Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.109.101105

Genomewide Analysis Reveals Novel Pathways Affecting EndoplasmicReticulum Homeostasis, Protein Modification and Quality Control

Alenka Copic,*,1 Mariana Dorrington,*,1 Silvere Pagant,*,1 Justine Barry,* Marcus C. S. Lee,†

Indira Singh,‡ John L. Hartman, IV‡ and Elizabeth A. Miller*,2

*Department of Biological Sciences, Columbia University, New York, New York 10027, †Department of Microbiology,College of Physicians and Surgeons, Columbia University, New York, New York 10032, and

‡Department of Genetics, University of Alabama, Birmingham, Alabama 35294

Manuscript received February 15, 2009Accepted for publication May 1, 2009

ABSTRACT

To gain new mechanistic insight into ER homeostasis and the biogenesis of secretory proteins, wescreened a genomewide collection of yeast mutants for defective intracellular retention of the ERchaperone, Kar2p. We identified 87 Kar2p-secreting strains, including a number of known components insecretory protein modification and sorting. Further characterization of the 73 nonessential Kar2pretention mutants revealed roles for a number of novel gene products in protein glycosylation, GPI-anchor attachment, ER quality control, and retrieval of escaped ER residents. A subset of these mutants,required for ER retrieval, included the GET complex and two novel proteins that likely function similarlyin membrane insertion of tail-anchored proteins. Finally, the variant histone, Htz1p, and its acetylationstate seem to play an important role in maintaining ER retrieval pathways, suggesting a surprising linkbetween chromatin remodeling and ER homeostasis.

THE endoplasmic reticulum (ER) is a centralorganelle in the biogenesis of secretory and

membrane proteins. In the model eukaryote, Saccharo-myces cerevisiae, fully one-third of the proteome is deliv-ered to the ER en route to various destinations withinthe cell (Ghaemmaghami et al. 2003). Newly synthe-sized secretory proteins enter the ER as linear poly-peptide chains, which are subsequently acted on bya variety of resident enzymes that promote proteinfolding through chaperone action, glycosylation, anddisulfide bond formation. In addition to these protein-oriented events, the ER is also an important site of lipidbiosynthesis, housing a suite of enzymes that function invarious steps of fatty acid elongation and desaturation,ergosterol synthesis, and attachment of glycosylphospho-inositol (GPI) lipids to a class of lipid-tethered membraneproteins. Proteins and lipids destined for downstreamcompartments of the secretory pathway are exported fromthe ER via transport vesicles that are sculpted from thedonor membrane by the action of a set of cytoplasmiccoat proteins, known as the COPII coat (Gurkan et al.2006; Lee et al. 2004).

In maintaining ER function and homeostasis, a centralevent is the efficient homeostasis of the resident proteins

that perform the essential functions of protein and lipidbiogenesis. Several pathways contribute to this process.First, ER resident proteins lack the positive sorting signalsthat mediate efficient capture into ER-derived COPIIvesicles (Barlowe 2003; Otte and Barlowe 2004),making them poor clients for forward transport. How-ever, nonspecific export of proteins may result fromstochastic sampling of the ER membrane and lumenduring vesicle formation in a process known as ‘‘bulkflow’’ (Klumperman 2000; Lee et al. 2004). Unchecked,this nonspecific ER egress could deplete the ER of rel-atively abundant proteins that would be packaged intotransport vesicles at their prevailing concentrations. Thishighlights the importance of the second mechanism ofER retention: retrieval of escaped ER residents from theGolgi apparatus via retrograde COPI-coated transportvesicles (Lee et al. 2004). This process often employs atransmembrane receptor, Erd2p, that recognizes thecanonical ER retrieval motif, K/HDEL on soluble ERresidents (Townsley et al. 1994). The contribution ofeach of these pathways can be appreciated by consideringthe fate of the ER chaperone, Kar2p/BiP, that lacks itsHDEL retrieval signal; this protein is efficiently secretedfrom cells (Schuldiner et al. 2005; Semenza et al. 1990),suggesting that ER residents can access transport vesicles,presumably by nonspecific capture, but are efficientlyretrieved to maintain the correct steady state localization.

In addition to these pathways that regulate the lo-calization of ER residents, further mechanisms likelygovern the export of the large cast of client proteins that

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.101105/DC1.

1These authors contributed equally to this work.2Corresponding author: Columbia University, 1212 Amsterdam Ave.,

MC2456, NY, NY 10027. E-mail: em2282@columbia.edu

Genetics 182: 757–769 ( July 2009)

transit through the ER, a population of proteins thatmust be carefully managed (Ellgaard and Helenius

2003; Anelli and Sitia 2008). Improperly assembledproteins are prevented from premature egress, andterminally misfolded proteins are destroyed by a processknown as ER-associated degradation (ERAD). This ERquality control checkpoint ensures that aberrant pro-teins are not deployed within the cell, where they mayinterfere with normal protein function (Ron 2002).Although the mechanisms that contribute to ER re-tention of misfolded proteins are not well understood,the improper presentation of sorting signals and re-trieval from post-ER compartments may play importantroles (Caldwell et al. 2001; Vashist et al. 2001; Kincaid

and Cooper 2007). Furthermore, an aberrant form ofthe viral protein, VSV-G, is excluded from the dedicatedER exit sites that give rise to transport vesicles, suggest-ing additional layers of regulation function to segregateproteins prior to ER egress (Mezzacasa and Helenius

2002). Should the ER become overburdened withmisfolded proteins, a transcriptional pathway knownas the unfolded protein response (UPR) is activated,which functions to upregulate both ER chaperones andthe degradation machinery (Travers et al. 2000). Thesemultiple mechanisms interface with each other tocontribute to ER homeostasis; when Erd2p-mediatedretrieval is crippled, the UPR can compensate to main-tain viability by increasing the levels of ER chaperones(Beh and Rose 1995). However, the extent of cross-talkbetween these processes is unclear, and the full spec-trum of components that maintain the fidelity of pro-tein biogenesis and the integrity of ER function remainunknown.

We aimed to identify components that act in ERhomeostasis using an unbiased, genomewide approachfocused on protein processing and transport throughthe ER. We reasoned that mutants that secrete ERresident proteins to the cell surface could have defectsin either retention of ER resident proteins or in thequality control process that contributes to the fidelity ofprotein secretion. We screened the yeast genome formutants that secrete significant amounts of the ERchaperone, Kar2p (orthologous to mammalian BiP) tothe cell surface, and identified 87 yeast mutants thatsecrete at least twofold more Kar2p than wild-type (WT)cells. We classified each of these mutants according totheir involvement in protein folding, protein glycosyla-tion, maturation of GPI-anchored proteins, ER qualitycontrol, and ER retrieval to define a number of novelcomponents that function in each of these pathways.

