat-ment, which stimulates chitin synthesis in vivo (Bulik et al.2003; see Sugar nucleotides), had little effect on polymerchain length (Grabinska et al. 2007).

S. cerevisiae’s three chitin synthases are all stimulated upto a few fold in vitro by high concentrations of GlcNAc

Figure 7 Roles of chitin synthases II and III in chitin deposition duringbudding growth. (A) Chitin synthase III synthesizes a chitin ring (blue)around the base of the emerging bud. (B) The plasma membrane inva-ginates and chitin synthase II synthesizes the primary septum (red). Nochitin is made in the lateral walls of the bud. (C) Secondary septa (green)are laid down on the mother- and daughter-cell sides of the primaryseptum, and chitin synthase III starts synthesizing lateral wall chitin inthe bud (blue). (D) After cell separation, the bud scar (which is formedfrom the chitin ring made by Chs3), most of the primary septum made byChs2, as well as secondary septal material deposited on the mother cellside, remain on the mother cell. The birth scar on the daughter cellcontains residual chitin from the primary septum as well as secondaryseptal material. (E and F) Chitinase digestion of the primary septum fromthe daughter-cell side facilitates cell separation, and lateral wall chitinsynthesis continues as the daughter cell grows. Figure is adapted fromCabib and Duran (2005).

790 P. Orlean

(Sburlati and Cabib 1986; Orlean 1987). Possible explana-tions are that GlcNAc serves as a primer or allosteric activa-tor in the chitin synthase reaction (see File S6).

S. cerevisiae’s chitin synthases and auxiliary proteins:Chitin synthase I: Most, if not all, Chs1 activity is detectablein vitro only after pretreatment of membranes or extensivelypurified Chs1 with trypsin (Duran and Cabib 1978; Kanget al. 1984; Orlean 1987). Proteolytically activated Chs1has the highest in vitro activity of the chitin synthasesassayed in membranes from wild-type cells (Sburlati andCabib 1986; Orlean 1987), although Chs1 does not contrib-ute measurably to chitin synthesis in vivo, even in the ab-sence of Chs2 and Chs3 (Shaw et al. 1991). Although trypsinactivation may mimic the effect of an endogenous activatingprotease, neither such an activator, nor an active, processedform of Chs1, have been identified.

Levels of protease-elicited Chs1 activity are the same inmembranes from logarithmically growing and stationary-phase cells (Orlean 1987), and levels of Chs1 show littlechange during the cell division cycle (Ziman et al. 1996).CHS1 transcription and in vitro CS I activity increase in re-sponse to mating factors, but elevated in vitro activity isdetectable only after trypsin activation (Schekman andBrawley 1979; Orlean 1987; Appeltauer and Achstetter1989). However, Chs1 does not contribute to pheromone-induced chitin synthesis (Orlean 1987).

chs1D cultures contain the occasional lysed bud, a phenotypemore pronounced in acidic medium but partially alleviatedwhen Cts1 chitinase is also deleted (Cabib et al. 1989). Twoexplanations, which are not mutually exclusive, are that Chs1may repair wall damage due to overdigestion of chitin by Cts1or that Chs1 participates in septum synthesis and makes chitinduring growth in acidic medium (Cabib et al. 1989; Bulawa1993). Chs1 promotes wall association of at least one proteinbecause small amounts of the GPI protein Gas1 are releasedinto the medium from chs1D cells (Rolli et al. 2009).

Although the contribution of Chs1 to chitin synthesis issmall, a wider role for the protein emerged from an analysisof the networks of genes that interact synthetically withCHS1 and CHS3 (Lesage et al. 2005). Most of the 57 genesin the CHS1 interaction network fell into two sets. One setcontained genes that, when mutated, impact cell integrity orthat themselves interact with genes involved in b1,3-glucansynthesis, indicating a role for Chs1 in buffering the wallagainst changes impacting its robustness. The other set con-tained genes involved in budding and in endocytic proteinrecycling, which in turn may impact Chs2 function, suggest-ing that Chs1 also buffers against deficiencies in Chs2. TheCHS1-interacting genes were mostly distinct from the genesin the network that impacts Chs3 function, and, moreover,mutations in CHS1 itself or in the genes in the CHS1 inter-action set do not trigger the Chs3-dependent chitin stressresponse. Chs1 and Chs3 therefore have distinct functionsand one does not buffer against defects in the other (Lesageet al. 2005).

Chitin synthase II and proteins impacting its localizationand activity: Chs2 makes no more than 5% of the chitin inbudding cells. Activity of endogenous Chs2 is detectableonly in membranes from growing cells and can be stimu-lated by treatment with trypsin (Sburlati and Cabib 1986)although, in some studies, membrane preparations as wellas partially purified Chs2 have significant in vitro activitywithout prior trypsin treatment, raising the possibility thatfull-size Chs2 makes chitin (Uchida et al. 1996; Oh et al.2012). A soluble fraction from growing yeast cells, whichstimulates Chs2 activity two- to fourfold but which must itselfbe pretreated with trypsin, has been described (Martínez-Rucobo et al. 2009). An endogenously activated, processedform of Chs2 has not been identified (File S6).

Levels of CHS2 expression and localization of the proteinare coordinated with synthesis of the primary septum (Fig-ure 8). CHS2 message levels peak just prior to primary sep-tum formation at the G2/M phase (Pammer et al. 1992; Choet al. 1998; Spellman et al. 1998), and levels of Chs2 and CSII activity then peak as the primary septum is made (Pammeret al. 1992; Choi et al. 1994a; Chuang and Schekman 1996).Upon completion of cytokinesis, levels of Chs2 and its mes-sage drop, indicating that both turn over rapidly.

Temporal and spatial localization of Chs2 is impacted atat least two stages by protein kinases. Chs2 is synthesized inthe ER during metaphase, but its release from the ER iscoordinated with exit of the cell from mitosis and triggeredupon inactivation of mitotic kinase by Sic1 (Zhang et al.2006). The mitotic kinase Cdk1 likely acts directly onChs2, which contains four CDK1 phosphorylation sites nearits N terminus, because mutation of the target Ser residuesto Glu leads to retention of Chs2 in the ER, whereas chang-ing the serines to Ala leads to constitutive release of themutant Chs2 even in the presence of high Cdk1 activity(Teh et al. 2009). Timed release of Chs2 from the ER afterchromosome separation and exit of the cells from mitosis istriggered by dephosphorylation of the Cdk1 sites by theCdc14 phosphatase, the terminal component of the mitoticexit network (MEN) cascade (Chin et al. 2012).

Exit of Chs2 from the ER and its delivery to the plasmamembrane at the mother cell–bud junction is effected byCOPII vesicles (Chuang and Schekman 1996; VerPlankand Li 2005; Zhang et al. 2006). Localization of Chs2 atthe bud neck, correct formation of the primary septum,and removal of Chs2 at the end of cytokinesis depend onphosphorylation of Chs2 by the mitotic exit kinase Dbf2, alsoa component of MEN (Oh et al. 2012). Inn1 and Cyk3,whose localization to the division site is also regulated byMEN, are also involved in activation of Chs2 for primaryseptum formation (Nishihama et al. 2009; Meitinger et al.2010; Oh et al. 2012). Overexpression of CYK3 leads to in-creased deposition of chitin at the division site in chs1Dchs3D cells, where Chs2 is the sole chitin synthase(Oh et al. 2012). Cyk3 has a transglutaminase-like domain(Nishihama et al. 2009), but the nature of Cyk3’s effect onChs2 is unclear and Inn1’s role in Chs2 activation is unknown.

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Additional phosphorylation sites are present in Chs2’s N-ter-minal domain (Martínez-Rucobo et al. 2009), but their rolesare unclear.

Chs2 resides at the site of primary septum formation foronly 7–8 min (Roh et al. 2002a; Zhang et al. 2006). Theprotein is degraded upon endocytosis and delivery to thevacuole (Chuang and Schekman 1996; Schmidt et al.2002; VerPlank and Li 2005), and optimal endocytic turnoverof Chs2 requires components of the endosomal sorting com-plexes required for transport (ESCRT) pathway (McMurrayet al. 2011).

Chitin synthase III and proteins impacting its localizationand activity: Chitin synthase III is responsible for thesynthesis of .90% of the chitin in unstressed vegetativecells, for the additional chitin made in the chitin stress re-sponse and in response to mating pheromones, and for thesynthesis of the chitin that is de-N-acetylated to chitosanduring ascospore wall formation. Cells deficient in CS IIIactivity are resistant to CFW (Roncero et al. 1988). Chs3 isthe transferase, but its function depends on its regulatedtransport from the ER to the plasma membrane, its removalfrom the plasma membrane and sequestration in intracellu-lar vesicles called chitosomes, and its remobilization fromchitosomes to the plasma membrane. A number of proteinsare required for regulated Chs3 trafficking and for enzymeactivity (Bulawa 1993; Trilla et al. 1999; Roncero 2002).

CS III [referred to as chitin synthase II by Orlean (1987)]is the major, if not only, activity detected in membrane frac-tions from logarithmically growing wild-type cells withoutprior treatment with trypsin and is trypsin sensitive (Orlean1987). CS III activity determined in this way is presumablyeither due to constitutively active Chs3 or to an endogenouslyactivated form of the protein. A pool of trypsin-activatableCS III was detected in detergent-treated membranes fromchs1D chs2D cells or from cells lacking Chs4, an activatorof CS III (Choi et al. 1994b; Trilla et al. 1997). The latterfinding, together with the observation that overexpression ofChs4 lowers the extent to which trypsin activates CS III, sug-gested that trypsin treatment might mimic Chs4-dependentprocessing of Chs3. However, because no endogenously pro-cessed forms of Chs3 have been detected (Santos andSnyder 1997; Cos et al. 1998), and because Chs4 does notresemble any protease, the apparent zymogenicity of Chs3in chs4D may be an artifact (Reyes et al. 2007).

Levels of CHS3 mRNA and Chs3 vary little during thebudding cycle (Choi et al. 1994a; Chuang and Schekman1996; Cos et al. 1998), indicating that CS III is regulated atthe post-translational level. Sequences involved include theC-terminal extracellular region containing the motif (S/T)WG(X)T(R/K), which is required for in vitro CS III activityand chitin synthesis in vivo (Cos et al. 1998).

