THE JOURNAL OF BIOLOGICAL. CHEMISTRY 0 1994 by The American Society for Biochemistry and Moleeular Biology, Inc.

Vol. 269, No. 31, Issue of August 5, pp. 19695-19700, 1994 Printed in U.S.A.

O-Linked Oligosaccharides in Yeast Glycosyl Phosphatidylinositol- anchored Protein gp115 Are Clustered in a Serine-rich Region Not Essential for Its Function*

(Received for publication, February 16, 1994, and in revised form, May 6, 1994)

Evelina Gatti+, Laura Popolo, Marina Vai, Nicola Rota, and Lilia Alberghina9 From the Dipartimento di Fisiologia e Biochimica Generali, Universita degli Studi di Milano, Sezione di Biochimica Comparata, Via Celoria 26, 20133 Milano, Italy

The protein gpll6 is an exocellular yeast glycoprotein modified by 0- and N-glycosylation and attached to the plasma membrane through a glycosylphosphatidylinosi- tol. The more remarkable structural feature in gpll5 is the presence of a 36-amino acid serine-rich region. Simi- lar sequences have been found in mammalian glyco- proteins, such as the low density lipoprotein receptor, the decay-accelerating factor, and the mucins, where they are targets of multiple sites of O-glycosylation. The modification of these regions greatly influences their conformation and gives rise to “rodlike” structures. In this work, we have deleted or duplicated the Ser-rich region of gp115. The analysis of the size and glycosyla- tion state of both mutant proteins indicates that about 62% of the total contribution of the O-glycosylation to the mass of the protein is concentrated in this region. The phenotype of ggpl null mutant expressing the mu- tant proteins was also analyzed to understand if this region is important for gp115 function. The defects of slow growth rate and resistance to zymolyase of the ggpl cells are completely complemented by both mutant proteins, suggesting that this region could be dispensa- ble for gpll6 function. A tentative model of gp116 struc- ture is presented on the basis of the obtained data.

Many proteins in the yeast Saccharomyces cereuisiae are modified by the attachment of N-linked oligosaccharides to as- paragine, O-linked mannose to serine or threonine, and glyco- sylphosphatidylinositol (GPI)’ membrane anchors (Herscovics and Orlean, 1993). As in all eukaryotes, glycoproteins are found in various intracellular compartments (endoplasmic reticulum, Golgi, vacuoles) and in the plasma membrane. Moreover, in yeast, glycoproteins are present in the periplasmic space and in the wall that surrounds the cell. The cell wall is composed mainly of a glucan layer that determines the rigidity and the shape of the cell, of chitin that is mostly localized in the septum structures, and of mannans, a class of glycoproteins with high mannose content (Cabib et al., 1992; Ballou, 1981). Mannans also appear responsible for the permeability properties of the

* This work has been partially supported by a grant from MURST (60%) (to L. A,) and by P. F. Ingegneria Genetica, Consiglio Nazionale delle Ricerche (to L. P.). The costs of publication of this article were

therefore be hereby marked “aduertisement” in accordance with 18 defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a fellowship from Eniricerche. 5 To whom correspondence should be addressed. Tel.: 39-2-70644808;

Fax: 39-2-70632811. The abbreviations used are: GPI, glycosylphosphatidylinositol; LDL,

low density lipoprotein; DAF, decay-accelerating factor; PAGE, poly- acrylamide gel electrophoresis; Endo F, endo-p-N-acetylglucosamini- dase F; Gsmp, Gp115 Ser minus protein; Gdsp, Gp115 duplicated Ser protein.

cell wall (Zlotnik et al., 1984; De Nobel and Barnett, 1991). Among the proteins present outside the cells, glycoproteins with hydrolytic or proteolytic activities, adhesion molecules, and enzymes involved in the biosynthesis and degradation of cell wall polymers have been characterized. The glycoprotein, gp115, is the first exocellular protein that has been found in yeast to be anchored to the plasma membrane by means of a GPI anchor (Conzelmann et al., 1988; Vai et al., 1990). This protein is also modified by N- and O-glycosylation, which over- all contribute to about 50% of the molecular mass of the pro- tein. The function of gp115 is still unknown. The gene (GGPl / GASl ) has been cloned (Nuoffer et a l . , 1991; Vai et aE., 1991). It is not essential for vegetative growth or for the processes of conjugation and sporulation. The phenotype of cells carrying a disrupted GGPl gene suggests a role in morphogenetic events occurring in the growing cells. In fact, ggpl null mutants show a reduced growth rate and defects in cell separation, which are enhanced upon entry into stationary phase and lead to an ab- normally high proportion of budded cells. Analysis of the cell wall properties has revealed that ggpl disrupted cells are strongly resistant to zymolyase during exponential growth phase, suggesting that the absence of gp115 affects the compo- sition or the arrangement of the cell wall polymers (Popolo et al., 1993).