MATERIALS AND METHODS

Yeast strains and growth: Cultures were grown at 30� instandard rich media (YPD: 1% yeast extract, 2% peptone, and2% glucose) or synthetic complete media (SC: 0.67% yeastnitrogen base and 2% glucose, supplemented with amino

acids appropriate for auxotrophic growth). Cells were trans-formed using standard lithium acetate protocols. The haploiddeletion and Tet-repressible strain collections were purchasedfrom Open Biosystems (Huntsville, AL). We employed asynthetic genetic array approach to systematically introduceErd2-GFP and the hac1D allele into the MATa collection ofKar2p secretors. The parental query strain [MATa,hmrD0TURA3 (C. albicans), leu2D0, his3D1, can1D0TPGAL1-TDH1-MFA1pr-his5 1 (S. pombe), lyp1D0] was constructed bytransforming Y15578-1.2b (Singh et al. 2009) with PCRproducts to integrate GFPTLEU2 at the ERD2 locus or replacethe HAC1 locus with the NATMX selection cassette to gener-ate LMY446 and LMY445, respectively. These query strainswere then mated to the haploid MATa strains that showedsignificant Kar2p secretion. Diploid cells were selected on SEmedium (0.17% yeast nitrogen base without amino acids orammonium sulfate; 1 g/liter monosodium glutamate; 2%glucose) supplemented with the appropriate amino aciddropout mix (Sunrise Science Products, San Diego, CA) andthe selective agents G418 (Fisher, Hanover Park, IL) and/ornourseothricin (Werner Bioagents, Jena, Germany), followedby replica plating onto sporulation medium. Haploid doublemutants were selected by virtue of the mating-type regulatedauxotrophy cassette allowing selection of MATa haploiddouble mutants following multiple rounds of replica platingonto appropriate selective media lacking histidine and argi-nine and containing canavanine, G418, and nourseothricin.In some cases, strains were also constructed by traditional yeastmating or PCR-mediated integration (supporting informa-tion, Table S3). Strains YM1729 (WT), YM1730 (htz1D),YM1530 (sir2D), YM1589 (hmrD), YM1536 (htz1Dsir2D), andYM1590 (htz1DhmrD) were the gifts of Hiten Madhani [Uni-versity of California at San Francisco (UCSF)]. Jasper Rine[University of California, Berkeley (UC Berkeley)] kindlyprovided JRY7981 (htz1DTHIS5, pHTZ1-3FLAG), JRY7985(htz1DTHIS5, pHTZ1-K3R-3FLAG), JRY7986 (htz1DTHIS5,pHTZ1-K8R-3FLAG), JRY7987 (htz1DTHIS5, pHTZ1-K10R-3FLAG), and JRY7988 (htz1DTHIS5, pHTZ1-K14R-3FLAG).

Plasmids: The UPRE-lacZ reporter plasmid was derivedfrom pMZ11 by EcoRI/XhoI digest and subcloning into pRS423to create pLM38. A HA-tagged form of CPY* was encoded onplasmid pCP258, originally from Peter Walter’s lab (UCSF).ILM1 was expressed from a multicopy plasmid, pLM550, whichwas generated by PCR amplification of the ILM1 gene plus1000 bp upstream of the open reading frame, followed bysubcloning into the EcoRI/SpeI sites of pRS426. The plasmidencoding GFP-Frt1p was a gift from Traude Beilharz (VictorChang Cardiac Research Institute, Sydney, Australia). Plas-mids encoding SEC22, BET1, and BOS1 on a 2m pRS426plasmid were from the Schekman lab strain collection(RSB1241, RSB1247, and RSB1248, respectively). A SED5overexpression construct was made by PCR amplification ofthe SED5 locus, including�350 bp upstream and downstreamfollowed by subcloning into the pRS426 vector.

Kar2p secretion assays: For genomewide screening purpo-ses, cells were grown from glycerol stocks in 96-well trays for�2 days. Saturated cultures (5 ml) were spotted onto YPDplates and incubated at 30� for 8 hr, at which point colonieswere overlaid with nitrocellulose membranes and incubationcontinued for 30 min. Nitrocellulose membranes were washedunder running water to remove any adhering cells and thenincubated sequentially with TBS, 5% milk powder, and anti-Kar2p antibodies (kindly provided by Randy Schekman,UCBerkeley) diluted 1/10,000 in TBS. In initial screens, secretedKar2p was detected with HRP-conjugated goat-anti-rabbitantibodies followed by ECL detection (Pierce, Rockford, IL).To quantify the amount of Kar2p secretion, we detected Kar2pusing IRDye800-conjugated anti-rabbit antibodies (Rockland

758 A. Copic et al.

Immunochemicals, Gilbertsville, PA) followed by imaging onthe LiCor Odyssey fluorescent scanner and analysis usingOdyssey software. The Kar2p secretion signal was normalizedto that observed in a wild-type strain to yield the Kar2psecretion index listed in Table 1. Kar2p secretion was alsomonitored from liquid cultures according to published pro-tocols (Belden and Barlowe 2001).

Network analysis: Relationships among Kar2p retentionmutants were examined and visualized using the BioGridonline resource (http://www.thebiogrid.org) and Osprey soft-ware (Breitkreutz et al. 2003).

Detection of UPR: Strains expressing the UPRE-lacZ re-porter plasmid (pLM38) were grown to midlog phase inselective medium and UPR measured by b-galactosidase ac-tivity assays as described (Ng et al. 2000). Data were normalizedto the appropriate BY4742 or BY4741 wild-type strains.

Pulse-chase analysis: Pulse-chase analysis of CPY and Gas1pmaturation was performed as described (Pagant et al. 2007).CPY and Gas1p were immunoprecipitated using antibodieskindly provided by Randy Schekman. To measure the acqui-sition of a-1,6-mannose, CPY* was first immunoprecipitatedusing anti-HA antibodies; the precipitated protein was re-leased from the beads with 1% SDS followed by dilution withIP buffer without SDS and a second round of immunoprecip-itation using antibodies against the a-1,6-mannose moiety, alsoprovided by Randy Schekman. The degree of a-1,6-mannosemodification was calculated relative to a parallel immunopre-cipitation that yielded the total CPY* present at each timepoint.

Microscopy: Strains expressing Erd2p-GFP were grown inYPD to midlog phase and imaged using a Nikon TE300inverted microscope (Melville, NY) with 1003 N.A. 1.4PlanApo optics and a Hamamatsu Orca-ERG charge-coupleddevice camera. Images were collected with the Openlab 5.0(Improvision, Waltham, MA) software system and analyzedusing Adobe Photoshop (Adobe Systems, Mountain View, CA).Strains expressing GFP-Frt1p were grown in selective mediumand shifted briefly to YPD, which exaggerated the mislocal-ization phenotype of Frt1p, prior to imaging as describedabove.