A number of proteins interact with Chs3 as it transits thesecretory pathway (Figure 9). Chs3 is palmitoylated in theER by Pfa4 (Lam et al. 2006; Montoro et al. 2011). pfa4Dmutants are CFW-resistant and accumulate Chs3 in the ER,indicating a role for palmitoylation in Chs3 export (Lam et al.

2006). Chs3 has two palmitoylation sites in a cytoplasmic do-main N-terminal to the proposed catalytic residues (Meissneret al. 2010). Exit of Chs3 from the ER also requires Chs7, anER chaperone with six or seven transmembrane domains(Trilla et al. 1999) that interacts with Chs3. The effects ofCHS7 deletion on chitin levels and CSIII activity are almostas severe as those of CHS3 deletion (Trilla et al. 1999). Chs3aggregates in the ER in chs7D cells (Lam et al. 2006), andChs7 is a limiting factor in export of Chs3 because simulta-neous overexpression of CHS3 and CHS7 leads to elevatedCSIII activity, whereas overexpression of CHS3 alone does not.Neither Pfa4 nor Chs7 is required for exit of Chs1 and Chs2from the ER (Trilla et al. 1999; Lam et al. 2006). ER-membraneproteins Rcr1 and Yea4 also impact Chs3-dependent chitinsynthesis in ways that are unclear (File S6).

Transport of new Chs3 from the trans-Golgi to the plasmamembrane, as well as Chs3 cycling from chitosomes to theplasma membrane, requires the peripheral Golgi membraneproteins Chs5 and Chs6 (Santos and Snyder 1997; Santos

Figure 8 Trafficking and regulation of Chs2. Cell cycle-regulated expres-sion of CHS2 peaks at the G2-M phase transition, and Chs2 is synthesizedat the ER. Phosphorylation of Chs2 by Cdk1 retains Chs2 in the ER. Uponchromosomal separation, Cdc14-dependent dephosphorylation of Chs2allows release of the protein from the ER and its transit to the mothercell–bud junction. Inn1 and Cyk3, localized at the division site, are in-volved in Chs2 activation. After primary septum formation is complete,Chs2 is endocytosed and degraded. Localization, function, and subse-quent removal of Chs2 when the primary septum is complete dependon phosphorylation by Dbf2. Figure is adapted from Lesage and Bussey(2006).

792 P. Orlean

et al. 1997; Ziman et al. 1998). Chs6 and its homologs Bch1,Bch2, and Bud7, referred to as Chs5-Arf1-binding proteins,join with Chs5 to form exomer complexes that transientlybind Chs3 to promote its incorporation into secretoryvesicles (Sanchatjate and Schekman 2006; Trautwein et al.2006; Wang et al. 2006). Although Chs5 and Chs6 act ina complex, the two have different impacts on Chs3 activityand transport. chs5D and chs6D mutants make 25 and 10%of wild-type amounts of chitin, respectively, but whereaschs5D membranes lack in vitro CS III activity, this activityis normal in chs6D membranes (Bulawa et al. 1993; Santoset al. 1997). This may be because, in chs5D cells, Chs3 accu-mulates in late Golgi vesicles (Santos and Snyder 1997),whereas, in chs6D mutants, it collects in chitosomes, whereit may encounter a chitosomal activator (Ziman et al. 1998).Exomer has a role in the transport of the chitin-b1,3-glucan

cross-linker Crh2 to the cell surface. Cotransport of Chs3and Crh2 would ensure colocalization of these proteins forefficient cross-linking of chitin to b1,3-glucan.

At the plasma membrane, Chs4 (Csd4/Skt5) interactswith Chs3 (DeMarini et al. 1997; Ono et al. 2000; Meissneret al. 2010) and has two roles apparently specific to Chs3.chs4D mutants lack in vitro CS III activity and make verylittle chitin (Bulawa 1993; Trilla et al. 1997). Overexpres-sion of CHS4, but not CHS3, raises in vitro CS III activity(Bulawa 1993; Trilla et al. 1997; Ono et al. 2000) as well aslevels of Chs3 in the plasma membrane (Reyes et al. 2007),suggesting that Chs4 is an activator of CS III. Stimulation ofCS III by Chs4 requires a region of Chs4 to bind Chs3 be-cause the ability of truncated forms of Chs4 to elicit CS IIIactivity correlates with the ability of Chs4 fragments to in-teract with Chs3 in a two-hybrid analysis (Ono et al. 2000;Meissner et al. 2010). Chs4 has a C-terminal farnesylationsite (Bulawa et al. 1993; Trilla et al. 1997; Grabinska et al.2007) whose roles are discussed in File S6.

Chs4 not only activates Chs3, but also mediates Chs3localization on the mother cell’s plasma membrane at thesite of formation of the chitin ring prior to bud emergence.There, it interacts with the scaffold protein Bni4, whichin turn associates with the septins (DeMarini et al. 1997;Kozubowski et al. 2003; Sanz et al. 2004). Absence of Bni4leads to mislocalized deposition of chitin (DeMarini et al.1997; Kozubowski et al. 2003; Sanz et al. 2004), and Chs4is absent from the base of buds in small-budded cells (Sanzet al. 2004). In contrast to chs4D mutants, chitin synthesisand CS III activity are not dramatically affected in bni4D cells,suggesting that Bni4 is not required for CS III activity per se(Sanz et al. 2004).

Chs3 and Chs4 are associated with the plasma membranejust before formation of the chitin ring at the site of budemergence and reside there in a ring at the base of thebud in many cells with small buds, then become scarcelydetectable in cells with medium-sized buds, only to reap-pear, in a Bni4-independent manner, at both sides of theneck in cells with large buds prior to cytokinesis (Chuangand Schekman 1996; DeMarini et al. 1997; Santos andSnyder 1997; Kozubowski et al. 2003; Sanz et al. 2004).In between, Chs3 is retrieved from the membrane to chito-somes in an endocytic process dependent on End4/Sla2(Chuang and Schekman 1996; Ziman et al. 1996, 1998),but is recruited back to the plasma membrane in a Chs6-dependent manner (Ziman et al. 1998; Wang et al. 2006).Chs3’s itinerary is consistent with the overall order of eventsin yeast cytokinesis.

Chitin synthesis in response to cell wall stress: Cells withmutations affecting the formation of b-glucan, mannan,O-linked glycans, and GPI anchors respond by depositingadditional chitin—as much as 10 times more than in wild-type cells—in their lateral walls in apparent compensationfor compromised cell integrity (Gentzsch and Tanner 1996;Kapteyn et al. 1997, 1999a; Popolo et al. 1997; Dallies et al.

Figure 9 Overview of Chs3 trafficking. Chs3, synthesized in the ER,requires palmitoylation by Pfa4 and association with Chs7 to exit theER. In the trans-Golgi, Chs3 association with exomer components Chs5and Chs6 facilitates incorporation of Chs3 into secretory vesicles for de-livery to the plasma membrane at the site of chitin ring formation. Local-ization and activation of Chs3 depends on association with Chs4, whoseassociation with the septin ring is mediated in turn via an interaction withBni4. In cells with medium-sized buds, Chs3 is retrieved from the plasmamembrane and sequestered in chitosomes in an endocytic processdepending on End4 and later recruited back to the neck region ina Chs6-dependent manner. During the cell wall stress response, Rho1and Pkc1 trigger mobilization of Chs3 from chitosomes to the plasmamembrane for synthesis of extra chitin in the lateral wall. Figure is adap-ted from Lesage and Bussey (2006).

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1998; Osmond et al. 1999; Garcia-Rodriguez et al. 2000;Valdivieso et al. 2000; Carotti et al. 2002; Lagorce et al.2002; Magnelli et al. 2002; Sobering et al. 2004; Lesageet al. 2005). This chitin stress response, which is accompa-nied by increased precursor supply (see Precursors and Car-rier Lipids), requires Chs3 and is dependent on Chs4, -5, -6,and -7 in gas1D cells (Valdivieso et al. 2000; Carotti et al.2002). The response does not involve upregulation of theCHS genes, but, rather, an altered distribution of Chs3,which was seen in the plasma membrane of buds of gas1Dand fks1D cells, and Chs4 was also delocalized (Garcia-Rodriguez et al. 2000; Valdivieso et al. 2000; Carotti et al.2002; Valdivia and Schekman 2003). Interestingly, gas1Dsuppressed the lysed bud phenotype of chs1D, suggestingthat the chitin stress response also repaired weakened budwalls (Valdivieso et al. 2000). The Chs3 making the stressresponse originates from chitosomes, and its translocation tothe plasma membrane is regulated by Rho1 and Pkc1, whichact early in the CWI pathway that triggers the chitin stressresponse (Valdivia and Schekman 2003).

Chitin synthase III in mating and ascospore wall forma-tion: Chitin synthase III is responsible for the extra chitinmade in response to mating pheromones and for formationof the chitosan of ascospore walls. MATa cells treated witha-factor show a three- to fourfold increase in chitin, which islaid down diffusely in the shmoo (Schekman and Brawley1979). Chs3 is necessary because no extra chitin is made inpheromone-treated chs3D cells, and the response is eitherabolished or much smaller in chs5D, chs6D, and chs4D cells,indicating that the machinery for trafficking and activationof Chs3 is required (Orlean 1987; Roncero et al. 1988;Bulawa 1993; Santos and Snyder 1997; Bulik et al. 2003).Consistent with its role in chitin deposition, Chs3 is localizedat the periphery of the mating projection, and it remainsthere because it is not subject to endocytic turnover as it isin budding cells (Santos and Snyder 1997; Sacristan et al.2012). Although the extra chitin synthesis in response toa-factor is presumably driven by the increased amountof UDP-GlcNAc made during the pheromone response(Orlean et al. 1985; Bulik et al. 2003), the mechanism be-hind pheromone-stimulated chitin synthesis by Chs3 is unclear.Levels of Chs3 increase sixfold upon a-factor treatment (Coset al. 1998), but neither CHS3 transcription nor levels ofchitin synthase III activity are elevated (Orlean 1987; Choiet al. 1994a). Factors that might limit total Chs3 activitymight include prevention of the mobilization of the proteinto the plasma membrane in shmoos or interference withinteractions between Chs3 and regulatory proteins (Choiet al. 1994a).

