Copyright Q 1997 by the Genetics Society of America

Saccharomyces cwm'siae HOCl, a Suppressor of pkcl, Encodes a Putative Glycosyltransferase

Aaron M. Neiman, Trijay Mhaiskar, Vladimir Manus," Francis Galibert" and Neta Dean

Department of Biochemist9 and Cell Biology, State University of New York, Stony Brook, Stony Brook, New York I 1 794-5215 and *Laboratoire de Biochimie et Biologie Moleculaire, Unite de Recombinaisons Genetique (UPR4l-CNRS),

Faculte de Medicine, Rennes, France

Manuscript received May 14, 1996 Accepted for publication November 21, 1996

ABSTRACT The Saccharomyces cerevisiae gene PKCl encodes a protein kinase C isozyme that regulates cell wall

synthesis. Here we describe the characterization of HOCl, a gene identified by its ability to suppress the cell lysis phenotype of pkcZ-?71 cells. The HOC1 gene (Homologous to OCHl) is predicted to encode a type I1 integral membrane protein that strongly resembles Ochlp, an a-1,6-mannosyltransferase. Immu- nofluorescence studies localized Hoclp to the Golgi apparatus. While overexpression of HOCl rescued the pkcl-371 temperature-sensitive cell lysis phenotype, disruption of HOCl lowered the restrictive tem- perature of the pkcl-371 allele. Disruption of HOCl also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOCl appears to be distinct from that of OCHl. Taken together, these results suggest that HOCl encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall.

T HE Saccharomyces cerevisiae protein kinase C isozyme encoded by PKCl is a key regulator of cell wall

synthesis (KLIS 1994). Mutants in pkcl display abnor- mally thin and brittle cell walls (PARAVICINI et al. 1992; LEVIN et al. 1994). This defect in cell wall structure manifests itself as a cell-cycle-specific cell lysis pheno- type (LEVIN and BARTLETT-HUEBUSCH 1992). That de- fects in the cell wall are the primary cause of lethality of pkcl mutants is shown by the fact that a deletion of PKCl can be rescued by the inclusion of osmotic stabilizers, such as sorbitol, in the growth medium (LEVIN and BARTLETT-HUEBUSCH 1992).

Pkclp is a cytoplasmic enzyme that appears to regu- late cell wall synthesis indirectly, via a signal transduc- tion pathway. Pkclp activity initiates signalling through a M A P kinase cascade consisting of the BCKl , MKKl or MKKZ, and MPKl gene products (LEE and LEVIN 1992; IRIE pt al. 1993; LEE et al. 1993). Overexpression of any of these downstream kinases can partially rescue the lysis phenotype of a pkcl mutant. Mutations of bckl, mpkl , or an mkkl mkk2 double mutant all result in cell wall defects producing a temperature-sensitive cell lysis phenotype (LEE and LEVIN 1992; IRIE et al. 1993; LEE et al. 1993).

In addition to regulation of this kinase cascade, Pkclp may have a second function in cell wall synthesis (LEE and LEVIN 1992). The nature of this function has not yet been elucidated, but one potential regulatory

Cell Biology, SUNY Stony Brook, Stony Brook, NY 117945215. E-mail: ndean@mcbsgi.bio.sunysb.edu

Cmresponding author: N. Dean, Department of Biochemistry and

Genetics 145 697-645 (March, 1997)

target is the KRE6 gene product. Kre6p is required for synthesis of P-l,&glucan. Overexpression of KRE6 will partially rescue the cell lysis defect of a pkc l mutant, but not of an mpkl mutant or an mkkl, mkk2 double mutant (ROEMER et al. 1994). These observations sug- gest a defect in pkcl mutants, suppressible by KREG, which is not present in strains mutant in the down- stream kinases (ROEMER et al. 1994).

The cell wall itself is constructed of an outer mannan layer and an inner layer consisting largely of P-1,6- and P-1,3-glucans as well as some chitin. The P-l,%glucan and chitin polymers are synthesized in the periplasm, while the P-1,6-glucans are assembled in the endoplas- mic reticulum (ER) and Golgi (=IS 1994). The man- nan layer consists primarily of heavily glycosylated man- noproteins that have oligosaccharide chains attached via asparagine (Nlinked) or serine and threonine (0 linked) linkages. These chains are assembled as the proteins transit through the secretory pathway (HERS COVICS and ORLEAN 1993).