RESULTS AND DISCUSSION

To uncover novel mechanisms that contribute to ERhomeostasis, we aimed to employ a genomewide ap-proach to identify S. cerevisiae mutants that improperlysecrete the ER lumenal chaperone, Kar2p (Figure 1A).Previous attempts to isolate such mutants used atraditional genetic selection approach whereby thesecreted enzyme invertase was appended with an HDELER retention signal, yielding two mutants, erd1 and erd2(for ER retention defective), which showed secretedinvertase activity (Pelham et al. 1988; Semenza et al.1990). We designed a colony immunoblotting assay toscreen a genomic collection of haploid MATa deletionstrains that contains individual disruptions in eachnonessential gene (Winzeler et al. 1999). In prelimi-nary screens we identified a number of strains thatsecreted more Kar2p than wild-type cells; secondaryscreening with fluorescent secondary antibodies al-lowed quantification of Kar2p secretion in each of thesestrains, yielding a Kar2p secretion index that representsthe fold change of Kar2p secretion over wild-type cells

(Table S1). To further validate our collection of Kar2pretention mutants, we also screened a second collectionof deletion strains, composed of the MATa set of haploiddeletants. There was a high degree of overlap betweenthe two collections, with only a few strains unique toeither collection (Table S1). We tested the genedisruptions for this nonoverlapping set of mutants andidentified three cases in which there were anomalies inthe insertion of the deletion cassette. In the MATailm1D

background (which gave a Kar2p secretion phenotype)the KanMX marker was correctly integrated whereas inthe corresponding MATa strain, the locus was wild type,consistent with the absence of Kar2p secretion in thisstrain (data not shown). In two cases, pmt1D and pro1D,the strain that gave a Kar2p secretion phenotype con-tained the correct gene deletion whereas in the oppo-site mating type there appeared to be both a wild-typelocus and a KanMX-disrupted locus, suggesting that achromosomal duplication masked the effect of the genedeletion. In the other strains that gave a Kar2p secretionphenotype in a mating-type-dependent manner, bothstrains contained the correct genetic disruption, sug-gesting either mating-type specific effects on the endo-plasmic reticulum or the presence of spontaneoussuppressor mutations. This latter effect has been pre-viously reported as a particular problem for the getmutants (Schuldiner et al. 2008). These discrepanciesserve to highlight the utility of performing genomewidescreens in multiple collections to identify false negativesand generate an independent data set to lend confi-dence in the original screen.

We selected a Kar2p secretion index of 2 as a cutoff forfurther characterization: we consider this a conservativethreshold since strains with a secretion index of 1.5 alsoshowed consistently higher Kar2p secretion than wild-type cells, albeit with more variability from experimentto experiment. Our final analysis yielded 73 strains thatsecreted at least twofold more Kar2p than wild-type cells(Table 1). To similarly investigate genes that are lethal ifperturbed in a haploid cell, we also screened a straincollection that contains �800 essential genes under thecontrol of a tetracycline-regulated promoter. We testedKar2p secretion following an 8-hr period of generepression by doxycycline and identified another 14strains that secrete Kar2p (Table 2 and Table S2).Among these were several strains that secreted Kar2peven in the absence of doxycycline, likely because alteredexpression from the Tet-responsive promoter perturbscellular function. These combined screens yielded anumber of expected components with known functionsin Kar2p retention, including the KDEL receptor, Erd2p,as well as many mutants of unknown or poorly definedfunction that may represent novel components in ERhomeostasis.

We considered that cell wall defects might cause celllysis during the colony immunoblotting procedure,yielding a Kar2p secretion signal that was in fact derived

ER Homeostasis in Yeast 759

from intracellular pools of Kar2p. We tested this bymonitoring Kar2p secretion in the presence of osmoticsupport (1 m sorbitol) during the growth and colonyoverlay phases of the experiment (data not shown). Wetested the majority of MATa Kar2p retention mutantsunder these conditions and only one strain, she10D,showed a rescue of Kar2p secretion on sorbitol medium,suggesting that Kar2p secretion in most mutants did notresult from cell lysis. We also aimed to determinewhether the Kar2p secretion phenotype applied morebroadly to other HDEL-containing ER residents. To thisend we probed a subset of mutants for secretion ofanother ER chaperone, Pdi1p. In a qualitative manner,we saw similar trends in the degree of Pdi1p secretion;however, the relatively low affinity and specificity of thea-Pdi1p antibodies precluded more quantitative analy-sis (data not shown).

We focused our efforts on further characterizing thenonessential Kar2p retention mutants. This set of

mutants was enriched in components with definedfunctions in ER-Golgi traffic and various aspects ofsecretory protein biogenesis (Figure 1B). Within thefunctional category of ER-Golgi transport, we identifiedmultiple examples of shared phenotypes among distinctmembers of the same protein complex, including theGET complex (Get1p, Get2p, and Get3p), the p24family (Emp24p, Erv25p, Erp1p, and Erp2p), and theBug1p/Grh1p complex. Furthermore, this subset ofmutants showed extensive genetic interactions witheach other as defined by systematic genetic analysis(Schuldiner et al. 2005), suggesting that the geneproducts function in shared pathways (Figure 1C). Oneunexpected functional category that was overrepre-sented in our set of Kar2p retention mutants waschromatin assembly, including components of the SWR1-complex that deposit the variant histone, Htz1p, itselfalso identified (Figure 1D). Moreover, additional mem-bers of the SWR1 complex and two essential subunits of

Figure 1.—Overview of theKar2p retention screen and cate-gorization of mutants. (A) Thehaploid deletion collection wasgrown on YPD (left) and secretedKar2p was quantified using fluo-rescent secondary antibodies(right). Colonies secreting two-fold more Kar2p than WT cellswere selected for further charac-terization. (B) Functional distri-bution of Kar2p secretors andthe number of genes isolated ineach category. (C) Genetic inter-actions among Kar2p secretorswith annotated functions in ER-Golgi traffic. Components thathave been isolated as physicalcomplexes are shown in gray. Ag-gravating interactions are shownin blue, alleviating interactionsin red. (D) Subunit architectureof the SWR1 and NuA4 com-plexes. Kar2p secretors are shownin red, components that showed aless significant Kar2p secretionphenotype are shown in orange,essential genes are annotatedwith an asterisk. Adapted fromKobor et al. (2004).

760 A. Copic et al.

TABLE 1

Kar2p retention mutants identified from haploid deletion collections

Gene ORF Functional categorya

Kar2p secretionindexb

Synthetic sick/lethalwith hac1Dc

Kar2p secretionin hac1Dd

Erd1 YDR414C Ambiguous 9.0 � 1

Emp24 YGL200C ER-Golgi traffic 9.0 1 NDBst1 YFL025C O-linked glyc./GPI 8.8 1 NDErv25 YML012W ER-Golgi traffic 8.4 1 NDLhs1 YKL073W Protein maturation 6.8 1 NDGup1 YGL084C O-linked glyc./GPI 6.7 � �Per1 YCR044C O-linked glyc./GPI 6.1 � �Erp1 YAR002C-A ER-Golgi traffic 5.9 � 1