The gp115 sequence of 559 amino acids reveals the presence of a serine-rich region close to the COOH-terminal determi- nants for attachment of GPI. This particular structural feature is also shared by other glycoproteins such as the low density lipoprotein (LDL) receptor (Davis et al., 19861, the decay-accel- erating factor ( D M ) (Medof et al., 19871, and the mucins of mammalian cells (Carlstedt et al., 1985). This Ser-rich region has been the object of the present work. We have constructed mutant forms of gp115 in which the Ser-rich region has been deleted or duplicated, and we tested the possibility that the Ser-rich region could be the site of extensive O-glycosylation. Then, we determined if this region is important to the function of gp115 by analyzing the ability of mutant forms to comple- ment the phenotype of the null mutant.

MATERIALS AND METHODS Strains and Growth Conditions-Escherichia coli JM109 (endAl

recAl gyrA96 thi hsdRl7 (rk-, mk+) relAl supE44 A- A (Zac-prom) [F’ traD36 proAB ZacIqZ AM151) was the host strain for recombinant DNA manipulations. The S. cerevisiae haploid strain WB2d (ggpl::LEU2) was generated from the wild type strain W303-1B (MAT a ade2-1 canl- 100 ura3-1 Zeu2-3,112 tripl-1 his3-11,15) by the one-step gene disrup- tion procedure (Rohstein, 1983). Strain 6210 (Zeu2-3, 112 ura3-52 his3- A200 lys2-801 trpl-A901 suc2-A9) and its derivative carrying the kre2::TRPl disruption were kindly provided by Dr. H. Bussey. Yeast cells were grown in batches at 30 “C in YEPD medium containing 1% yeast extract, 2% Bacto-peptone, and 2% glucose or in Difco yeast ni- trogen base without amino acids medium (6.7 ghter), 2% glucose, and supplemented with the required amino acids. For the buffered medium

19695

19696 Clustered 0-Linked Glycans in East Protein gp115

(pH 6.8), precalculated amounts of 0.2 M monobasic and 0.2 M dibasic sodium phosphate solutions were mixed and added to yeast nitrogen base without amino acids medium to obtain the required pH. Cell num- ber was determined using a Coulter counter, and cell volume distribu- tions were obtained as previously described (Vanoni et al., 1983). The percentage of budded cells was determined on formalin-fixed samples, and morphologies were examined after staining with calcofluor (Vanoni et al., 1983).

DNA Manipulation Procedures and Oligonucleotide-directed Muta- genesis-Standard protocols were used for recombinant DNA manipu- lation and bacterial and yeast transformations (Sambrook et al., 1989; Hill et al., 1991). To delete or duplicate the Ser-rich region of the gp115, the 0.4-kilobase SalI-Hind111 fragment containing the 3' terminus of GGPl gene (Vai et al., 1991) was subcloned in the pSELEC'P-1 vector (Promega, Madison, WI), and a 28-mer oligonucleotide (5'"MTATCT- TCTAGTI'CGAAGAATGCTGCC-3') was used to introduce an SfuI site in the second codon upstream from AsdZ8 in the gp115 sequence. Mu- tagenesis was carried out using the Altered Sitesa in vitro mutagenesis system (Promega), and mutagenized DNAs were verified by sequencing (Sanger et al., 1977). For the deletion construct, we excised the 100-base pair ScaI-SfuI fragment after having removed the 5"protruding end of the SfuI site. For the duplication construct, we substituted the 100-base pair SfuI-Hind111 fragment with the 200-base pair wild type ScaI- Hind111 fragment after having filled in the SfuI site. Both constructs were sequenced to verify the preservation of correct reading frame. The 0.4-kilobase SalI-Hind111 fragments from each plasmid described above were substituted for the same fragment of the placN8 plasmid, obtain- ing pSSz6gp115, pGds, and pGsm, respectively. placN8 was obtained by cloning the 4.5-kilobase NcoI-NdeI fragment, containing the whole GGPl gene and its flanking regions, into the YCplac33 ARS-CEN shuttle vector (Gietz and Sugino, 1988).