The chitosan of the ascospore wall is initially synthesizedas chitin by Chs3 (Pammer et al. 1992) and is then de-N-acetylated by chitin deacetylases Cda1 and Cda2, of which Cda2has the dominant role (Mishra et al. 1997; Christodoulidouet al. 1999). From the sporulation defects in mutants in proteinsinvolved in Chs3 trafficking in vegetative cells, Chs6 and Chs7,

but not Chs5, have as-yet-undefined roles in ascospore matura-tion (Santos et al. 1997; Trilla et al. 1999). A Chs4 homolog,Shc1, has a regulatory role in chitosan synthesis (Sanz et al.2002; File S6). Ascospore wall structure and assembly arereviewed by Neiman (2011).

b 1,3-Glucan

De novo b1,3-glucan synthetic activity is associated withmembers of the Fks family, of which Fks1 and Fks2 requirethe soluble Rho1 GTPase as a regulatory subunit. In vitroactivity is membrane-associated, uses UDP-Glc as donor, isstimulated by GTP via Rho1, and yields a product with achain length of 60–80 glucoses (Shematek et al. 1980; Kangand Cabib 1986; Drgonová et al. 1996; Mazur and Baginsky1996; Qadota et al. 1996). In vitro b1,3-glucan synthaseactivity is inhibited by acylated cyclic hexapeptides of theechinocandin group and by papulocandins, acylated deriva-tives of b1,4-galactosylglucose (Debono and Gordee 1994;Georgopapadakou and Tkacz 1995).

Fks family of b1,3-glucan synthases: Fks1 (Cwh53/Etg1/Gsc1/Pbr1), Fks2, and Fks3 are in GT family 48, which alsocontains proteins implicated in callose sythesis in plants(Verma and Hong 2001). Fks1 has an N-terminal cytoplas-mic domain that is followed by 6 transmembrane helices,a large cytoplasmic domain, and then 10 transmembranehelices (Inoue et al. 1995; Mazur et al. 1995; Qadota et al.1996; Dijkgraaf et al. 2002; Okada et al. 2010). Three func-tional domains, mutations in which separately affect in vivoand in vitro b1,3 glucan synthetic activity, as well as cellpolarity and endocytosis, have been distinguished (Okadaet al. 2010; File S7). The phenotypes of fks1 mutants mayin part reflect the involvement of the protein in processesother than b1,3-glucan biosynthesis. For example, muta-tions in both FKS1 and FKS2 result in lowered b1,6-glucansynthesis (Dijkgraaf et al. 2002). Fks1 is localized to theplasma membrane at sites of polarized growth and cell wallremodeling throughout the cell cycle, and this localizationcoincides with that of actin patches (Qadota et al. 1996;Dijkgraaf et al. 2002; Utsugi et al. 2002). Fks1 transits thesecretory pathway because it accumulates intracellularly invesicular transport mutants and its activity is sensitive tophytosphingosine levels in the ER (El-Sherbeini and Clemas1995; Abe et al. 2001; File S7).

Roles of the Fks proteins in b1,3-glucan synthesis: The Fksproteins show a degree of specialization. Deletion of FKS1leads to slow growth, a 75% reduction in b1,3-glucan, andlow in vitro b-1,3-glucan synthase activity, whereas thein vitro activity of fks2D membranes is nearly that of wild-type membranes and the disruptants have no defect in veg-etative growth (Inoue et al. 1995; Mazur et al. 1995). Al-though this suggests that Fks1 is the major contributor tob1,3-glucan synthesis in budding cells, fks1D fks2D nullmutants are inviable, indicating that Fks1 and Fks2 haveoverlapping functions (Inoue et al. 1995; Mazur et al.

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1995). Consistent with this, overexpression of either FKS1or FKS2 can partially correct the defects caused by deletingthe other of the two genes (Mazur et al. 1995; Dijkgraafet al. 2002), and, furthermore, the two proteins colocalizein sites of polarized growth in budding cells, although Fks1is the most abundant (Dijkgraaf et al. 2002).

FKS1 and FKS2 show different expression patterns. FKS1is expressed during budding growth and transcript levelspeak in the late G1 and early S phases (Mazur et al. 1995;Ram et al. 1995; Lesage and Bussey 2006). FKS2 mRNA, incontrast, cannot be detected in budding cultures grown inglucose, but appears when glucose becomes depleted; whencells are grown on acetate, glycerol, or galactose; when cellsare treated with a-factor or Ca2+; in fks1 mutants andmutants defective in the synthesis of other wall polymers;and when cells are stressed by shift to high temperature(Mazur et al. 1995; Zhao et al. 1998; Lesage and Bussey2006). Induction of FKS2 is mediated via the PKC CWIand calcineurin pathways (Mazur et al. 1995; Ram et al.1995; Zhao et al. 1998; Lagorce et al. 2003).

Fks2 is important in sporulation because fks2D fks2D dip-loids have a severe defect in this process (Mazur et al. 1995;Huang et al. 2005; Ishihara et al. 2007). Homozygous fks3Dfks3D diploids also form abnormal spores, indicating a rolefor Fks3 in ascopore wall formation, although Fks3’s role insporulation does not overlap with Fks2’s. It was proposedthat Fks2 is primarily responsible for synthesis of b1,3-glucanin the ascospore wall and that Fks3, rather than functioningas a synthase, modulates glucan synthesis during ascosporewall formation (Ishihara et al. 2007; File S7).

After their export through the plasma membrane, b1,3-glucan chains can be cross-linked to chitin by Crh1 and Crh2(Cabib 2009), and the polymer can be extended through theaction of Gas1 family b1,3-glucanosyltransferases (Mouynaet al. 2000), and side-branching b1,6-linked glucoses as wellas PIR proteins may be attached (Ecker et al. 2006; see In-corporation of PIR proteins into the cell wall and Exg1, Exg2,and Ssg/Spr1 exo-b1,3-glucanases).

Deficiencies in Fks1 are compensated for by Chs3-dependentchitin synthesis (Garcia-Rodriguez et al. 2000; Valdiviesoet al. 2000; Carotti et al. 2002), and fks1D shows syntheticinteractions with chs3D, chs4D, chs5D, chs6D, and chs7D(Osmond et al. 1999; Lesage et al. 2004), but correct syn-thesis of other wall constituents is also necessary whenb1,3-glucan synthesis is compromised (Lesage et al.2004). Analyses of the genome-wide responses to FKS1deletion revealed upregulation of a “cell wall compensa-tory cluster” of 79 coregulated genes whose products in-clude a range of proteins involved in wall synthesis andremodeling (Terashima et al. 2000; Lagorce et al. 2003).An overlapping set of genes, whose products function inthe biosynthesis of chitin, b1,6-glucan, and mannan, aswell as in the function of the secretory pathway and inmaintenance of cell polarity, was identified in an analysisof the synthetic genetic interactions of fks1D (Lesage et al.2004). This study showed that FKS2 made interactions

only with FKS1 and that FKS3 made no interactions, consis-tent with differential expression of FKS2 and FKS3 (Lesageet al. 2004).

Rho1 GTPase, a regulatory subunit of b1,3-glucan synthase:The essential Rho1 GTPase, which activates Pkc1 in the CWIpathway and is required for cell cycle progression and po-larization of growth (Drgonová et al. 1999; Levin 2011), hasa distinct role as a regulatory subunit of b1,3-glucan syn-thetic complexes containing Fks proteins. Evidence for this isthat (i) Fks1 and Rho1 colocalize and coimmunoprecipitate,(ii) membranes from a temperature-sensitive rho1 mutanthave a thermolabile b1,3-glucan synthase activity that canbe corrected by adding back purified Rho1, (iii) membranesfrom cells expressing a consitutively active rho1 allele haveGTP-independent b1,3-glucan synthase activity, and (iv) in-activation of Rho1 by ADP ribosylation eliminates thein vitro b1,3-glucan synthase activity of membranes fromfks1D and fks2D strains (Drgonová et al. 1996; Mazur andBaginsky 1996; Qadota et al. 1996). Moreover, there arerho1 mutations that affect regulation of b1,3-glucan synthe-sis, but not other Rho1 functions, and the amino acids af-fected are different from those whose mutation causes cellcycle and polarization defects (Saka et al. 2001; Roh et al.2002b). The amino acid changes in the b1,3-glucan synthesis-specific rho1 mutants might impact binding to Fks proteins,but the interacting domains on the regulatory and catalyticsubunits have not been defined. The Rho1-Fks interaction atthe cytoplasmic face of the plasma membrane, as well asactivation of b1,3-glucan synthesis, requires Rho1 to be ger-anylgeranylated at its C terminus (Inoue et al. 1999).

b1,6-Glucan

Mutations in genes with products localized along the secretorypathway impact formation of b1,6-glucan (Shahinian andBussey 2000; Lesage and Bussey 2006), but the biochemistryof b1,6-glucan synthesis is unclear. In vitro synthesis of b1,6-glucan is hard to detect, and no fungal enzyme has yet beenshown to catalyze formation of a b1,6-glucosidic linkage usingUDP-Glc as donor, although the linkage can be generated bythe Bgl2 protein in a transglycosylation reaction (Goldman et al.1995). Synthesis of b1,6-glucan is normal in alg5D mutants,indicating that Dol-P-Glc is not involved in formation of thispolymer (Shahinian et al. 1998; Aimanianda et al. 2009).

In vitro synthesis of b1,6-glucan

Because b1,6-glucan is a linear polymer with side brancheson average every fifth Glc (see b-glucans), it could be gen-erated by a processive, UDP-Glc-dependent b1,6-glucan syn-thase and then branched or by assembly of shorter repeatunits, whose glucoses originate from UDP-Glc. Detection ofUDP-Glc-dependent formation of b1,6-glucan is complicatedby the fact that UDP-Glc is also the donor in the synthesis ofb1,3-glucan, glycogen, and glucolipids.