Arv1 YLR242C Lipid 5.6 1 NDTed1 YIL039W ER-Golgi traffic 5.6 � 1

Slp1 YOR154W Ambiguous 5.0 � �Get1 YGL020C ER-Golgi traffic 5.0 � 1

Ics3 YJL077C Ambiguous 4.7 � �Get3 YDL100C ER-Golgi traffic 4.6 � 1

Scj1 YMR214W Protein maturation 4.3 1 NDYHR078W Unknown 4.2 � 1

YOR164C Unknown 4.1 � 1

Get2 YER083C ER-Golgi traffic 4.1 � �She10 YGL228W ambiguous 3.8 � 1

Alg3 YBL082C N-linked glyc. 3.8 1 NDPps1 YBR276C Cell cycle 3.7 � �Mnn11 YJL183W N-linked glyc. 3.7 1 NDCsf1 YLR087C Ambiguous 3.7 1 NDPho88 YBR106W Ambiguous 3.7 1 NDEos1 YNL080C N-linked glyc. 3.7 1 NDErv26 YHR181W ER-Golgi traffic 3.7 � �Sec22 YLR268W ER-Golgi traffic 3.5 1 NDSum1 YDR310C Chromatin 3.4 � 1

YLR065C Unknown 3.4 1 NDYMR031W-A Unknown 3.4 1 ND

Mdy2 YOL111C Ambiguous 3.3 � 1

YBL083C Unknown 3.2 1 NDCcw12 YLR110C Ambiguous 3.2 � �Arp6 YLR085C Chromatin 3.2 � 1

Htz1 YOL012C Chromatin 3.2 � 1

Vps71/Swc6 YML041C Chromatin 3.1 � 1

Cwc21 YDR482C Ambiguous 3.1 � 1

YLR374C Unknown 3.1 � 1

Ubx2 YML013W ERAD 3.1 1 NDErp2 YAL007C ER-Golgi traffic 3.1 � 1

YML013C-A Unknown 3.0 1 NDMga2 YIR033W Lipid 3.0 � �Hoc1 YJR075W N-linked glyc. 3.0 � 1

Yaf9 YNL107W Chromatin 3.0 N/D NDSte24 YJR117W Protein maturation 3.0 1 ND

YER140W Unknown 3.0 � 1

Pin4 YBL051C Cell cycle 2.9 � �Tcb2 YNL087W Ambiguous 2.8 � �Pro1 YDR300C Amino acid biosynth. 2.8 � �

YLR111W Unknown 2.7 � �Vps72/Swc2 YDR485C Chromatin 2.6 � 1

Swc3 YAL011W Chromatin 2.6 � 1

Ost3 YOR085W N-linked glyc. 2.6 1 NDPmt1 YDL095W O-linked glyc./GPI 2.6 � �Vps74 YDR372C Intra-Golgi traffic 2.5 � �Ilm1 YJR118C Ambiguous 2.5 1 ND

(continued )

ER Homeostasis in Yeast 761

the NuA4 histone acetylation machinery that modifiesHtz1p also secreted Kar2p when mutated, albeit to alesser extent than our twofold cutoff (Table S1). Someof these chromatin assembly mutants also showed avacuolar protein sorting (VPS) phenotype, suggestingpleiotropic defects in secretory pathway function thatmay stem from disregulation of a specific subset ofgenes.

UPR-dependence of the Kar2p secretion phenotype:Secretion of Kar2p can result from activation of theunfolded protein response (UPR), which dramaticallyupregulates ER chaperones, among other targets.Mutations that cause the accumulation of misfoldedproteins and subsequent activation of the UPR mayinduce secretion of Kar2p simply as a result of Kar2poverexpression (Belden and Barlowe 2001). Con-versely, mutations that result in defective ER retentionmay in turn lead to an activated UPR; inappropriateleakage of chaperones would deplete the essentialfolding factors within the ER such that protein foldingis impaired and the UPR induced. Thus, simply moni-toring UPR activation in these strains is unlikely toresolve whether Kar2p secretion causes or results fromER stress. To overcome this conundrum and determinewhich mutants secrete Kar2p largely because of an activeUPR, we introduced a deletion allele of HAC1, thetranscription factor that mediates the UPR, into each ofthe Kar2p retention mutants. For viable double mu-tants, we quantified Kar2p secretion relative to a hac1D

single mutant. Approximately 40% of Kar2p retentionmutants exhibited significant Hac1p-independent se-cretion of Kar2p, designated as a Kar2p secretion signalin the double mutant at least twofold greater than thatof a hac1D strain (Table 1). We propose that thesemutants correspond to proteins with roles in the re-tention and/or retrieval of Kar2p rather than in thematuration of secretory proteins. It is somewhat surpris-ing that these mutants that secrete a key ER chaperoneare still viable absent the ability to upregulate thesynthesis of additional ER residents via the UPR. Thesestrains may be able to survive with lowered steady-statelevels of Kar2p, or may be able to upregulate ER chaper-ones via a Hac1p-independent pathway.

Conversely, a number of mutants no longer secretedKar2p in the absence of functional Hac1p, suggesting acontribution of the UPR to Kar2p secretion in theoriginal single mutant strain. Indeed, several of thesemutants showed a constitutive UPR as measured by a b-galactosidase reporter assay (Table S1). Finally, a subsetof mutants exhibited a severe synthetic growth defectwhen combined with the hac1D allele (Table 1), suggest-ing a requirement for the UPR to maintain viability inthe single mutants. Under conditions of impaired pro-tein folding, the UPR can become essential to cope withthe increased burden on the ER (Spear and Ng 2003),and several of the Kar2p retention mutants previouslyidentified as synthetic sick/lethal in combination withhac1D also possess a constitutive UPR (Schuldiner et al.

TABLE 1

(Continued)

Gene ORF Functional categorya

Kar2p secretionindexb

Synthetic sick/lethalwith hac1Dc

Kar2p secretionin hac1Dd

Van1 YML115C N-linked glyc. 2.5 � 1

Gsg1 YDR108W ER-Golgi traffic 2.4 � 1

Fat1 YBR041W Lipid 2.4 � �Mnn10 YDR245W N-linked glyc. 2.4 � �Ioc3 YFR013W Chromatin 2.3 � �Rce1 YMR274C Protein maturation 2.3 � 1

Swr1 YDR334W Chromatin 2.3 � 1

Sir4 YDR227W Chromatin 2.3 ND NDLas21 YJL062W O-linked glyc./GPI 2.2 1 NDGrh1 YDR517W ER-Golgi traffic 2.2 � 1

Mrpl16 YBL038W Mitochondria 2.2 1 NDSdc1 YDR469W Chromatin 2.2 � 1

YJL123C Unknown 2.2 � 1

Gds1 YOR355W Ambiguous 2.1 � �Mih1 YMR036C Cell cycle 2.1 � 1

Nem1 YHR004C Ambiguous 2.1 � 1

Bug1 YDL099W ER-Golgi traffic 2.0 � �

ND, not determined.a Functional category assigned from SGD annotations.b Kar2p secretion index was determined by quantitative immunoblotting and represents a fold increase over wild type.c Synthetic genetic interactions between individual mutations and a hac1D allele (1 indicates significant synthetic growth de-

fect).d Kar2p secretion was determined in the hac1D/yfgD double mutant strains by quantitative immunoblot; a twofold increase over

a hac1D strain was considered significant secretion (1).