Extract Preparation, Electrophoretic Procedure, and Immuno- blotting-Total protein extracts of S. cerevisiae cells prepared as previ- ously described (Vai et al., 1986) and membrane fractions according to Ulaszewski et al. (1983) were resolved by SDS-PAGE on 8% polyacryl- amide slab gels. Electrophoretic transfer of proteins and immunodeco- ration of blots were carried out as reported (Popolo et al., 1988).

Assay of Zymolyase Sensitivity-Exponentially growing cells were treated with zymolyase as previously described (Popolo et al., 1993). Spheroplast lysis after dilution in water was followed by absorbance measurements at 660 nm.

Phosphatidylinositol Phospholipase C Deatment-Membrane pro- teins in the Triton X-114 detergent phase were treated with phospha- tidylinositol phospholipase C from Bacillus cereus (Boehringer Mann- heim, Germany) as described (Bordier, 1981; Vai et al., 1990).

Endo F Deatment and a-Mannosidase Deatments-Endo-p-N- acetylglucosaminidase F (Boehringer Mannheim) treatment was ap- plied to cell lysates (corresponding to 2 x lo8 cells) as previously re- ported (Vai et al., 1990) except that lysis was carried out in 200 1.11 of 100 mM sodium acetate (pH 5), 50 mM EDTA, 0.8% SDS, 1% 2-mercaptoeth- anol, and 4.8% Triton X-100. The Endo F-treated sample was then split in two aliquots that were incubated overnight a t 37 "C in the absence or presence of 2 units of jack bean a-mannosidase (Oxford Glycosystems, United Kingdom), both in the presence of 2 mM ZnSO, and inhibitors of proteases. All the samples were then lyophilized, resuspended in SDS sample buffer, and denatured.

Labeling with [3H]Mannose and Analysis of 0-Linked Oligosac- charides-Cells were grown overnight in minimal medium containing 2% galactose and then diluted to lo7 cells/ml in 1% galactose as indi- cated by Byrd et al. (1982). After 2.5 h, 5 ml of culture were transferred to 50-ml tubes in which 1.2 mCi of [2-3Hlmannose had been lyophilized. Cells were labeled for 2.5 h. The total radioactivity incorporated was determined on aliquots of the culture. Labeled cells were collected by centrifugation, washed with ice-cold distilled water, and frozen a t -80 "C. Total extracts and two-dimensional gel electrophoresis were performed as described (Popolo et al., 1984). About 6 x lo6 cpm were loaded on the first dimension gel. After the second dimension run, proteins were transferred to nitrocellulose filters that were air-dried and exposed to 3H-sensitive films for 8 days. In parallel, filters carrying non-radioactive extracts were processed for immunoblotting to confirm the identity of the labeled spots with the proteins of interest. For p-elimination, spots were excised from the blots, cut into smaller pieces, and incubated overnight at 30 "C in 150 pl of 0.1 M NaOH. The released radioactivity was determined. The solution was neutralized with 10 pl of 10% acetic acid and lyophilized. The separation of oligosaccharides by descending chromatography and silver staining of sugars as performed as described (Orlean et al., 1991).

A

N 7

c gpllS

S 1 A S S N L I Y I S S S I S N S G S ~ ~ ~ S g ~ A ~ Q S S ~ - 490 642

? S S G S 5 5 ~ S S S S A S ~ ~ K ~ ~ ~ ~ ~ l ~ ~ ~ ~ ~ ~ V

513 191

Gdop

FIG. 1. A, schematic illustration of wild type gp115 and mutant forms

minal hydrophobic domain (black shading), and the NH,-terminal sig- Gsmp and Gdsp. The Ser-rich region (uertical stripes), the COOH-ter-

nal sequence (diagonal stripes) are indicated. The arrow indicates the site of attachment of GPI AS^^^^). B, amino acid sequence of the COOH- terminal region of gp115 and of deleted and duplicated forms with the Ser-rich region, the hydrophobic domain, and the substitution of Lys526 with Ser due to the mutagenesis procedure, from which the protein S5"gp115 originates.