S. cerevisiae Cell Wall 795

Two assays of the formation of b1,6-glucan using UDP-Glc as donor have been described. In the first, formation ofb1,6-glucanase-sensitive polymer by membranes was de-tected by dot-blot assay using an anti-b1,6 glucan antibody(Vink et al. 2004). The reaction was distinguished fromb1,3-glucan synthase because membranes from kre5 mu-tants, which make little b1,6-glucan but have normalb1,3-glucan synthetic capability, made little b1,6-glucanin vitro but had nearly wild-type b1,3-glucan synthase ac-tivities. Comparisons of the activities of wild-type and b1,6-glucan synthesis-defective strains revealed that levels ofb1,6-glucan formed de novo correlated with the reductionin b1,6-glucan synthesis in vivo. It was proposed that thedot-blot assay measured b1,6-glucan chain extension andthat higher rates of Glc transfer reflected the presence ofmore acceptor (Vink et al. 2004). The reaction was stimu-lated by GTP and higher b1,6-glucan synthetic activity wasdetected in membranes from cells overexpressing Rho1GTPase, suggesting that b1,6-glucan synthase, like b1,3-glucansynthase, is Rho1-dependent (Vink et al. 2004).

In the second approach, formation of b1,6-glucan wasmeasured in cells permeablized by osmotic shock and incu-bated with radiolabeled UDP-Glc (Aimanianda et al. 2009).The insoluble, radiolabeled b1,6-glucan formed was chem-ically identical to the branched b1,6-linked glucan isolatedfrom cell walls, and radioactivity was distributed throughoutthe in situ product, indicating that de novo polymerization ofb1,6-glucan had occurred (Aimanianda et al. 2009). Consis-tent with their severe in vivo defects in b1,6-glucan synthe-sis, permeabilized kre5 and kre9 mutants showed no in situb1,6-glucan synthetic activity, but made b1,3-glucan. Theb1,6-glucan synthetic activity in permeabilized cells wasnot stimulated by GTP. However, because b1,3-glucan syn-thesis mutants make less b1,6-glucan, and vice versa, for-mation of the two polymers may be coordinated in anotherway (Dijkgraaf et al. 2002; Aimanianda et al. 2009; see TheFks family of b1,3-glucan synthases).

Proteins involved in b1,6-glucan assembly

Mutants defective in b1,6-glucan synthesis were identifiedin screens for resistance to K1 killer toxin, which uses b1,6-glucan as its receptor (Hutchins and Bussey 1983), and inscreens for CFW sensitivity (Ram et al. 1994; Lussier et al.1997b; Orlean 1997; Shahinian and Bussey 2000; Pagéet al. 2003). In these mutants, levels of alkali-insoluble b1,6-glucan were lowered to different extents, and the propor-tions of b1,6- and b1,3-linked Glc residues in the alkali-insoluble glucan fraction were often altered. The finding thatthe proteins implicated in b1,6-glucan assembly were local-ized in the ER, Golgi, or plasma membrane, together withdemonstrations of epistasis relationships and genetic interac-tions, led to the notion of a secretory pathway-based pathwayfor b1,6-glucan elaboration (Boone et al. 1990; reviewed byOrlean 1997 and Shahinian and Bussey 2000). However,b1,6-glucan is not detectable intracellularly (Montijn et al.1999), and the roles of most of the proteins so far implicated

are indirect. Proteins affecting the formation of b1,6-glucanwill be discussed in the order of their location along thesecretory pathway.

ER proteins: Homologs of the UGGT/calnexin protein qualitycontrol machinery: Four homologs of proteins involved in theUGGT/calnexin protein quality control system (see N-glycanprocessing in the ER and glycoprotein quality control) are re-quired for formation of normal amounts of b1,6-glucan(Jiang et al. 1996; Abeijon and Chen 1998; Shahinianet al. 1998; Simons et al. 1998). These are diverged UGGThomologs Kre5, Gls1/Cwh41, Gls2/Rot2, and Cne1, ofwhich Kre5 has the most important role because kre5mutants make no more than 5% of normal amounts of b1,6-glucan (Meaden et al. 1990; Montijn et al. 1999; Levinsonet al. 2002; Aimanianda et al. 2009). The contributions ofthe glucosidases and calnexin are likely indirect ones inmaintaining normal levels of unknown components of theb1,6-glucan assembly machinery in the secretory pathway(Shahinian et al. 1998; Lesage and Bussey 2006). The es-sential function of Kre5 is other than as a UGGT in proteinquality control because kre5D remained lethal in an alg8Dgls2D background in which all N-glycans stayed monoglu-cosylated, thereby bypassing the need for UGGT activity(Shahinian et al. 1998). Kre5 could be a glucosyltransferasewith a specialized role in quality control of b1,6-glucanassembly proteins (Levinson et al. 2002; Herrero et al.2004; Lesage and Bussey 2006), or it could glucosylatethe GPI glycan of future GPI-CWPs to generate a signalor attachment point for subsequent transfer to b1,6-glucan(Shahinian and Bussey 2000). S. cerevisiae has the neces-sary ER UDP-Glc transport activity to supply the donor(Castro et al. 1999).

N-glycosylation is important for wild-type levels of b1,6-glucan to be made. For example, stt3 mutants have a severedefect in b1,6-glucan synthesis and are synthetically lethalwith kre5 and kre9 (Chavan et al. 2003b). This may reflecta requirement for N-glycosylation of one or more b1,6-glu-can synthetic proteins or for an N-glycan to serve as acceptorfor initiation of a b1,6-glucan chain (Lesage and Bussey2006). Interestingly, mutations such as och1 andmnn9, whichaffect synthesis of the a1,6-mannan backbone, and mnn2,which blocks addition of the first, a1,2 side-branching Man,show elevated levels of b1,6-glucan (Magnelli et al. 2002;Pagé et al. 2003), suggesting that a balance is normally main-tained between these two polymers.

Fungus-specific ER chaperones required for b1,6-glucansynthesis: Mutations in genes encoding the ER-localized,fungus-specific membrane proteins Rot1, Big1, and Keg1 allcause a b1,6-glucan synthetic defect. ROT1 and BIG1 nullmutants grow only with osmotic support, and even thenvery slowly, and in this and in the severity of their b1,6-glucan synthetic defect—a 95% reduction—they resemblekre5D strains (Bickle et al. 1998; Azuma et al. 2002; Machiet al. 2004). Levels of b1,3-glucan and chitin are elevated inrot1D and big1D. The b1,6-glucan defect in keg1D cells is

796 P. Orlean

similar to that in kre6D—about a 50% reduction (Nakamataet al. 2007).

Rot1, Big1, and Keg1 are small proteins that show nosimilarity to one another or to carbohydrate-active enzymes(Lesage and Bussey 2006). They seem to function as ERchaperones with varying degrees of importance for the stabil-ity of proteins involved in b1,6-glucan synthesis and may insome cases cooperate. Observations supporting this notionand indicating a relationship to Kre5 are discussed in File S8.

More widely distributed secretory pathway proteins: Kre6and Skn1: Kre6 and Skn1 are homologous type 2 membraneproteins in GH family 16 of b-1,6/b-1,3-glucanases (Henrissatand Davies 1997; Montijn et al. 1999). kre6D cells make halfnormal amounts of b1,6-glucan, whereas skn1D cells makeb1,6-glucan normally and have no growth defect. Expressedat high copy, Skn1 restores almost normal levels of b1,6-glucan to kre6D cells, and kreD skn1D double mutants areinviable or very slow growing, depending on the strain back-ground, and make no more than 10% of normal amounts ofb1,6-glucan (Roemer et al. 1993). From this, Kre6 and Skn1seem to be functional homologs, with Kre6 normally havingthe dominant role in b1,6-glucan synthesis. As hydrolases ortransglycosylases, Kre6 and Skn1 could act on a structurethat serves as a precursor or acceptor in elaboration of b1,6-glucan or on a glycoprotein involved in b1,6-glucan synthe-sis (Lesage and Bussey 2006), but enzyme activity has yet tobe demonstrated for these proteins. Much of Kre6 is ER-localized, where it interacts with Keg1, but the protein isalso detectable in the Golgi, in secretory vesicles, and atthe plasma membrane at sites of polarized growth (Liet al. 2002; Nakamata et al. 2007; Kurita et al. 2011; FileS8). Localization of Skn1 has not been explored in detail.Skn1 also has a role in the formation of mannosyl diinosi-tolphosphoryl ceramide [M(IP)2C], because skn1D, but notkre6D strains, is defective in M(IP)2C (Thevissen et al. 2005;File S8).

Kre6 has been implicated in the mode of action of a pyr-idobenzimidazole derivative identified in a screen for inhib-itors of cell wall incorporation of a reporter GPI-CWP(Kitamura et al. 2009). Because cells treated with this com-pound showed lowered incorporation of radiolabeled Glcinto a b1,6-glucan fraction, and because a resistant mutanthad an amino acid substitution in Kre6, it was proposed thatthe compound is an inhibitor of b1,6-glucan synthesis andthat Kre6 is its likely target (Kitamura et al. 2009).

Kre9 and Knh1: Kre9 and Knh1 are 30 kDa, soluble, fun-gus-specific, O-mannosylated proteins that are secreted intothe medium when overproduced (Brown and Bussey 1993;Dijkgraaf et al. 1996). The two are functional homologs,with Kre9 having the dominant role. kre9 nulls are slowgrowing and show an 80% reduction in b1,6-glucan, andthe residual b1,6-glucan in them has about half the molec-ular mass as the wild-type polymer and is altered in its pro-portion of b1,6 and b1,6 linkages (Brown and Bussey 1993).The size and structure of b1,6-glucan made in knh1D cells is

normal. Overexpression of KNH1 corrects the growth andb1,6-glucan defects of kre9D, but the kre9 skn1 combinationis synthetically lethal (Dijkgraaf et al. 1996). kre9D, but notknh1D, is also synthetically lethal with kre1D (see below)and kre6D, but not with skn1D. KRE9’s genetic interactionsindicate that its product has a pleiotropic impact on b1,6-glucan formation, although its effects must be exerted afterKre5’s because the kre5D kre9D double null has the samephenotype as kre5D (Dijkgraaf et al. 1996; Shahinian andBussey 2000). Neither Kre9 nor Knh1 shows similarity toproteins of known function. If they are not enzymes, Kre9and Knh1 may serve to anchor b1,6-glucan in the cell wall(Lesage and Bussey 2006), but this must be reconciled withthe finding that kre9 mutants have no UDP-Glc-dependentb1,6-glucan synthetic activity (Aimanianda et al. 2009).