762 A. Copic et al.

2005). We demonstrated that the UPR was activated ineach of the strains that showed a synthetic lethalinteraction with hac1D (Table S1), suggestive of rolesin protein folding and/or degradation within the ER.Some of these mutants (eos1D, lhs1D, alg3D, mnn11D,

and ubx2D) have known defects in protein glycosylationor folding, whereas several mutants (pho88D, csf1D,mrpl16D, and ylr065cD) correspond to novel candidatesfor similar roles. Furthermore, another recent genome-wide analysis of mutations that induce the UPR identi-fied a large number of these strains as havingconsititutive UPR, consistent with a role in proteinfolding and/or biogenesis ( Jonikas et al. 2009). Addi-tional characterization of secretory pathway functionprovided more direct support for roles for threeproteins (Csf1p, Mrpl16p, and Pho88p) in the matura-tion of secretory proteins (Figure 2) and for the finalprotein, Ylr065cp, in protein quality control (Figure 3).

A subset of Kar2p secretors exhibit defects in post-translational modification of secretory proteins: Wetested secretory pathway function in each of the Kar2pretention mutants by monitoring the in vivo maturationof the cell wall GPI-anchored protein, Gas1p and thevacuolar hydrolase, CPY. Both of these model proteinsare modified upon delivery to the Golgi, yielding aneasily monitored marker for forward transport throughthe secretory pathway during a pulse-chase experiment.We identified a subset of mutants that showed glycosyl-ation defects in CPY, resulting in the appearance ofunderglycosylated forms of the mature protein (Figure2A). Alg3p and Ost3p are known components of the N-linked glycosylation machinery (Lehle et al. 2006), anda dubious ORF, YBL083C, partially overlaps with ALG3.The remaining mutants, ics3D, pho88D, mdy2D, andmrpl16D, have not previously been described as de-

TABLE 2

Kar2p retention mutants identified from a Tet-repressiblecollection of essential genes

Gene ORF Functional categorya

Kar2p secretionindexb

Erd2 YBL040C ER-Golgi traffic 8.0Ost2 YOR103C Protein maturation 5.6Iqg1 YPL242C Ambiguous 5.0Pgi1 YBR196C Gluconeogenesis 4.4Gpi1 YGR216C O-linked glyc./GPI 4.2Alg11 YNL048W Protein maturation 4.2Srp72 YPL210C Protein maturation 4.0Gpi17 YDR434W O-linked glyc./GPI 3.8Rft1 YBL020W Protein maturation 3.6Sec39 YLR440C ER-Golgi traffic 3.4Mot1 YPL082C Transcription 2.6Sec17 YBL050W ER-Golgi traffic 2.4Gpi19 YDR437W O-linked glyc./GPI 2.4Abd1 YBR236C mRNA processing 2.2

a Functional category assigned from SGD annotations.b Kar2p secretion index was determined by quantitative im-

munoblotting following an 8-hr period of treatment withdoxycycline and represents a fold increase over wild type.

Figure 2.—Maturationof CPY and Gas1p is de-layed in some Kar2p secre-tion mutants. (A) CPYmaturation from ER (p1)to Golgi (p2) and vacuolar(p3) forms was monitoredby pulse-chase analysis. Sev-eral mutants accumulatedunderglycosylated forms ofCPY (marked with *). (B)Maturation of the GPI-anchored protein, Gas1p,from ER-localized precur-sor (p) to Golgi-modifiedmature (m) forms was sim-ilarly monitored. A numberof mutants showed a delayin the appearance of ma-ture Gas1p. Only thosestrains exhibiting defectsin protein maturation areshown.

ER Homeostasis in Yeast 763

fective in processing of secretory proteins. SinceMrpl16p is localized to mitochondria and has a struc-tural role in the mitochondrial ribosome, the effect onglycosylation of secretory proteins is likely to be in-direct, perhaps by influencing the synthesis of lipid-linked oligosaccharide precursors. Conversely, Pho88pis localized to the ER, putting it in a prime positionto participate directly in N-linked glycosylation. Fur-thermore, the synthetic lethality of pho88D and mrpl16D

strains with hac1D further supports a role in pro-tein folding through post-translational modificationof secretory cargoes. The precise functions of theseputative glycosylation components remain to be fullycharacterized.

We also identified a number of mutants with a delay inmaturation of the cell wall GPI-anchored protein, Gas1p(Figure 2B). Some of the more severely affectedmutants correspond to previously described compo-nents of the GPI-anchor attachment and remodelingmachinery (Bst1p, Gup1p, and Per1p; Orlean andMenon 2007), or as putative cargo receptors for Gas1p(Emp24p; Muniz et al. 2000). Furthermore, relatedmembers of the p24 family of proteins were similarlyaffected; Erv25p, Erp1p, and Erp2p form a physicalcomplex with Emp24p and play roles in both anterog-rade and retrograde traffic between the ER and Golgi(Belden and Barlowe 1996; Marzioch et al. 1999;Aguilera-Romero et al. 2008). The remaining mutantscorrespond to proteins with poorly described molecularfunctions in vivo: Ted1p, which may regulate the p24proteins (Haass et al. 2007); Arv1p, involved in in-tracellular distribution of lipids (Kajiwara et al. 2008);and Csf1p, an uncharacterized protein with a singlepredicted transmembrane domain. Finally, several mu-tants showed less severe delays in the maturation ofGas1p: Lhs1p is an ER chaperone (Craven et al. 1996);Sec22p functions in protein localization in the earlysecretory pathway by mediating vesicle fusion (Bennett

1995); Fat1p and Nem1p play distinct roles in lipidbiosynthesis, functioning in fatty acid transport andregulation, respectively (Zou et al. 2002; Santos-Rosa

et al. 2005). Proteins that function in various aspects oflipid biosynthesis and trafficking likely impact Gas1pbiogenesis through alterations in the attachment of theGPI anchor or the ability of Gas1p to enter into anappropriate lipid environment prior to forward trans-port. This accumulation of immature GPI-anchoredproteins in the ER could overwhelm the protein foldingand degradation machinery, causing induction of theUPR and subsequent upregulation of Kar2p. Similarly,failure to appropriately glycosylate CPY could result inUPR activation and Kar2p secretion. Indeed, many ofthese glycosylation and GPI-anchor mutants were syn-thetic lethal when combined with a hac1D null allele,suggesting they depend upon the UPR to maintain afunctional secretory pathway (Table 1).

ER quality control is defective in a subset of Kar2psecretors: An important function of the endoplasmicreticulum is to regulate protein secretion such thatmisfolded or unassembled proteins are not deployedwithin the cell (Ellgaard and Helenius 2003). Wereasoned that Kar2p secretion from cells may resultfrom a breakdown in this quality control checkpoint,either because this pathway also functions in the re-tention of ER residents or alternatively, because Kar2pmay be bound to misfolded proteins that are improperlypackaged into COPII vesicles and thereby secreted. Weexamined ER quality control in our panel of mutants bymonitoring the glycosylation state of a model misfoldedprotein, CPY*, which is normally targeted for ERAD andthus does not acquire Golgi-specific modifications. Weintroduced an HA-tagged form of CPY* into each of theKar2p retention mutants and measured the acquisitionof a-1,6-mannose during a pulse-chase experiment,indicative of Golgi delivery of the aberrant protein(Figure 3). In wild-type cells, very little CPY* was

Figure 3.—Quality control of a mis-folded protein in Kar2p retention mu-tants. (A) Kar2p retention mutantsexpressing HA-CPY* were subjected topulse-chase analysis (inset) and the de-gree of acquisition of a-1,6-mannosewas quantified by sequential immuno-precipitation. Only those strains exhib-iting defects in protein quality controlare shown.