RESULTS

Muta t iona l Analysis of GGPl Gene in Ser-rich Region-A serine-rich region is located in the COOH-terminal portion of gp115 between residues 489 and 525 in the amino acid se- quence deduced from the GGPl gene. As shown in Fig. lA, this region is close to the site of attachment of GPI, proposed to be the Asn528 and indicated by an ar row (Nuoffer et al., 1991). The region that has been manipulated spans from residues 491 to 526 and contains 25 serines and 1 threonine in a 36-amino acid stretch (Fig. 1B). Different methods of analysis of secondary structure concordantly predict an unordered form for this re- gion. A program that analyzes the chain flexibility of the pro- tein (Karplus and Schulz, 1985) predicts an extended highly flexible region in correspondence with the Ser-rich region (Fig. 2, upper panel) . Since serine and threonine residues could pro- vide multiple sites for 0-glycosylation, the conformation of the segment could be stabilized in the extended conformation by this modification. To investigate this possibility, we constructed two mutant forms of gp115. In the Gsmp protein (Gp115 Ser minus), the Ser-rich region was deleted, whereas in the Gdsp protein (Gp115 duplicated Ser), the Ser-rich region was dupli- cated in tandem. A scheme of these constructs is reported in Fig. 1B. The flexibility profiles indicate that these mutations indeed alter the more extended flexible region predicted in the protein (Fig. 2, central and lower panels). To obtain these mutations, a restriction site was introduced

by site-directed mutagenesis, which caused a transition of Lys526 to Ser (Fig. 1B). This modification does not alter the processing or the functionality of the mutant protein (S526gp115) with respect to gp115, and we refer to this protein as the control.

Since GGPl is not an essential gene, the mutant forms can be directly expressed in the null mutant and herein tested for their effects. The mutant forms of gp115 were expressed from an ARS-CEN vector (YCplac33), under the control of the natural promoter, in a ggpl null mutant (strain WB-Sd, ggpl::LEU2) derived from the strain W303-1B. As described in detail below, the phenotype of this null mutant is similar to that of mutants previously characterized (Popolo et al., 1993).

Clustered 0-Linked Glycans in Yeast Protein gp115

ael I 8 0 160 2 4 0 320 400 4 e o

eo 160 2 4 0 320 400 4RO

as-240 e o ' 320 400 4 0 0 j10 ' R e s 1 due number

FIG. 2. Flexibility plots. Profiles of the predicted side chain flexibil- ity for gp115 and mutant proteins, according to Karplus and Schulz (1985), are shown. Shaded areas indicate the Ser-rich region analyzed in this work.

Characterization of Mutant Proteins Gsmp and Gdsp-To test the expression of mutant forms, we analyzed total extracts from transformed cells by immunobloting (Fig. 3). Since more resolutive conditions in SDS-PAGE were adopted, gp115 mi- grates with a molecular mass of 130 kDa instead of that re- ported of 115 kDa, which referred to separation on two-dimen- sional gels where the protein was initially identified (Popolo and Alberghina, 1984). As shown in Fig. 3, the SSz6gp115 mi- grates as a 130-kDa band as gp115, whereas Gsmp and Gdsp migrate as 105- and 165-kDa bands, respectively. This behavior was obtained for different independent transformants. The 105- and 165-kDa bands represent the fully glycosylated Gsmp and Gdsp proteins and are unique, suggesting that their matu- ration is as efficient as for gp115. As a control, WB-2d cells were also analyzed, and neither gp115 nor a truncated product has been detected. In all of the lanes, a 60-kDa band is present and is due to a cross-reaction of the antibodies. The difference in mobility of Gsmp and Gdsp, with respect to gp115, is -25 and +35 kDa, considerably higher than the 24 kDa expected on the basis of the number of amino acids present in the manipulated segment. This is the first indication that the Ser-rich region may be the target of an extensive modification.