Plasma membrane protein Kre1: Kre1, a GPI protein, func-tions at the plasma membrane or in the wall. kre1D cellsmake 40% of wild-type levels of b1,6-glucan, but this glucanis smaller and its b1,3 side branches are not extended(Boone et al. 1990; Roemer and Bussey 1995). GPI attach-ment is necessary for Kre1’s function and cell surface local-ization (Breinig et al. 2004). The hydrophilic portion of Kre1shows no similarity to known enzymes. Kre1 has a structuralrole and becomes cross-linked to other wall proteins (Breiniget al. 2004), and it also serves as a receptor for K1 killertoxin (File S8).

How might b1,6-glucan be made?: Obstacles to identifyingthe b1,6-glucan synthase gene might be an inherent difficultyin obtaining hypomorphic alleles of an essential synthasegene or the existence of multiple redundant synthase geneswhose individual mutation gives no phenotype (Lesage andBussey (2006). Furthermore, there are no precedents in otherorganisms that could be exploited in bioinformatics-basedapproaches to b1,6-glucan synthesis. Although b1,6-glucanis widely distributed in the Fungi (Lesage and Bussey2006), it is very rare elsewhere. The bacterium Actinobacillussuis makes a lipopolysaccharide containing a b1,6-glucan ho-mopolymer (Monteiro et al. 2000), but the proteins involvedin its formation are unknown. Some bacteria have GT2 familytransferases that make polymers of b1,6-linked GlcNAc(Gerke et al. 1998; Itoh et al. 2008), but these enzymes re-semble the S. cerevisiae Chs proteins. If b1,6-glucan is indeedformed directly from UDP-Glc, the b1,6-glucosyltransferasewould represent a new GT family. Further possibilities arethat a known yeast GT may also form b1,6-glucosidic linkagesusing UDP-Glc as donor or that b1,6-glucan is generatedsolely by transglycosylation.

Remodeling and Cross-Linking Activities at the CellSurface

Order of incorporation of components into the cell wall

CWPs delivered by the secretory pathway meet up with chitinand b-glucans at the outer face of the plasma membrane and

S. cerevisiae Cell Wall 797

undergo cross-linking reactions that incorporate them intothe wall. The order in which wall components are assembledhas been inferred from analyses of the material formedwhen spheroplasts regenerate their walls and from the wallcompositions of mutants unable to make a particular com-ponent (Kreger and Kopecká 1976; Roh et al. 2002b). Thestarting component is b1,3-glucan, which is necessary forincorporation of both b1,6-glucan and mannoproteins. Be-cause b1,6-glucan was still attached to b1,3-glucan whenGPI anchoring was inhibited (Roh et al. 2002b), and becauseincorporation of GPI-CWPs is lowered in b1,6-glucan syn-thesis mutants (Lu et al. 1995; Kapteyn et al. 1997), GPI-CWPs are likely incorporated after b1,6-glucan. Because chi-tin became detectable in the walls of daughter cells onlyafter cytokinesis (Shaw et al. 1991), it was concluded thatchitin is the last component to be incorporated into the wall(Roh et al. 2002b). The sequence b1,3-glucan/b1,6-glucan/mannoprotein must be able to accommodatechanges in expression or assembly of individual componentsdictated by the cell cycle, cell wall stress, mating, or sporu-lation, as well as remodeling of individual polysaccharides.For example, a compensatory incorporation of PIR proteindirectly attached to b1,3-glucan is seen in b1,6-glucan syn-thesis mutants (Kapteyn et al. 1999b).

The model for the order of incorporation of wallcomponents needs to be reconciled with the model fora bilayered wall, during whose formation CWPs are pro-pelled to the cell surface, leaving polysaccharides nearer theplasma membrane. Furthermore, surface CWP may not beretained at the surface of wild-type cells. Thus, wild-typediploids expressing a Sag1-GFP fusion released a significantbasal level of that glycoprotein into the medium (Gonzaleset al. 2010). CWP may therefore routinely be shed duringvegetative growth, perhaps upon digestion of the wall be-tween mother cell and bud, along with secreted proteinssuch as chitinase and invertase (Kuranda and Robbins1991). Cross-linking and remodeling reactions will be de-scribed next, and hydrolases of known or unknown func-tions, as well as nonenzymatic wall proteins are discussedin Cell Wall-Active and Nonenzymatic Surface Proteins andTheir Functions.

Incorporation of GPI proteins into the wall

The v(2) region of a GPI protein (see Identification of GPIproteins) influences whether the protein will be retained inthe plasma membrane in lipid-anchored form or whether itcan be transferred into the wall (Caro et al. 1997; Hamadaet al. 1998a, 1999; De Sampaïo et al. 1999; Frieman andCormack 2004). If this region includes two basic aminoacids, the protein will be mostly retained in the plasmamembrane (Caro et al. 1997; Frieman and Cormack 2003),but if basic residues are absent or replaced with hydrophobicones, the predominant location is the wall (Hamada et al.1998b, 1999; Frieman and Cormack 2003). However, havingtwo basic amino acids in the v(2) region does not guaranteemembrane localization because the additional presence of

a longer stretch of amino acids rich in Ser and Thr will overridethe dibasic motif and shift the protein to the wall (Friemanand Cormack 2004). Furthermore, not all wall-anchored GPIproteins have the amino acids suggested to promote incor-poration into the wall (De Groot et al. 2003). In general, GPIproteins are partitioned between the membrane and wallto varying extents, and none may be restricted to only onelocation (Gonzales et al. 2009).

The nature of a GPI-protein’s mode of cell surface attach-ment can be critical. Ecm33, which is required for growth atelevated temperature (see Sps2 family), occurs mainly asa plasma membrane-anchored GPI protein, and this locali-zation is required for in vivo function. Replacement of v(2)amino acids v-1-13 of Ecm33 with the corresponding aminoacid sequences from wall-localized proteins resulted in in-creased cross-linking of Ecm33 to the wall, but also in loss ofthe protein’s ability to support growth at high temperature(Terashima et al. 2003).

The lipid-to-wall transfer reaction could be a one-steptransglycosylation in which the GPI glycan is cleaved and itsreducing end transferred to b1,6-glucan, or it could involveseparate GPI cleavage and transglycosylation steps. Candi-dates for cross-linkers are Dfg5 and Dcw1, an essential, re-dundant pair of homologous GPI proteins that resemble ana1,6-endomannanase and are in GH Family 76 (Kitagakiet al. 2002). Single dfg5D and dcw1D mutants are viable,although dcw1D is hypersensitive to Zymolyase, but thecombination of dfg5D and dcw1D is lethal (Kitagaki et al.2002). Depletion of Dfg5 or Dcw1 by repressing their ex-pression in the double-null background led to cell enlarge-ment, delocalized chitin deposition, and secretion of a GPI-CWP protein into the growth medium (Kitagaki et al. 2002).dcw1D was also recovered in a screen for impaired cross-linking of GPI proteins (Gonzalez et al. 2010). The defectscaused by loss of Dfg5 and Dcw1, together with the proteins’resemblance to an a-endomannanase, are consistent withtheir having a role in GPI cleavage and/or transglycosyla-tion. Homozygous DFG5 nulls are defective in filamentousgrowth (Mosch and Fink 1997).

GPI-CWP can be used to display heterologous proteins onthe yeast cell surface (Schreuder et al. 1993; Van der Vaartet al. 1997; Gai and Wittrup 2007; Shibasaki et al. 2009). Inone such system, heterologous proteins are fused to theAga2 subunit of the a-mating agglutinin (see Flocculinsand agglutinins), which is disulfide-linked to its partner,the GPI-CWP Aga1 (Boder and Wittrup 1997).

Incorporation of PIR proteins into the wall

The internal repeat (PIR) sequences of PIR proteins arerequired for the alkali-labile linkage that joins these proteinsto b1,3-glucan (see Wall Composition and Architecture). De-letion of all PIR sequences from Pir1 and Pir4 leads to re-lease of these proteins from the cells (Castillo et al. 2003;Sumita et al. 2005), indicating that the repeats are necessaryfor wall association. The more repeats, the stronger thebinding: deletion of increasing numbers of Pir1’s repeats

798 P. Orlean

led to release of increasing amounts of Pir1 into the medium(Sumita et al. 2005).

Studies of Pir4/Ccw5, which has one PIR sequence andneeds it for cell wall anchorage, revealed that the alkali-labile linkage was an ester between the g-carboxyl groupof glutamate and the hydroxyl groups of b1,3-glucan. Thelinkage was generated in a transglutaminase reaction withQ74 in the PIR sequence SQIGDGQ74[V/I]QAT[T/S] (Eckeret al. 2006). In addition to substitutions of Q74, individualmutations of Q69, D72, and Q76 also resulted in loss of wallanchorage of the protein, indicating that these residues haveroles in the reaction. No transglutaminase has yet been iden-tified, but Ecker et al. (2006) point out that, because the freeenergy of hydrolysis of the amide is high enough to driveformation of the ester linkage, the PIR proteins could cata-lyze their own attachment to b1,3-glucan.

The glucan attachment sequence of PIR repeats is alsofound in the GPI-CWP Tip1, Tir1, Cwp1, and Cwp2 (Van derVaart et al. 1995). In the case of Cwp1, the PIR repeat maybe used as an additional wall anchorage point because theprotein is attached to the wall by both an alkali-labile anda GPI-dependent linkage (Kapteyn et al. 2001). Like GPI-CWP, PIR proteins can be used as carriers to direct surfaceexpression of heterologous proteins fused to them (Andréset al. 2005; Shimma et al. 2006).