764 A. Copic et al.

immunoprecipitated with antibodies against the a-1,6-mannose moiety; however, in a small number of mutantswe detected significant Golgi modification of thismisfolded protein.

Among the mutants with impaired ER retention werethe p24 family members, emp24D, erv25D, erp1D, anderp2D and the putative p24 regulator, ted1D. Similarlyaffected were two components of the GPI-anchorremodeling machinery, bst1D and gup1D. Mutation ofeither EMP24 (also known as BST2) or BST1 has beenpreviously described as causing defects in ER retentionof another misfolded secretory protein, the S11 form ofinvertase, consistent with broad defects in ER retentionin these related sets of mutants (Elrod-Erickson andKaiser 1996). Several other strains that showed moremoderate defects in the ER retention of CPY* corre-spond to particularly interesting candidates in ERquality control. Yer140wp is a predicted membraneprotein of unknown localization that physically interactswith Slp1p, another poorly characterized membraneprotein (Collins et al. 2007). Ylr065cp is yet anotherpredicted membrane protein that, when mutated, issynthetic lethal with hac1D, indicative of a role inprotein folding and quality control. We propose thatthese proteins represent novel players that contribute tothe fidelity of protein sorting in the ER.

Kar2p secretion can result from Golgi-ER retrievaldefects: Release of Kar2p to the cell surface could resultfrom impaired retention of the ER population, or byaltered retrieval of escaped Kar2p from the Golgi via theHDEL receptor, Erd2p (Townsley et al. 1994). Weaimed to distinguish between defects in retention andretrieval by examining the localization of Erd2p, whichwe fused to GFP as integrated insertion into the ERD2locus. This integration supports viability, suggesting thatit functionally complements erd2 disruption. In wild-type cells grown in rich medium, Erd2p localizes largelyto the ER (Schuldiner et al. 2005), with a minorpopulation in punctate foci that likely correspond tothe cis-Golgi (Figure 4A). In the majority of Kar2pretention mutants, Erd2p was similarly localized; how-ever, there were several mutants that accumulatedErd2p-GFP in a single larger spot that may represent adistended Golgi structure, suggestive of a failure torecycle proteins back to the ER. These mutants includethe ER-Golgi SNARE mutant, sec22D; SNARE proteinsmediate the fusion of vesicles with the correct targetcompartment and Sec22p functions both in anterog-rade ER-Golgi and retrograde Golgi-ER traffic (Burri

et al. 2003; Dilcher et al. 2003). Perturbation of Sec22pfunction likely directly impedes the retrieval of Erd2p-GFP, resulting in its steady-state Golgi localization.

Figure 4.—Golgi-ER re-trieval is perturbed in someKar2p retention mutants.(A) Subcellular localizationof Erd2p-GFP was exam-ined in each of the Kar2pretention mutants. Unlikethe ER localization seen inWT cells, a number of mu-tants accumulated Erd2p-GFP in punctate foci. Inilm1D cells, Erd2p-GFP ac-cumulated in distendedER structures (arrowheads).(B)TheseErd2p localizationmutants show a variety ofinteractions, including phe-notypic enhancement orsynthetic lethality (PE/SL;blue), phenotypic suppres-sion (PS; red), yeast 2-hybrid(Y2-H; green), and physicalassociation (gray). (C) Sub-cellular localization of thetail-anchored protein, GFP-Frt1p, in WTcells and Kar2pretention mutants.

ER Homeostasis in Yeast 765

Erd2p-GFP was similarly mislocalized in mutants thatcorrespond to all three members of the GET complex,Get1p, Get2p, and Get3p, which function in the ERintegration of tail-anchored proteins (Schuldiner

et al. 2008), including SNAREs, and thereby indirectlyaffect Golgi-ER retrieval. The get mutants have beenpreviously described as defective in Erd2p localization(Schuldiner et al. 2005), among other pleiotropicphenotypes that are consistent with broad defects inmany aspects of cellular function (Schuldiner et al.2008). Five additional mutants, mdy2D, mga2D, rce1D,htz1D, and yor164cD, exhibited a similar aberrant distri-bution of Erd2p-GFP, albeit less striking than the get1D,get2D, get3D, and sec22D strains. Finally, the ilm1D strainshowed a distinct Erd2-GFP localization pattern, withincreased fluorescence in a distended structure thatappeared to be continuous with the perinuclear ER andmay represent a distended karmella-like domain. Theknown genetic interactions among the group of mu-tants that showed defective Erd2p-GFP localization areconsistent with overlapping roles in Golgi-ER retrieval;many components show synthetic sick or aggravatinginteractions with the get mutants, suggesting distinctfunctional roles that are more deleterious when com-bined than when present as single mutations (Figure4B). Conversely, genetic interactions among the getmutants are neutral or alleviating, consistent withshared roles in a single pathway (Boone et al. 2007).

Five of the mutants that show defective Erd2plocalization correspond to proteins that have beenisolated as two separate physical complexes: Get1p,Get2p, and Get3p form an ER-localized protein com-plex (Schuldiner et al. 2005), and Mdy2p copurifieswith Yor164cp (Fleischer et al. 2006; Krogan et al.2006). High throughput yeast two-hybrid analysisidentified an interaction between Get3p and Yor164cp(Ito et al. 2001), suggesting a link between these twocomplexes that may explain the shared Erd2p defect.Get3p has been proposed to function as a cytosolicreceptor that binds the hydrophobic domains of newlysynthesized tail-anchored proteins that subsequentlydocks with the ER localized receptors, Get1p andGet2p. The physical association of Yor164cp with Mdy2p,which in turn interacts with ribosomes (Fleischer et al.2006), is suggestive of an intermediate role for theYor164cp/Mdy2p complex linking the GET machinerydirectly to the ribosome. We examined the localizationof a tail-anchored protein, GFP-Frt1p, in the yor164cDand mdy2D strains to determine if membrane integra-tion of this class of proteins is perturbed. In wild-typecells, GFP-Frt1p localizes to punctate foci within theendoplasmic reticulum; in get mutant strains, we ob-served a more diffuse, cytosolic localization, with signif-icant accumulation in a single large puncta, similar tothat observed for other tail-anchored proteins in thesemutants (Schuldiner et al. 2008). Similarly, in yor164cDand mdy2D cells, GFP-Frt1p, displayed diffuse cytosolic

staining with a single focus (Figure 4C). Other Erd2pretrieval mutants did not show the same phenotype andclosely resembled wild-type cells (data not shown),suggesting a specific role for the Yor164cp and Mdy2pproteins in this process. The precise function thatYor164cp and Mdy2p perform in protein insertion andhow they interface with both the ribosome and Get3premain to be explored. However, we propose that theKar2p secretion phenotype of the mdy2D and yor164cDmutants stems largely from defective insertion ofSNARE proteins that, like in the getD mutants, resultsin the impaired retrograde traffic of Golgi-localizedErd2p, thereby causing inefficient retrieval and secre-tion of Kar2p. Similar findings identifying Yor164cp andMdy2p, newly renamed Get4p and Get5p, respectively,as components of the tail-anchored insertion pathwayhave recently been published (Jonikas et al. 2009).