The membrane association of the mutant proteins was also investigated. The crude membrane and soluble fractions ob- tained from transformed cells were analyzed by immunoblot- ting. S526gp115, Gsmp, and Gdsp were found to be associated with the membrane fraction (Fig. 4A). Since the same amount of proteins was loaded on each lane, the steady-state levels of the mutant proteins (from the intensity of the bands) appear unchanged with respect to SSz6gp115. This suggests that dele- tion and duplication do not bring about significant changes on the stability of the proteins.

To ascertain if the mutant proteins are associated with the membrane through a GPI, cell proteins were extracted with Triton X-114. The extracts were phase-partitioned at 32 "C, and the detergent phases, containing GPI proteins, were subjected to phosphatidylinositol phospholipase C treatment. After enzy- matic treatment, single bands corresponding to the mutant proteins were mostly detected in the aqueous phase in all the extracts (Fig. 4B 1. This points to an association of mutant pro- teins with the membrane through a GPI.

Ser-rich Region Is a Cluster of 0-Linked Oligosaccharide Chains-Previous experiments have suggested that gp115 is also modified by 0-glycosylation (Vai et al., 1991). This is shown by several criteria. The mobility of gp115 undergoes a shift of about 10 kDa in a kre2 mutant (Fig. 5A). In kre2/mntl mu- tants, the elongation of 0-linked oligosaccharides is blocked a t the addition of the third mannose (for a review, see Herscovics and Orlean (1993)).

To analyze the state of 0-glycosylation of gp115 and of the

1 2 3 4 5

165 kD-w 130 kD - 105kD-

19697

FIG. 3. Western analysis of wild type and mutant forms of gp115. Total extracts from exponentially growing cells (2.5 x 10') of WB-2d strain (ggpl-disrupted cells) expressing S526 gp115 (lane 2) , Gsmp (lane 3). and Gdsp (lane 4 ) were separated by SDS-PAGE and after blot transfer immunodecorated with anti-gpll5 antiserum and

1) and from WB-2d null mutant (lane 5) are also shown. The bund with ""I-labeled protein A. Total extracts from W303-1B wild type cells (lane

lower mobility is a cross-reaction of the antiserum.

A S5"gp115 Gsm p Gds P

"5-2

130kD+ I-165kO

I-105kD

B P Z 6 gp115 Gsm p Gds p

-r-"l- ."." c 165kO

130 kD +

105kD+

PhaseA D A D A D A D A D A D PI-PLC - - + + " + + - - + + FIG. 4. Membrane association of Gdsp and Gsmp proteins. A,

crude membrane (M) and soluble (S) fractions were obtained according to Ulaszewski et al. (1983) from exponentially growing transformed cells and analyzed by immunoblotting as described in Fig. 3. B, immu- noblot analysis of the aqueous (A) and detergent (Dl phases obtained after a phase separation with Triton X-114. The detergent phases were also treated with phosphatidylinositol phospholipase C (PI-PLC) from B. cereus, re-extracted, and analyzed after partitioning. Molecular masses are indicated.

mutant forms, an Endo F treatment was carried out on cellular lysates to remove the N-linked oligosaccharide chains, followed by treatment with a-mannosidase to remove the 0-linked chains. Immunoblot analysis is shown in Fig. 5B. Endo F shifts the molecular mass of S526gp115 (130 kDa), Gdsp (165 kDa), and Gsmp (105 kDa) to deglycosylation products of 95,130, and 75 kDa, respectively. From these findings, a contribution of N-glycosylation of approximately 35 kDa for S526gp115, 30 kDa for Gsmp, and 40 kDa for Gdsp has been determined. The discrepancies could be attributed to deletion or duplication of an N-oligosaccharide chain attached to one potential glyco- sylation site located in the Ser-rich region ( A m 4 " ) (Vai et al., 1991). All of these deglycosylated products are susceptible to mannosidase treatment. Bands of about 60-62 kDa, which

19698 Clustered 0-Linked Glycans in East Protein gp115

..

QP115 n

“...r - i- + I - i- 4- I - i- + EndoF

KREP KrePA - - I + - - + - - + “Man ”.