One critical protein in the extension of Nlinked oli- gosaccharide chains is the OCHl gene product (NAKA- YAMA et al. 1992). OCHl encodes an cY-l,&mannosyl- transferase that resides in the Golgi. Ochlp catalyzes the addition of an a-l,&linked mannose to the core oligosaccharide that is attached to the protein in the ER (NAKANISHI-SHINDO et al. 1993). In the absence of Ochlp, extension of Nlinked oligosaccharide chains is blocked. In this report we describe the isolation and characterization HOCI, a high copy suppressor of a tem- perature-sensitive pkcl mutation. HOCl encodes a puta- tive glycosyltransferase related to Ochlp.

638 A. M. Neiman et a l .

TABLE 1

Strains used in this study

Strain Genotype Source

SEY62 1 0 M7iu um3-52 his3-A200 tqlA901 lys2-801 ~ u r 2 - A Y h 2 - 3 , 1 1 2 S . EMU RSE55 MA ?h um3-52 lm2-3, I 12 R. SCI-WKMAN 371-4C-5C-4D MATa uru3 tq l ku2 pkrl-371 This study VMY2 As RSE55 plus, hocl::IJX? This study VMY2-"'LD MA722 urn3 leu2 fiklkcl-371 /zocl::IEU2 This study VMY2"iD kUTa um3 lm2 pkcl-371 hocl::LEU2 This study

MATERIALS AND METHODS

Strains and media: S. crrrcvi,sicte strains used in this study are listed in Table 1. Yeast were routinely grown on WAD (1% yeast extract, 2% bacto-peptone, 50 mg/l adenine, 2% glucose) or SD (0.67% Dif'co yeast nitrogen base, 2% glucose) containing appropriate nutritional supplements. Sensitivity to calcofluor white (CFW) (Sigma) was tested by streaking yeast onto WAD plates supplemented with 1.5 mg/ml CFW ( R A M rt nl. 1994). Hygromycin B sensitivity was tested on WAD plates supplemented with 50 pg/ml hygronycin B (Boeh- ringer) (BAI.I.OU et al. 1991).

Plasmids: Plasmid p371B is a library clone of PKCl in the plasmid pLWl (2p lJKA3). Plasmid p371A is the original clone of IfOCl in the library plasmid p366 ((EN LEU2). p371A HzndlIIn was made by digestion of p371A with Hh i I I I and religation of the backbone fragment. Similarly, p371A Bnm- HIA was constructed by digestion of p371A with RnmHI and religation of the backbone. Plasmid pRS425:: HOCl was made by cloning the 2.5-kb HindIII-HnmHI genomic fragment encoding the HOCl gene into HindIII-BumHI-cut plasmid pRS425. Similarly, plasmid YEp352:: HOCl was constructed by cloning the same 2.5-kb HindIII-BamHI genomic fragment into HindlII-BnmHI-cut plasmid YEp352. pCDCl1 (from J . KONOIW) carries the COCll gene under control ofthe ADHI promoter.

Plasmid pVM3 was constructed as follows: first, convenient restrictit)n sites were placed at the 5' and 3' ends of the HOC1 coding sequence by PCR. Primer 1 (5'-CTCCGAATTCAGCA- CATAGATATAT-3') introduced an EcoN site before the initi- ation codon and primer 2 (5'-GACGATC;C;TCTAGATGCATT- T(:T(;<:T(;CAC(:TTTC3') introduced NsiI and HnmHI sites at the 3' end of the coding sequence. The PCR fragment was digested with /<coRI and BumHI and cloned into EcoRI-BnmHI- cut pBlueScript SK-, generating the plasmid pVM1. A HiJindIII- N d fragment from pVMl was then ligated into a plasmid containing the e-my epitope, generating plasmid pVM2. This places the e-myc epitope at the 3' end o f the HOC1 coding sequence, changing the carboxy-terminal amino acid se- quence from PKEVQK to PKEVQK~;MES~LISEEDLNSAE- AARL. pCN2 was cut with RnmHI, treated with Klenow to fill in the rnds, and then digested with ~lindlll. The HindIII- blunt end fragment carrying I i O C l q r was subcloned into a liindlIl-.SmnI-cut deriviative o f Ylp56X (PEI.HAM 1'388), an integrating plasmid that carries the URA3 gene with a XhoI site irlserred into it and the constitlltive triose phosphate iso- merase (71'4 promoter. This construct (pVM3) places the HOC;I-?nyr scquence under control of the TPZ promoter. Be- fore transformation o f yeast strains, pVM3 was cut at the unique Xhol site within the ZIRA3 gene to target the integra- tion.