Evidence for modulation of Kar2p secretion by thehistone variant Htz1p: One surprising set of mutantsthat was markedly enriched in our collection of Kar2pretention mutants corresponds to the SWR1 complexand the variant histone, Htz1p, that it acts on. Many ofthese mutants have pleiotropic defects associated withvarious deficiencies in the secretory pathway, includingvacuolar protein sorting defects (Bonangelino et al.2002), membrane remodeling defects (Wright et al.2003), and sensitivity to exogenous lipids (Lockshon

et al. 2007). Having added an additional defect, ERretrieval, to the list of htz1D phenotypes, we aimed tofurther dissect the nature of this mislocalization. Onefunction of Htz1p is to antagonize the silencing activi-ties of Sir2p and HMR at telomeres and the mating loci(Meneghini et al. 2003). We reasoned that if Erd2pmislocalization and Kar2p secretion resulted from in-appropriate silencing in these regions, then normalfunction could be restored by deletion of either SIR2or HMR in the htz1D background. However, Kar2psecretion was equivalent in the htz1D, htz1Dsir2D, andhtz1DhmrD mutants (Figure 5A), suggesting that thespecific regulatory function of Htz1p is likely to resultfrom binding to individual promoters of candidategenes rather than a broader anti-silencing function.

The shared Kar2p secretion phenotype of the SWR1and NuA4 complexes (Figure 1D) led us to determinewhether acetylation of Htz1p is required for normal ERretention and homeostasis (Babiarz et al. 2006; Keogh

et al. 2006). We examined Kar2p secretion in strainswhere Htz1p was replaced with mutant forms that areunable to be acetylated on each of four key lysineresidues, K3, K8, K10, and K14 (Babiarz et al. 2006).Compared with an isogenic wild-type strain, only theK14R variant of Htz1p showed significant Kar2p secre-tion (Figure 5B), suggesting a requirement for acetyla-tion of K14 on Htz1p to maintain ER homeostasis ororganization. To determine whether the Kar2p secre-tion phenotype of the htz1D strain stems from down-regulation of any of the 87 known Kar2p retention

766 A. Copic et al.

mutants, we examined existing microarray data sets forgenes that coincided with our collection of mutants(Meneghini et al. 2003). Only one gene that wasdownregulated in the htz1D and htz1Dsir2D mutantsalso showed a Kar2p retention defect when mutated:ILM1. We tested the ability of ILM1 to rescue the Kar2secretion and Erd2p-GFP localization phenotypes whenoverexpressed in the htz1D strain, but were unable todetect any rescue (data not shown). We were concernedthat overexpression of Ilm1p, an ER membrane protein,would induce the UPR and thereby upregulate Kar2p

expression, masking any rescue of the phenotype. Werepeated the ILM1 suppression experiment in a hac1D

background, such that the UPR would be inactivatedand Kar2p secretion would stem directly from the htz1D

defect. Similar to wild-type cells, extracellular Kar2p wasnot abundant in hac1D cells, whereas hac1Dhtz1D doublemutants showed significant Kar2p secretion, which wasnot rescued by overexpression of ILM1, suggesting thatdownregulation of ILM1 is not the primary cause ofKar2p secretion in htz1D cells (Figure 5C). Precisely howmany genes contribute to the Kar2p secretion pheno-type of the htz1D mutant remains to be determined, andIlm1p disregulation may still play a part in this process.The pleiotropic phenotypes of the htz1D and SWR1-complex mutants suggest a relatively broad impact onthe secretory pathway, consistent with a complex pat-tern of disregulation that would perturb a variety ofprocesses.

Such broad defects in secretory pathway function inhtz1D mutants might indicate a role in tail-anchoredprotein insertion, where multiple steps of membranefusion would be impaired due to altered abundance ofthe SNARE fusion machinery. Using a variety of tail-anchored proteins, we did not observe any defect inintracellular distribution in htz1D mutant cells (data notshown). However, overexpression of the SNARE,Sec22p, which functions both in anterograde andretrograde traffic between the ER and Golgi, caused aredistribution of Erd2p-GFP to the ER (Figure 4D) anddiminished the Kar2p secretion phenotype in htz1D

cells (data not shown). Similar overexpression of ad-ditional anterograde SNAREs, Bet1p, Bos1p, andSed5p, had no effect (Figure 4D and data not shown),consistent with a retrograde trafficking defect in thehtz1D mutant. We speculate that in the absence ofnormal Htz1p function, retrograde vesicles are ren-dered less fusion competent and that upregulation ofthe SNARE machinery alleviates this defect. How Htz1pinfluences membrane trafficking events remains to bedetermined, but the pleiotropic phenotypes of htz1D

cells, including aberrant formation of proliferated ERdomains known as karmellae (Wright et al. 2003), acold-sensitive growth phenotype and sensitivity to thepresence of the fatty acid, oleate (Lockshon et al. 2007),are consistent with altered lipid metabolism that couldlead to broad defects in secretory pathway function.

CONCLUSIONS

To define the cellular machinery that contributes toER homeostasis, we employed a genomewide approachto identify mutants that are defective in the intracellularretention of the abundant lumenal chaperone, Kar2p.We identified 73 nonessential genes and 14 essentialgenes as candidate mediators of ER retention. This setof mutants encompassed known components of proteinfolding, post-translation modification, and protein traf-

Figure 5.—Htz1p contributes to Kar2p secretion via Golgi-ER retrieval. Secretion of Kar2p was detected in the indicatedstrains by immunoblotting of the extracellular medium (top)and total cellular protein was monitored by immunoblottingof the plasma membrane protein, Yor1p (bottom). (A) Kar2psecretion in htz1D cells was equivalent regardless of sir2D andhmrD deletion. (B) Secretion of Kar2p correlates specificallywith the K14R acetylation-deficient mutant of Htz1p. (C)Kar2p secretion in an htz1D mutant was not rescued by over-expression of ILM1. Cells expressing the empty vector,pRS426, serve as a negative control. (D) The punctate distri-bution of Erd2p-GFP in htz1D cells (left) was restored to an ERlocalization by overexpression of SEC22 (middle), but notBET1 (right).

ER Homeostasis in Yeast 767

ficking pathways and identified novel candidates ofprotein glycosylation, GPI biogenesis, and ER qualitycontrol (Figure 6). Our characterization of the misloc-alization of Erd2p-GFP led to the identification of anovel pair of regulators of tail-anchored protein in-sertion. Like the get mutants, yor164cD and mdy2D

accumulate tail-anchored proteins in a large punctatespot that likely stems from aggregation of the hydro-phobic domains when membrane insertion fails. SinceMdy2p associates with the ribosome, we propose thatthe Mdy2p/Yor164cp complex binds tail-anchored pro-teins as they come off the ribosome, working inconjunction with Get3p, which binds to the hydropho-bic membrane domains to deliver them to the ER-localized Get1p/Get2p complex. Although the order ofbinding events and the precise role of each componentremains to be fully elucidated, our findings expand ourunderstanding of the machinery that facilitates bio-genesis of this important class of membrane proteins.