205

116

97

66

45

FIG. 5. Analysis of the glycosylation state of the mutant pro- teins. A , immunoblot analysis of gp115 in KREZ and kre2A strains is shown. About 40 pg of total extracts from exponentially growing cul- tures were separated by SDS-PAGE, and proteins were blotted to ni- trocellulose filters. B, deglycosylation of gp115 and mutant proteins are shown. Lysates from 2 x 10* exponentially growing transformed cells were divided in three aliquots and incubated overnight in the presence (+) or absence (-) of Endo F as described under “Materials and Meth- ods.” Two of the Endo F-treated aliquots were incubated in the presence (+I or absence (-) of a-mannosidase (a-Man). Immunoblot analysis was carried out as in Fig. 3.

comigrate with the bovine serum albumin carrier, are poorly detected, probably due to the instability of the completely de- glycosylated protein or to low reactivity with the polyclonal antibodies, which have been raised against the fully glyco- sylated protein.

In Table I, a rough estimation of the 0-glycans has been obtained by subtracting from the mass of the N-deglycosylation products the molecular mass of the polypeptide precursor and that of GPI (about 3 kDa). Thus, the contribution of O-glyco- sylation to the molecular mass of the proteins amounts to 31 kDa for S526gp115, roughly doubles for Gdsp, and is only 15 kDa for Gsmp. This finding indicates that 16 out of 31 kDa (52%) of 0-glycans present in gp115 are clustered in the Ser-rich region, and only 15 kDa are located in the remaining part of the pro- tein. However, this is an approximate evaluation of the carbo- hydrate moiety since it is well known that the determination of glycoprotein molecular weight is not accurate by SDS-PAGE.

To show more directly the presence of 0-linked chains, trans- formed cells were labeled with [3H]mannose. Equal amounts of total extract were subjected to two-dimensional gel electro- phoresis. Given the acidic PI and high molecular weight, the mutant proteins were well separated and isolated by this tech- nique. The spots corresponding to the three proteins of interest were excised from the blots and treated with mild alkali to release the 0-linked carbohydrate. The radioactivity released was determined. The results are shown in Fig. 6A and indicate that Gdsp releases 150% of radioactivity, whereas Gdsp re- leases only 25% with respect to S526gp115 taken as 100%. This is in good agreement with the results of Table I. The released material was also qualitatively analyzed by descending paper chromatography. The profiles shown in panels B , C, and D of Fig. 6 indicate the presence of mannose, oligomers of 2, 3, and 4 mannoses, and only a tiny amount of chains with 5 mannoses.

Phenotypical Analysis of Strains Expressing Gsmp or Gdsp- The ability of the mutant forms of gp115 to suppress the effects of gp115 deficiency was analyzed. The ggpl mutant (WB-28 shows a phenotype similar to that previously described (Popolo et al., 1993) for other null mutants generated in a different genetic background. The main defects are elongation of dupli- cation time, resistance to zymolyase, a persistence of cells with one or two buds during the stationary phase, loss of the ellip- soidal shape, and enlargement of cell volume. Cultures were

TABLE I Approximate estimation of 0-linked mannosylation of

mutant forms of gp115

Product Pol egtide Obseyed Observed- O-Glycans &, M r expected

SSz6gp115 61,000 95,000 34,000 31,000 Gsmp 57,000 75,000 18,000 15,000 Gdsp 65,000 125,000 60,000 57,000

Calculated from the deduced amino acid sequence. * Determined by SDS-PAGE of the Endo F-treated polypeptides fol-

lowed by immunoblotting as shown in Fig. 5.

u1 s #PIIS Cdrp Csmp

1000 I d

t man-4 m a n 4

750

500

250

0

750 ’

500 ’

250

0 0 10 20 30 40 50

Fractions

were labeled with L3HImannose as described under “Materials and FIG. 6. Analysis of 0-linked chains of the mutant proteins. Cells

Methods.” Spots excised from two-dimensional gel blots were treated with NaOH. A , counts/min released from each spot taking the radioac- tivity released by SSz6gp115 as 100% (83,000 cpm). B, C , and D, profiles of oligosaccharide chains of SSz6gp115, Gdsp, and Gsmp.

monitored from exponential to stationary phase. Growth pa- rameters obtained from several independent experiments are shown in Table 11. Duplication time of ggpl cells doubles with respect to wild type cells (W303-1B) whereas that of the trans- formant strains expressing Gsmp or Gdsp is quite similar to the value of wild type. We have also grown the cells in minimal medium buffered at pH 6.8 since this condition enhances the elongation of the duplication time of the disrupted strain. The