The disruption plasmid pVM4 was made as follows. Plasmid pVMl was cut at the unique PstI site in the HOC1 sequence and blunt-ended by mung bean nuclease Lreament. The Ld<lJ2 gcnc was isolated by BnmHI digestion of plasmid YDp12 (BFK- IWN rl nl. l W I ) , hlunt-cndccl b y Klenow trcamcni, and ligated

to the linearized pVM1. This fusion interrupts the HOCl cod- ing sequence after amino acid residue 45, separating the transmembrane domain from the region homologous to OCH1. The disruption fragment was liberated by BnmH1- Hind111 digestion.

To construct pTiOCH-HA, the OCHI gene was first cloned in-frame to three tandem copies of the influenza hemaggluti- nin epitope (HA) tag. A fragment containing the OCHI OW, lacking the stop codon and flanked by a 5' Hind111 and a 3' L%21 site was isolated by PCR. This OCH1 fragment was cloned into Hindlll-Pstl digested pSK-HAS, a Bluescript SK- deriva- tive (Stratagene). pSK-HA3 carries a 108-bp fragment COII-

taining sequences that encode three tandem copies of the HA epitope (YPYDWDY) cloned between the lJ.s/I and X h I sites. Thus, the SKOCH1-HA construct contains an in-frame fusion of the three copies of the HA epitope to the carboxy terminus of OCHI. Finally, pTiOCH-HA was contructed by isolating the HindIII (blunted with Klenow)-XbuI fra, ument containing the triple HA-tagged OCHl gene and cloning i t into YIp56X digested with EcoRI (blunted with Klenow) and XbnI. This plasmid carries the HA-tagged OCHl gene under control of the TPI promoter.

Cloning and sequencing the HOCl gene: Strain 371-5C-4C:- 1D was transformed by the lithium acetate method ( I T 0 rt nl. 1983) with a genomic library in the CKh' AIL? IBU2 vector p366 (from PHIL HIETRK). Plates selective for transformants were placed at 23" overnight and then shifted to 37" to select fi)r plasmids suppressing the temperature-sensitive pheno- type. One clone of HOC1 was obtained from -5000 trans- forrnants.

Sequence alignments were performed using the Megalign progr-am (DNASTAR). The sequence of the cosmid carrying HOC1 was obtained as follows: total cosmid DNA was nebu- lized and treated with T 4 DNA poIymerase and Klenow, the blunt end fragments werc then cloned into the l h R V site o f M13mp90, and sequencing was performed using an AB1373 sequencer and dye primers. The sequence of HOC1 is avail- able in GenBank, accession number Z49575.

Integration and disruption of the HOCl gene: One-step gene disruption using plasmid pVM4 and the haploid strain RSY255 was performed as described by ROTHSTEIN (1983) t o give strain VMW. Disruption of the genomic copy of HOf:l was confirmed by PCR analysis o f total yeast DNA using the primers used for PCR amplification of HO(:l. That disruption of I . f O c 1 directly in a haploid had not resulted in the isolation of suppressor mutations was confirmed by back-cross Of V" to a wild-type strain (data not showlj).