From the perspective of maintaining ER function, oneof the more interesting subsets of mutants we identifiedwere those with defective ER quality control. Theeffective retention of misfolded proteins plays an im-portant role in maintaining accurate cellular function;

however, an overzealous quality control checkpoint canhave detrimental consequences. For example, cysticfibrosis can be caused by the stringent ER retentionand degradation of mutant forms of CFTR, a membraneprotein that can partially function if delivered to the cellsurface (Welch 2004). The mutants that we identified asdefective in the ER retention of CPY* represent candi-dates for broad regulators of ER quality control. Inparticular, several poorly characterized membrane pro-teins, Slp1p, Yer140wp, and Ylr065cp, have clear humanorthologs and warrant further characterization withrespect to additional misfolded proteins. Together, thesuite of mutants that we have defined here represents astrong starting point from which we hope to gain furtherinsight into the interrelated processes of secretory pro-tein biogenesis, ER export, and protein quality control.

We thank Randy Schekman, Jasper Rine, Hiten Madhani, Liza Pon,Traude Beilharz, and Roy Buchanan for contributing strains, plasmids,and antibodies. Many thanks to Michael Wolfe for his early efforts inthe screening process and to David Fidock for providing access to themicroscopy facility. This work was supported in part by the NationalInstitutes of Health grants GM-078186 (to E.A.M.) and GM-078855 (toM.D.).

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Communicating editor: M. D. Rose

ER Homeostasis in Yeast 769

Supporting Information http://www.genetics.org/cgi/content/full/genetics.109.101105/DC1

Genomewide Analysis Reveals Novel Pathways Affecting Endoplasmic Reticulum Homeostasis, Protein Modification and Quality Control

Alenka Copic, Mariana Dorrington, Silvere Pagant, Justine Barry, Marcus C. S. Lee, Indira Singh, John L. Hartman, IV and Elizabeth A. Miller

Copyright © 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.101105

A. Copic et al. 2 SI

TABLE S1

Non-essential mutants - compiled data for the non-essential haploid mutant screen

Table S1 is available as an Excel file at http://www.genetics.org/cgi/content/full/genetics.109.101105/DC1.

A. Copic et al. 3 SI

TABLE S2

Essential mutants - Kar2p secretion data for the Tet-repressible essential mutant screen

gene Kar2 secretion index

erd2 7.92

ost2 5.60

iqg1 4.98

pgi1 4.36

gpi1 4.23

alg11 4.17

srp72 4.00

gpi17 3.80

rft1 3.58

sec39 3.42

mot1 2.59

sec17 2.42

gpi19 2.38

abd1 2.22

pmi40 1.97

psa1 1.92

tif35 1.91

sec21 1.88

vrg4 1.85

mrs6 1.79

alg7 1.71

sec20 1.70

alg13 1.69

syf1 1.64

dsl1 1.63

epl1 1.63

yjr046w 1.60

gpi19 1.57

uso1 1.55

esa1 1.54

sar1 1.54

snu71 1.51

A. Copic et al. 4 SI

TABLE S3

Strain construction details for the generation of double mutant alleles

gene hac1 double Erd2-GFP

bst (cross to

LMY288)

YDR414C Erd1 cross/SGA SGA cross

YGL200C Emp24 cross/SGA PCR cross

YFL025C Bst1 cross/SGA SGA cross

YML012W Erv25 SGA SGA cross

YKL073W Lhs1 SGA PCR cross

YGL084C Gup1 cross N/D cross

YCR044C Per1 cross/SGA PCR cross

YAR002C-A Erp1 cross/SGA SGA cross

YLR242C Arv1 cross/SGA SGA cross

YIL039W Ted1 cross/SGA SGA cross

YOR154W Slp1 cross/SGA SGA

YGL020C Get1 cross/SGA SGA

YJL077C Ics3 cross N/D

YDL100C Get3 SGA SGA

YMR214W Scj1 SGA SGA

YHR078W ORF cross/SGA SGA

YOR164C ORF cross/SGA SGA

YER083C Get2 SGA SGA cross

YGL228W She10 cross/SGA SGA

YBL082C Alg3 SGA SGA cross

YBR276C Pps1 cross/SGA SGA

YJL183W Mnn11 cross/SGA SGA

YLR087C Csf1 cross/SGA SGA

YBR106W Pho88 cross/SGA SGA

YNL080C Eos1 SGA PCR

YHR181W Erv26 cross PCR

YLR268W Sec22 cross/SGA SGA

YDR310C Sum1 cross/SGA SGA

YLR065C ORF cross/SGA SGA

YMR031W-A dubious SGA SGA

YOL111C Mdy2 cross/SGA SGA

YBL083C dubious SGA SGA

YLR110C Ccw12 SGA SGA

YLR085C Arp6 SGA SGA

YOL012C Htz1 cross/SGA SGA/PCR

YML041C Vps71/Swc6 SGA SGA

YDR482C Cwc21 cross/SGA SGA

A. Copic et al. 5 SI

YLR374C dubious cross/SGA SGA

YML013W Ubx2 cross/SGA SGA

YAL007C Erp2 cross/SGA SGA cross

YML013C-A dubious SGA SGA

YIR033W Mga2 cross PCR

YJR075W Hoc1 cross/SGA SGA

YNL107W Yaf9 N/D PCR

YJR117W Ste24 cross/SGA SGA

YER140W ORF cross/SGA SGA

YBL051C Pin4 cross/SGA SGA

YNL087W Tcb2 cross/SGA SGA

YDR300C Pro1 cross N/D

YLR111W dubious SGA SGA

YDR485C Vps72/Swc2 cross/SGA SGA

YAL011W Swc3 SGA SGA

YOR085W Ost3 SGA SGA

YDL095W Pmt1 SGA SGA

YDR372C Vps74 cross/SGA SGA

YJR118C Ilm1 cross PCR

YML115C Van1 cross PCR

YDR108W Gsg1 cross/SGA SGA

YBR041W Fat1 cross/SGA SGA cross

YDR245W Mnn10 cross/SGA SGA

YFR013W Ioc3 cross/SGA SGA

YMR274C Rce1 cross/SGA SGA

YDR334W Swr1 cross/SGA SGA

YDR227W Sir4 N/D PCR

YJL062W Las21 SGA SGA

YDR517W Grh1 cross/SGA SGA

YBL038W Mrpl16 cross/SGA PCR

YDR469W Sdc1 cross/SGA SGA

YJL123C ORF SGA SGA

YOR355W Gds1 SGA SGA

YMR036C Mih1 cross/SGA SGA

YHR004C Nem1 cross/SGA SGA cross

YDL099W Bug1 cross/SGA SGA

Strain construction details: cross (traditional mating and tetrad dissection); SGA (mating with SGA query strain followed by haploid double mutant selection); PCR (transformation with PCR integration product)