Clustered 0-Linked Glycans in Yeast Protein gp115 19699 TABLE I1

Kinetic parameters and zymolyase sensitivity

Exponential Strains T". TupH *

Stationary Zymolyase

B' pBd B PB sensitivity (tn)'

min min % % min

W303-1B 110 105 74 22 5 WB-2d (ggpl::LEU2) 240 360 74 14 65 15 >300

WB-2d [pGd~l 120 120 83 9 44 10 30 WB-2d [pGsml 120 120 78 5 34 4 30

WB-2d [pS526gp1151 115 120 77 22 10

Duplication time measured as increase in cell number. * Duplication time in buffered media, pH 6.8.

Total budded cells. Pluribudded cells (two buds or more).

e Time required for 50% reduction in A,,, " ~ . The treatment was carried out on exponentially growing cells.

results indicate that the mutant forms of gp115 complement the defect of slow growth rate of the disrupted strain.

As previously shown (Popolo et al., 1993), ggpl stationary cells do not uniformly arrest in the unbudded phase but main- tain 65% budded cells. Cells producing Gsmp and Gdsp appear to complement this defect since the percentage of budded cells decreases, although it does not reach the value of the control cells.

Cell wall sensitivity to hydrolytic enzymes was assayed by treating cells collected during the exponential growth phase with zymolyase. The time required to reach a 50% reduction in absorbance after lysis in water (t lJ is reported in Table 11. As previously shown, the disrupted cells are almost completely resistant to the lysis, whereas wild type cells are very sensitive. Cells expressing S526gp115 are also very sensitive as are control cells and those expressing Gdsp and Gsmp.

The cell volume distributions were analyzed (Fig. 7). During exponential growth, no differences were detected among S526gp115-, Gdsp-, and Gsmp-expressing cells, whereas ggpl cells are slightly larger (Fig. 7A). During the stationary phase, ggpl cells show a very broad distribution due to the presence of many large cells with two buds and a cluster of cells that do not separate by sonication. These clusters are counted as single units in this type of analysis. The cell volume profiles of Gsmp- and Gdsp-producing cells and microscopic analysis (data not shown) indicate a complementation of these effects (Fig. 7B).

DISCUSSION

In this work, we have focused our attention on the serine-rich region of gp115. The analysis of the glycosylation state of the mutant proteins has provided indications that this segment is the major site of 0-glycosylation. Although in this 36-amino acid stretch only 22% of the total serines (+ threonines) are clustered, about 52% of the overall contribution of O-glyco- sylation to the molecular mass of the protein is concentrated. Apparently, the rest of the 0-linked chains are dispersed in the molecule. Since the predominant 0-linked chain length is 3 and/or 4 sugars, and 15 Ser + 1 Thr are present in this region, we can figure out that almost all of the residues are equally modified.

It is not clear what the function is of 0-glycosylation. This type of modification has been less intensively studied than the more common N-glycosylation. The presence of many O-oligo- saccharide chains in a short peptide sequence has been pro- posed to influence greatly the conformation of the peptide se- quence (Jentoft, 1990). Heavily 0-glycosylated domains of some glycoproteins have a length of 20-70 amino acids, and the car- bohydrate contribution ranges from 65 to 85% (Medof et d . , 1987; Lopez et al., 1987). The steric interactions of the oligo- saccharide chains should hinder secondary and tertiary struc- tures predicted for the unmodified segments. Physico-chemical studies on mucins support the idea that heavily 0-glycosylated

1 -I

-"- I B /

C H W L " B E R FIG. 7. Cell volume distributions of cells expressing mutant

proteins. Cells were grown on glucose minimal medium at 30 "C.