Cross-linkage of chitin to b1,6- and b1,3-glucan

Related Crh1 and Crh2 generate cross-linkages between thereducing ends of chitin chains and both the nonreducing endof b1,3-glucose side branches on b1,6-glucan and the non-reducing ends of b1,3-glucan chains (Cabib et al. 2007; Cabib2009), and their homolog Crr1 likely does so during asco-spore wall assembly. These proteins are in GH family 16, andCrh2 and Crr1 also have a chitin-binding module (Rodriguez-Pena et al. 2000; Cabib et al. 2008). Crh1 and Crh2 are GPIproteins (Caro et al. 1997; Hamada et al. 1998a) whose lo-calization matches that of Chs3. Crh1-GFP fusions are detect-able at the site of bud emergence and later in the neck regionbetween mother cell and bud, and Crh2-GFP is seen in theneck region throughout the budding cycle, as well as in thelateral wall (Rodriguez-Pena et al. 2000, 2002). Single crh1and crh2 null mutants show Calcofluor White and Congo Redsensitivity, phenotypes enhanced in the double null, suggest-ing that Crh1 and Crh2 have a common wall-related function.Single crh mutants have a higher ratio of alkali soluble- toalkali-insoluble glucan, and this ratio is higher still in crh1Dcrh2D, indicating a role for Crh1 and Crh2 in linking b-glucanand chitin (Rodriguez-Pena et al. 2000).

Evidence that Crh1 and Crh2 are transglycosylases camefrom elegant studies by Cabib and coworkers, who used fluo-rescent, sulforhodamine-conjugated b1,3-gluco-oligosaccharidesas acceptors and showed that they became cross-linked tochitin in bud scars and the lateral walls of live cells (Cabibet al. 2008). Fluorescent labeling was very weak in crh1Dcrh2D cells or in cells lacking Chs3, which makes the chitinnormally bound to b1,3- and b1,6-glucan (Cabib and Duran

2005). The entire process of chitin polymerization and cross-linking could be reconstituted in detergent permeabilized,protease-treated cells. Cross-linking of fluorescent b1,3-gluco-oligosaccharides depended on the addition of UDP-GlcNAc (Cabib et al. 2008). Interestingly, the nascent chitinwas generated in situ by Chs1, which is highly active inpermeabilized cells, rather than by Chs3.

CRR1 shows sporulation-specific expression. Crr1-GFPfusions are localized on the surface of ascospores, and ho-mozygous crr1D diploids have ascospore wall abnormalities,with irregular deposition of the outer dityrosine and chito-san layers over the inner b-glucan layer (Gómez-Esqueret al. 2004). Ascopores from Crr1-deficient diploids showincreased sensitivity to heat shock and lytic enzymes, andthese defects are exacerbated when the chitin deacetylasesCda1 and Cda2 are also absent. These findings suggesta role for Crr1 in generating cross-links between the b-glucanand chitosan or chitin during ascospore wall maturation(Gómez-Esquer et al. 2004).

Cell Wall-Active and Nonenzymatic Surface Proteinsand Their Functions

Secreted, membrane, or wall proteins with known or con-jectured roles in wall biogenesis, adhesion, and nutrition aresurveyed here. The primary division is according to whetherproteins have or are likely to have enzymatic activity orwhether they are nonenzymatic, structural proteins. Bothgroups contain GPI proteins. Cell wall proteins have beenreviewed by Klis et al. (2002, 2006), De Groot et al. (2005),Lesage and Bussey (2006), and Gonzalez et al. (2009), glyco-sylhydrolases by Adams (2004), agglutinins by Dranginiset al. (2007), and flocculins by Goossens and Willaert(2010). Additional information about these proteins is pre-sented in File S9.

Known and predicted enzymes

Chitinases: S. cerevisiae has two chitinases, Cts1 and Cts2.Cts1, an endochitinase, has an N-terminal catalytic domain,followed by a heavily O-mannosylated Ser/Thr-rich region,and lastly, a C-terminal chitin-binding domain (Kurandaand Robbins 1991). Cts1 is periplasmic, but much of it issecreted into the medium of cells grown in rich medium(Correa et al. 1982; Kuranda and Robbins 1991). Cts1 hasa key role in cell separation because cts1D strains form aggre-gates of cells that remain joined at their chitin-containingsepta, a phenotype mimicked when cells are treated withthe chitinase inhibitor dimethyallosamidin (Kuranda andRobbins 1991). Cts1’s chitin-binding domain contributes tothe enzyme’s localization in the septal region because Cts1truncations lacking it only partially complement the cts1Dseparation defect (Kuranda and Robbins 1991). Cts2 mayhave a role in sporulation (Dünkler et al. 2008).

b1,3-glucanases: Exg1, Exg2, and Ssg/Spr1 exo-b1,3-glucanases:Exg1 is disulfide-linked (Cappellaro et al. 1998) whereas

S. cerevisiae Cell Wall 799

Exg2 is a surface-anchored GPI protein (Larriba et al. 1995;Caro et al. 1997). Single- or double-null mutants in EXG1and EXG2 have no overt defects, although exg1D cells haveslightly elevated levels of b1,6-glucan, and EXG1 overex-pressers lower amounts of that polymer, suggesting rolesfor Exg1 and Exg2 in b-glucan remodeling (Jiang et al.1995; Lesage and Bussey 2006). Ssg1/Spr1 is a sporulation-specific protein (File S9).

Bgl2, Scw4, Scw10, and Scw11 endo-b1,3-glucanases:These are GPI-less secretory proteins. Bgl2 has endo-b1,3-glucanase activity in vitro (Mrša et al. 1993), but it can alsocreate a b1,6 linkage between the reducing end that it gen-erates by cleaving a b1,3-gluco-oligosaccharide and the non-reducing end of another b1,3-glucan chain (Goldman et al.1995), and so could function as a b1,3-glucan branchingenzyme. No enzymatic activity has been shown for Scw4,Scw10, or Scw11, although mutation of predicted catalyticresidues in Scw10 abolished in vivo function (Sestak et al.2004). Scw4, Scw10, and Bgl2 are wall-associated via disul-fides (Cappellaro et al. 1998), but some Scw4 and Scw10can also be linked to b1,3-glucan (Yin et al. 2005).

These proteins have roles in maintaining normal walls.bgl2D, scw4D, and scw10D strains grow like wild-type cells,but show CFW sensitivity and slightly increased chitin levels(Klebl and Tanner 1989; Cappellaro et al. 1998; Kalebinaet al. 2003; Sestak et al. 2004), and bgl2D walls have elevatedlevels of alkali-soluble glucan (Sestak et al. 2004). Strainslacking both Scw4 and Scw10 are CFW-hypersensitive andmorphologically abnormal, have doubled chitin content andincreased alkali-soluble glucan, and show alterations inb1,3-glucan structure and in cross-linking of mannoproteinsto the wall (Cappellaro et al. 1998; Sestak et al. 2004).The growth and morphological defects of scw4D scw10Dare exacerbated by deletion of CHS3 or FKS2 (Sestak et al.2004). From the phenotypes of strains expressing differ-ent relative amounts of Bgl2 and Scw10, it was proposedthat levels of Bgl2 and Scw10 need to be balancedto ensure wall stability (Klebl and Tanner 1989; Sestaket al. 2004; File S9). Cells lacking Scw11 have a separationdefect, and, consistent with this, Scw11 is a daughter cell-specific protein (Cappellaro et al. 1998; Colman-Lerneret al. 2001).

Eng1/Dse4 and Eng2/Acf2 endo-b1,3-glucanases: Theserelated proteins have endo-b1,3-glucanase activity in vitro,but different localizations. Eng1 is a GPI protein (Baladronet al. 2002; De Groot et al. 2003), whereas Eng2 is likelyintracellular. Mutants lacking one or both proteins makenormal walls, but eng1D cells have a separation defect, con-sistent with Eng1’s localization to the daughter side of theseptum (Colman-Lerner et al. 2001; Baladron et al. 2002).ENG2 expression increases during sporulation, althougheng2D diploids are not defective in that process (Baladronet al. 2002). Surprisingly, loss of multiple exo- and endo-b1,3-glucanases is not catastrophic because cells lackingExg1, Exg2, Eng1, Eng2, and Bgl2 grow well and show onlythe eng1D separation defect (Cabib et al. 2008).

Gas1 family b1,3-glucanosyltransferases: This family hasfive members, all of which have GPI attachment sites(Fankhauser et al. 1993; Caro et al. 1997; Popolo and Vai1999; De Groot et al. 2003). Gas1, Gas3, and Gas5 can alsobe covalently linked to the wall (De Sampaïo et al. 1999; Yinet al. 2005). Gas proteins have b1,3-glucanosyltransfer activ-ity: they cleave b1,3-glucosidic linkages within b1,3-glucanchains and then transfer the newly generated reducing endof the cleaved glycan to the nonreducing end of anotherb1,3-glucan molecule, thereby extending the recipientb1,3-glucan chain (Mouyna et al. 2000; Carotti et al.2004; Ragni et al. 2007b; Mazan et al. 2011; File S9).

Gas1 has a major role in vegetative wall biogenesis.gas1D mutants are CFW-hypersensitive (Ram et al. 1994)and have less b1,3-glucan but more chitin and mannan intheir walls (Ram et al. 1995; Popolo et al. 1997; Valdiviesoet al. 2000). gas1D cells release b1,3-glucan to the medium(Ram et al. 1998), and Gas1’s b1,3-glucan elongase activitymay therefore be necessary for incorporation of b1,3-glucaninto the wall. In addition, analyses of the synthetic interac-tion network of gas1D revealed that survival in the absenceof Gas1 requires correct assembly of b1,6-glucan (Tomishigeet al. 2003; Lesage et al. 2004). Gas1 is detectable in thelateral wall, in the chitin ring in small-budded cells, andnear the primary septum and remains in the bud scar aftercell separation, and its localization is dependent on the pres-ence of its GPI-attachment sequence (Rolli et al. 2009).

Gas3 and Gas5 likely have wall-related functions in veg-etative cells (File S9). GAS2 and GAS4 are expressed onlyduring sporulation, and diploids lacking both Gas2 and Gas4have a severe sporulation defect. The inner glucan layer ofthe wall of double homozygous gas2 gas4 null spores wasdisorganized and detached from chitosan, suggesting thatthe b1,3-glucanosyltransferase activity of Gas2 and Gas4generates b1,3-glucan chains that associate optimally withchitosan (Ragni et al. 2007a).

Yapsin aspartyl proteases: GPI-anchored aspartyl proteasesof the yapsin family have roles in the turnover of CWPs andwall-localized enzymes. Yps1, Yps2/Mkc7, Yps3, and Yps6are mostly plasma membrane-associated, whereas Yps7 ispredicted to be wall anchored (Krysan et al. 2005; Gagnon-Arsenault et al. 2006). Yapsins cleave their substratesC-terminally to Lys or Arg or pairs of these residues (Olsenet al. 1998; Komano et al. 1999) and themselves undergoproteolytic processing to generate active enzyme (File S9).

Individual yapsin null mutants are sensitive to variouswall-disrupting agents, and loss of multiple YPS genes leadsto osmotically remedial, temperature-sensitive lysis defects,findings that indicate that the yapsins are involved in wallmaintenance (Krysan et al. 2005). Walls from yps1D yps2Dand yps1D yps2D yps3D yps6D yps7D null mutants showedlowered b1,3- and b1,6-glucan and elevated chitin levels,whereas mannan levels were unchanged, with the wallalterations being most pronounced in the quintuple mutant(Krysan et al. 2005). The b-glucan defects were due to

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decreased incorporation of these polymers into the wall,because synthesis of the two b-glucans was normal in thedeletion strains. These findings suggest that yapsins act onwall hydrolases and transglycosidases, thereby regulatingactivity of the latter, and hence, incorporation of glucansinto the wall (Krysan et al. 2005). Support for this camefrom identification of Gas1, Pir4, and Msb2 as Yps1 sub-strates (Gagnon-Arsenault et al. 2008; Vadaie et al. 2008).In addition to degrading or shedding proteins during wallremodeling, yapsins also have roles in mediating release ofaberrantly folded or overexpressed GPI proteins that induceER stress (Miller et al. 2010).

Nonenzymatic CWPs

Structural GPI proteins: There are three families of GPI-CWP and several individual GPI-CWP that do not resembleknown enzymes. Strains lacking one or more of these GPI-CWP have wall defects, and expression of some of theseproteins can vary with cell cycle stage or be induced duringmating or sporulation in response to cell wall stress or whenoxygen levels are low. In general, GPI-CWPs have a collectiverole in maintaining cell wall stability (Lesage and Bussey2006; Ragni et al. 2007c).

Sps2 family: This group comprises Ecm33, Pst1, Sps2, andSps22 (Caro et al. 1997). Of these, Ecm33 has an importantrole in vegetative walls. ecm33D cells are temperature-sensitiveand sensitive to various wall-perturbing agents, have a dis-organized wall with a thin or absent mannoprotein layer,and shed b1,6-glucan-linked mannoproteins and Pir2 thatis possibly linked to b1,3-glucan more than normal in themedium (Lussier et al. 1997b; Pardo et al. 2004). pst1D cellshave no obvious phenotype, but ecm33D pst1D double nullsshow exacerbated sensitivity to various wall stresses.Ecm33’s and Pst1’s functions partially overlap, but the pro-teins are not fully redundant because overexpression ofPST1 only weakly suppresses the ecm33D defects (Pardoet al. 2004). Sps2 and Sps22 are a redundant pair requiredfor normal ascospore wall formation. Diploids lacking themform spores with abnormal b-glucan, chitosan, and dityro-sine layers (Coluccio et al. 2004). Sps2 and Sps22 likely actat a similar stage in ascospore wall formation as Gas2, Gas4,and Crr1 in the formation of the b-glucan layer.

Tip1 family: Tip1, Cwp1, Cwp2, Tir1, Tir2, Tir3, Tir4, andDan1/Ccw13 are mostly small, Ser- and Ala-rich GPI-CWPthat show differential expression during the cell cycle andduring aerobic or anaerobic growth and can be localizeddifferently on the cell surface. Cwp2 also contains a PIR re-peat and so could be linked to b1,3-glucan (Klis et al. 2010).

Cwp1, Cwp2, Tip1, and Tir1 have roles in the vegetativewall. Deletion of their genes individually leads to CFW hy-persensitivity (Van der Vaart et al. 1995), and cwp1D cwp2Ddouble mutants show increased permeability to DNA-bindingagents relative to the single nulls (Zhang et al. 2008). Inaddition, the walls of cwp2D and cwp1D cwp2D cells arethinner than those of the wild type (Van der Vaart et al.1995; Zhang et al. 2008). Localization of these proteins

correlates with their expression. Tip1 is expressed in G1

and found in mother cells only, whereas Cwp1, Cwp2, andTir1 are expressed during the S-to-G2 transition, Cwp2 beingfound in small-to-medium-sized buds (Caro et al. 1998;Smits et al. 2006). Localization of Cwp2 and Tip1 is deter-mined by the timing of their expression in the cell cycle(Smits et al. 2006; File S9). Tip1 and Tip2 are also heat-and cold-shock-inducible, and Tir1 and Tir4 are induced bycold shock (Kowalski et al. 1995; Abramova et al. 2001).

CWP1 and CWP2 are downregulated upon shift to anaer-obic conditions, whereas Tip1, Tir1, Tir2, Tir3, Tir4, Dan1/Ccw13, and Dan4 are induced (Abramova et al. 2001). Ofthese, the Dan proteins are strongly repressed by oxygen.Strains lacking Tir1, Tir3, or Tir4 do not grow under anaer-obic conditions. Shift to anaerobiosis therefore leads toremodeling of the wall (Abramova et al. 2001), althoughit is not clear how the anaerobically induced CWPs permitanaerobic growth.

Sed1 and Spi1: These are two related, Ser/Thr-rich GPI-CWP whose expression is induced by nutrient limitation andstress. Sed1 is releasable from walls by treatment withb-glucanases or proteases (Shimoi et al. 1998). Associationof Sed1 with the wall is dependent on Kre6 (Bowen andWheals 2004), consistent with anchorage involving b1,6-glucan. SED1 expression is induced in the stationary phase,a time when the wall becomes thicker and more resistant tolytic enzymes (De Nobel et al. 1990). Consistent with a pro-tective role in stationary-phase walls, sed1D cells becomemore sensitive to Zymolyase in that growth phase (Shimoiet al. 1998). Elevated Sed1 expression is also part of thecompensatory response made by cells lacking multipleGPI-CWP (Hagen et al. 2004; File S9). Expression of SPI1is induced by weak organic acids, and Sps1 is a major con-tributor to the b1,3-glucanase resistance that arises in re-sponse to this stress (Simoes et al. 2003). Low external pHalso leads to formation of new alkali-labile linkages betweenGPI-CWPs and b1,3-glucan (Kapteyn et al. 2001).

Ccw12: Ccw12 is a small, heavily glycosylated, Ser/Thr-rich GPI-CWP with two C-terminal repeats of an amino acidsequence critical for its function (File S9). Ccw12 is releas-able by b1,3-glucanases (Mrša et al. 1999), but also has thepotential to make disulfide cross-links because it has a three-Cys motif found in several S. cerevisiae flocculins (see Floc-culins and agglutinins) and in wall proteins of other yeasts(Klis et al. 2010). Ccw12 is likely abundant because its genehas a very high codon adaptation index (Klis et al. 2010).Ccw12 has a major role in the wall, because cells lacking itare hypersensitive to CFW and other wall stressing agentsand rounder than wild type, with thick, disorganized walls,lysis-prone buds, and elevated levels of chitin (Mrša et al.1999; Hagen et al. 2004; Ragni et al. 2007c; Shankarnarayanet al. 2008). Man-to-Glc ratios in ccw12D cells are unchanged,but levels of alkali-soluble relative to alkali-insoluble glucanare higher, indicating altered organization and cross-linkageof wall components (Ragni et al. 2007c). Ccw12 localizes tosites of active wall synthesis, including the future bud site,

S. cerevisiae Cell Wall 801

the septum, the lateral walls of enlarging daughter cells, aswell as the tips of mating projections, but then turns over,suggesting that it may stabilize walls as daughter cells andthat mating projections are being formed (Ragni et al.2011). Loss of Ccw12 alone activates the CWI pathway-me-diated chitin stress response (Ragni et al. 2007c, 2011; seeChitin synthesis in response to cell wall stress), but deletion ofadditional GPI-CWP genes forces cells over a threshold thatleads to triggering of a new compensatory response to lossof multiple GPI-CWP that depends on Sed1 and the non-GPI-CWP Srl1 (see File S7).

Other nonenzymatic GPI proteins: Ccw14 (Ssr1/Icwp) isa b1,3-glucanase-extractable, Ser-rich GPI-CWP that has beenlocalized to the inner cell wall (Moukadiri et al. 1997; Mršaet al. 1999; File S9). The protein has an eight-Cys-containingCFEM domain found in various fungal surface proteins(Kulkarni et al. 2003; De Groot et al. 2005) and, hence, a po-tential for disulfide formation. CCW14/SSR1 null mutantshave no obvious growth defects, but show increased sensitiv-ity to CFW, Congo Red, and Zymolyase. Overexpression ofCCW14/SSR1 also leads to increased CFW and Congo Redsensitivity, although not Zymolyase sensitivity, suggesting thatlevels of Ccw14/Ssr1 relative to one or more other wall com-ponents need to be balanced (Moukadiri et al. 1997).

Dse2 and Egt2, which are unrelated to one another, aredaughter cell-specific proteins with roles in cell separation.In haploids, Dse2 is concentrated in regions connectingmother and daughter cells (Colman-Lerner et al. 2001; Doolinet al. 2001), and Egt2 is localized to the septum (Fujitaet al. 2004). Of these two GPI proteins (Hamada et al.1998a; Terashima et al. 2002; De Groot et al. 2003), Egt2’slocalization also depends on Gpi7 (Fujita et al. 2004). dse2Dhaploids show no defects, but homozygous DSE2 nulls showunipolar budding and form chains of cells. egt2D cells haveseparation defects similar to those of eng1D cells, a pheno-type exacerbated in the double null, indicating that the twoproteins act in parallel pathways involved in cell separation(Kovacech et al. 1996; Baladron et al. 2002).

The related Ser/Thr-rich GPI proteins Fit1, Fit2, and Fit3(Hamada et al. 1999) have a nutritional role. Their expres-sion is induced by iron limitation, and the proteins normallyretain iron bound to ferrichrome because Zymolyase treat-ment of FIT-deletion mutants releases less iron from cells(Protchenko et al. 2001). Fit1 localizes to the wall, whereit, Fit2, and Fit3 concentrate siderophore iron and facilitatesubsequent uptake of the metal, highlighting a role of thewall in nutrient acquisition (Protchenko et al. 2001).