WB2d cells (....I are as indicated. A, exponential growth phase; B, S526gp115 (- - -), Gdsp (-1, Gsmp (- - - - -1, and host null mutant

stationary phase.

regions assume a stiff and extended conformation. So far, the presence of this structure has been associated with two func- tions. These segments could form a connecting spacer between the membrane anchor and the bulk of the protein. This is the case of some mammalian glycoproteins such as DAF (Medof et al., 1987), the platelet glycoprotein Ib (Lopez et al., 1987), the sucrase-isomaltase complex of the small intestinal brush bor- der (Hunziker et al., 1986), and the human LDL receptor (Davis et al., 1986). In some cell types, this structure could be relevant to the peptide chain to traverse the glycocalyx (Vitala and Janerfelt, 1985). To understand the role of the 0-linked carbo- hydrate cluster, Davis et al. (1986) have removed the clustered 0-linked sugar domain of 48 amino acids from the human LDL receptor. This deletion does not affect the stability, function, and recycling of the receptor expressed in Chinese hamster ovary cells.

The second function proposed is that 0-glycosylation is im- portant for resistance to the proteases. This is supported by studies on LDL receptor and DAF expressed in a Chinese ham-

19700 Clustered 0-Linked Glycans in Zast Protein gp115

1 Oglycosylated

Ik, 4 region

membrane g y T , FIG. 8. Model of a115 structure. The GPI anchors the protein to

the plasma membrane through a link with the COOH-terminal amino acid. The location of multiple 0-glycosylation sites is indicated, and the possible extended conformation of the region is represented for simplic- ity by a cylinder. The remaining part of the protein, represented as an

most of the N-linked oligosaccharide chains ( not shown). ellipsoidal sphere, is predicted to form a compact region and contains

ster ovary line in which the 0-glycosylation of the whole pro- tein can be reversibly blocked. Unglycosylated LDL receptor and DAF have a decreased stability at the cell surface since they are proteolytically released into the medium. Moreover, the function of the LDL receptor is impaired when overall 0- glycosylation of the protein is inhibited (Kozarsky et al., 1988; Reddy et al., 1989).

0-Mannosylation in yeast differs from that of animal and plant cells since only linear chains of 1-5 residues of mannose are attached to serine or threonine and no complex sugars are present (Herscovics and Orlean, 1993). Yeast 0-glycosylated proteins include chitinase (Kuranda and Robbins, 19911, the FUSl gene product (Truehart and Fink, 1989), a- and a-agglu- tinins (Cappellaro et al., 1991; Lipke and Kurjan, 19921, and KEX2 protease (Wilcox and Fuller, 1991). So far, a cluster of 0-linked sugars has been detected in the NH,-terminal domain of the FUSl gene product. To the best of our knowledge, no Ser-rich regions have been altered in yeast glycoproteins so far.

The presence of this region in gp115 allows us to postulate a "lollipop on a stick" model (Fig. 8) similar to that proposed for the DAF and LDL receptors (Jentoft, 1990). The 0-glycosylated region can be considerably longer than that predicted for the random-coil conformation (Jentoft, 1990). Thus, the 36-amino acid region could form an extended structure. The rest of the protein would have a compact folded conformation stabilized by disulfide bridges (14 Cys are present). In this respect, the two parts of the protein could be considered rather independent.

The second aim of this work has been to assess if the 0- linked cluster is crucial for gp115 function. The proteins with a deleted or duplicated Ser-rich region show a good complemen- tation of the phenotype of the ggpl null mutant. These data indicate that this region is not crucial for gp115 function, which is similar to the results obtained for the LDL receptor (Davis et al., 1986) in mammalian cells. This further supports the inde- pendence of the stem and the folded domain proposed by our model.

The presence of the Ser-rich region allows us to speculate on the role of this structure in gp115. The hypothesis that this region is required to expose the functional domain to the cell

surface is less likely considering that the cell wall of yeast is 100-150-nm thick (Ballou, 1981). Rather, we propose that this structure contributes (although in a dispensable way) to a pos- sible role of gp115 as a space-filling protein involved in cell wall organization among the glucan fibrils. In particular, the hydro- philic side of the fibrils could interact with the hydroxyl groups of the carbohydrate chains that also could be narrowed and packed by means of the high mobility of the GPI. The functional domain could play a role in the microarchitecture of the cell wall, and this could explain the defects observed in the ggpl mutant (Popolo et al., 1993).

Acknowledgments-We thank Dr. Widmar Tanner for helpful sugges- tions in the labeling experiment and Dr. Bussey for the kre2 mutant. We are grateful to Antonio Grippo for preparing the figures.

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