Flocculins and agglutinins: GPI-CWP involved in cell–celladhesion are the related Flo1, Flo5, Flo9, Flo10, and Flo11/Muc1 flocculins, the Aga1 and Fig2 pair, and the a-agglutininSag1 (Roy et al. 1991; Cappellaro et al. 1994; Chen et al.1995; Caro et al. 1997; Erdman et al. 1998; Guo et al. 2000;Shen et al. 2001; Dranginis et al. 2007; Van Mulders et al.2009; Goossens and Willaert 2010).

Flo1, Flo5, Flo9, and Flo10 are modular proteins composedof 1100–1500 amino acids. Major features are N-terminal

PA14 domains that bind a-mannosides and mediate adhe-sion to adjacent cells, a central Ser/Thr-rich domain that isorganized in repeat sequences and heavily glycosylated,and two or three conserved three-Cys repeats toward theirC termini (Verstrepen and Klis 2006; Goossens and Willaert2010; Klis et al. 2010; Veelders et al. 2010; Goossens et al.2011). In addition, the Ser/Thr-rich domains of Flo1 andFlo11/Muc1 have short sequences enriched in Ile, Thr, andVal that are predicted to form intramolecular b-sheet-like interactions or amyloids, and both a soluble, GPI-lessportion of Flo11/Muc1 and a Flo1-derived form fibrillarb-aggregates in vitro (Ramsook et al. 2010). Amyloid forma-tion correlates with flocculation in vivo, for cells expressingFlo1 and Flo11/Muc1 that had been induced to flocculate inthe presence of Ca2+ stained more brightly with an amyloid-binding dye, and amyloid formation may be part of themechanism by which these proteins promote cell aggrega-tion (Ramsook et al. 2010). FLO1, FLO5, FLO9, and FLO10are not expressed in laboratory strains such as S288C be-cause of a mutation in the transcriptional activator Flo8.However, activation of individual FLO genes confers the abil-ity to flocculate (Guo et al. 2000; Van Mulders et al. 2009).Flo11/Muc1, a diverged Flo protein (Lambrechts et al. 1996;Lo and Dranginis 1996), is not involved in flocculation,but is required for pseudohypha formation by diploids, in-vasion of agar by haploids, and biofilm development (Loand Dranginis 1998; Guo et al. 2000; Reynolds and Fink2001; Dranginis et al. 2007; Bojsen et al. 2012).

Related Aga1 and Fig2 function in mating and localize tothe mating projection (Erdman et al. 1998; Guo et al. 2000;Jue and Lipke 2002). Aga1 is a component of a-agglutininthat displays the Aga2 subunit, which is disulfide linkedto it, and which confers binding specificity to a-agglutininSag1 (Orlean et al. 1986; Roy et al. 1991; Cappellaro et al.1994; Shen et al. 2001). Fig2, which like Aga1 is expressedin both mating types, is required for formation of matingprojections and maintenance of wall integrity during mating(Erdman et al. 1998; Guo et al. 2000; Zhang et al. 2002;File S9).

Sag1 has a long Ser/Thr-rich region in its C-terminal halfthat may hold up the N-terminal, Aga2-binding portionof the protein at the cell surface. Sag1’s N-terminal regioncontains three sequential domains that resemble variableimmunoglobulin-like folds (Chen et al. 1995; Shen et al.2001), the most C-terminal of which contains amino acids nec-essary for Aga2 binding (Wojciechowicz et al. 1993; Cappellaroet al. 1994; De Nobel et al. 1996).

Non-GPI-CWP: PIR proteins: Expression and localization ofPir1 (Ccw6), Pir2 (Ccw7/Hsp150), Pir3 (Ccw8), and Pir4(Ccw5/Cis3) is regulated during cell cycle progression andin response to stress. PIR1, PIR2, and PIR3 show peaks ofexpression in early G1, whereas PIR4 expression is highest inG2 (Spellman et al. 1998). PIR2 is also induced by heatshock, treatment with CFW or Zymolyase, and nitrogen lim-itation (Russo et al. 1993; Toh-e et al. 1993; Yun et al. 1997;

802 P. Orlean

Boorsma et al. 2004). Consistent with their upregulationupon wall stress, all four PIR genes show elevated expres-sion in an mpk1 mutant that constitutively activates the pro-tein kinase C-dependent CWI pathway, an effect eliminatedin mutants lacking the PKC pathway’s target transcriptionfactor, Rlm1 (Jung and Levin 1999).

PIR proteins localize to different parts of the surface ofbudding cells (Sumita et al. 2005; File S9). Pir1 and Pir2 arefound at bud scars of both haploids and diploids, Pir1 beinglocalized inside the chitin ring. Some Pir1 and Pir2 and mostPir3 are also present in lateral walls (Yun et al. 1997). Pir4has been reported be uniformly distributed at the cell sur-face or restricted to growing buds (Moukadiri et al. 1999;Sumita et al. 2005).

Strains lacking individual PIR proteins have subtlegrowth defects, but as more PIR genes are deleted, disrup-tants show a progressive increase in sensitivity to CFW,Congo Red, and heat shock, and cells become larger andirregularly shaped (Toh-e et al. 1993; Mrša and Tanner1999). pir1D pir2D pir3D pir4D mutants show a loss of vi-ability that is suppressed in osmotically supported medium(Teparic et al. 2004). These findings suggest a collective rolefor PIR proteins in maintenance of a normal wall. How theseproteins contribute is unclear, because the carbohydrate com-position of the quadruple PIR disruptant’s wall is unaltered,and the relative amounts of alkali-soluble and -insoluble glu-can and chitin show modest changes (Teparic et al. 2004;Mazan et al. 2008). PIR proteins, however, impact permeabil-ity of the wall because the pir1D pir2D pir3D mutant is hy-persensitive to membrane-active tobacco osmotin, whereasoverexpression of PIR1, PIR2, or PIR3 confers osmotin resis-tance on walled cells but not spheroplasts (Yun et al. 1997).The effects of PIR protein levels on wall permeability areconsistent with the role of these proteins in cross-linkingb1,3-glucans (see Mild alkali-releasable proteins).

Scw3 (Sun4): Haploids lacking this soluble cell wallprotein (Cappellaro et al. 1998) are larger than wild-typecells and have a separation defect and thickened septa(Mouassite et al. 2000). Scw3/Sun4 is a member of theSUN family of proteins, of which Sim1 and Uth1 are alsoreleased from cell walls by dithiothreitol treatment (Velourset al. 2002). Uth1 and Scw3/Sun4 additionally localize tomitochondria (Velours et al. 2002), but the significance ofthis distribution of the SUN proteins is unclear. The bio-chemical function of the SUN proteins is unknown as theyshow no similarity to known enzymes (File S9).

Srl1: This small Ser/Thr-rich protein is involved in thecompensatory response to loss of multiple GPI-CWP (Hagenet al. 2004; File S9). It rescues the lysis defects of strainsdefective in the function of the “regulation of Ace2 and po-larized morphogenesis” (RAM) signaling network when over-expressed (Kurischko et al. 2005), and some of it is tightlyassociated with the wall and released by b1,3-glucanase(Terashima et al. 2002). Slr1 localizes to the periphery ofsmall buds (Shepard et al. 2003). srl1D mutants have noobvious morphological defects and show modest Calcofluor

White sensitivity at 22�, but are hypersensitive to this agentat 37� (Kurischko et al. 2005). Mutants defective in RAMfunction are also suppressed by overexpression of Sim1 (seeabove) and Ccw12 (Kurischko et al. 2005), and slr1D andccw12D show a strong genetic interaction in the RAM-defective background. The srl1D ccw12D strain is CFW hy-persensitive at both 22� and 37�, and at 22�, but not at 37�,resembles mating pheromone-treated wild-type cells (Kurischkoet al. 2005). Srl1 and Ccw12 have been proposed to haveparallel functions in activation of a CWI pathway that oper-ates when RAM signaling is defective (Kurischko et al.2005).

What Is Next?

The biosynthesis of most individual yeast wall componentsis now understood in much detail and involves conservedpathways such as N-glycosylation and GPI anchoring andenzymes represented in other organisms, such as chitin andb1,3-glucan synthases. In contrast, b1,6-glucan formationand cross-linkage to GPI proteins and cross-linking of chitinto b-glucans are clearly restricted to certain yeasts and fila-mentous fungi, and the enzymes implicated in the latterprocesses, as well as certain CWP, are signatures of fungalcell walls, whose evolution of has been reviewed by Ruiz-Herrera and Ortiz-Castellanos (2010) and Xie and Lipke(2010).

Much of the work necessary to take both the conservedand the yeast-specific aspects of wall biogenesis to the nextlevel must be biochemical and analytical. These efforts willinvolve charting new biochemical territory, such as de-termining how b1,6-glucan is made and defining the func-tions of wall-active proteins such as Ccw12, Ecm33, Kre1,and Kre9, which have key roles in wall biogenesis, but showno resemblance to proteins of known function and may notbe enzymes. Other biochemical challenges are the mecha-nism and activation of chitin and b1,3-glucan synthases, themechanism and conjectured flippase activities of the multispan-ning glycosyltransferases of the dolichol, O-mannosylation, andGPI pathways, the functions of the Etn-P side branches onGPIs, and the biochemical activities of predicted enzymessuch as the Dcw1/Dfg5 pair, Kre5, Kre6, Scw4, and Scw10.The latter efforts require application of high-resolution tech-niques to analyze the fine structure and linkages of cell wallglycans (Magnelli et al. 2002; Aimanianda et al. 2009),which should highlight the reactions for which biochemistsneed to develop assays and screen mutants.

With the identification of so many proteins involved in cellwall biogenesis, and with ever-improving knowledge of wallcomposition, we can look forward to deepening our un-derstanding of the complexities of yeast cell wall biogenesis.

Acknowledgments

I thank my students for their many contributions to GPI andwall biosynthesis. I also acknowledge the contributions of

S. cerevisiae Cell Wall 803

the late Yoshifumi Jigami to our field. I am grateful to threereviewers for their helpful comments. Work in my laboratoryhas been supported by grant GM-46220 from the NationalInstitutes of Health and by a Burroughs Wellcome ScholarAward in Molecular Pathogenic Mycology.

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Communicating editor: J. Thorner

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