Detection of c-myc epitope-tagged Hoclp and invertase in protein extracts: Protein extracts of yYdS1 strains were pre- pared as described (POSTER and DEAN 1996). Protein samples werc boiled for 5 min before fractionation on arl 8% SDS polyacrylamide gel and transferred to nitrocellulose. The Hoc1 p protein was visualized by incubating nitrocelhilose blots with 1/10 diluted mouse monoclonal antibody 9E10 cu1tqn-t. sq>ernata?r (EVAN d d . 1985), f o l l 0 ~ ~ ~ 1 b y 1/3000

25" 37"

RESULTS

Isolation of HOCl a a dosage suppressor of a pkcl-371 temperature-sensitive mutant: A scrccn for mutants w i t h abnormal nuclcar mcmbrane morpholop was con- ducted by staining a collection o f temperature-sensitive strains (HARTWEI.I. 1967) with the lipophilic dye DiOC,; (KONING e/ (11. 1993; A. M. NI.:IIMN and R. $I-ERNGI;\S%,

unpublished data). Sequence of a complementing clone for onc of the mutants obtained in this scrccn showed the rescuing activity t o be the I X C I gene. Com- plementation tests of this mutant with previously identi- fied alleles of I'KCI demonstrated that the mutant is a new temperature-sensitive allele o f PKCI, designated pkrl-371 (tlata not shown).

I n the process of cloning I'KCI, a second lihrary plas- mid wits found that did not contain the PKCI gene but that rescued the temperattu-e-sensitive growth defect of a strain carning the /h-l-371 mutation (Figure 1). Subcloning of the genomic insert localized the sup- pressing activity to a 2..5-kb RnmHI-Hind111 fragment (Figure 2). A partial seqnence of the insert indicated that the genomic fragment came from the right arm of chromosome ?(. Analysis of the complete sequence of this region of chromosome X rcvealcd that the BnmHI- Hind111 fragmcnt contains the 3' end of the CDCII gene and a complete open reading frame that wc dcsig-

natecl HOCI. A clone containing the complete CDCI I gene but not HOCl did not rescue the temperature- sensitivity of {h l -371 (Figure 2).

The lethality of pkrl mutations has previously been shown to be caused by cell lysis due to defccts i n the cell wall (LEVIN and BARTIXIT-HUERUSCH 1992; PAM- \'I(:IXI r / crl. 1992). Two observations suggest that the temperature sensitivity of pkrl-371 is likely to result from a similar defect. First, jhl-371 mutants display abnormally thin cell walls when observed in the elec- tron microscope and second, the inclusion of 1 M sorbi- tol in the medium will allow growth of pkcl-371 strains at high temperature (data not shown). The rescue of /)ltrl-371 bv HOC1 therefore suggests a role for HOC1 either directly in cell wall metabolism or in the signal transduction pathway downstream of PKCI.

HOCl encodes a protein similar to the Ochlp glycosyl- transferase: The nucleotide and predicted amino acid sequence of HOCl is shown in Figure SA. HOCI is pre- dicted to encode a protein of 396 amino acids with a calculated molecular weight of 46.2 kDa. A search of the Genbank database using the Blastp algorithm (AI.TSCFIUI. e/ 01. 1990) revealed significant homology between Hoclp and the a-1 ,&mannosvltransferase en- coded by the S. rprplljsim OCHI gene. Over the entire length of the protein, Hoclp displavs 33% identity and 58% similarity to Ochlp (Fi,pre 4A), with the strongest homology Iving between residues 196 and 329 of Hoclp (50% identity and 71% similarity). In addition, Hoclp and Ochlp share more limited homology with the protl- ucts of the SUR1 gene, the S. cPrmi.Ti(ly chromosome I1 open reading frame YRR16lw and the . ~ r l ~ i z n . ~ ~ r r l ~ ~ ~ r n m ~ r r . s p n m h chromosome I open reading frame SPACl'iG8.11 (Figure 4B). SUR1 has been identified both by a mrm- tion that suppresses the starvation-sensitive phenotype o f an rral6l mutant (DESFARGES e/ rrl. 1993) and as a high copy suppressor of the Ca'+-sensitive mutant cls2 (T:\KI~~,\ e/ nl. 1995). The function of SURI, however, is unclear. No hmction has been reported for YRR16lw or for the S. ~ ~ n r n f w open reading frame.

Ochlp is predicted to be a type I1 integral membrane protein with a short cytoplasmic amino-terminal (lo- main and a lumenal carboxv-terminal catalytic domain (NAKAYAAIA et (11. 1992). This organization is characteris- tic of a variety of mammalian and yeast glvcosyltransfer- ases (PhUI.soN and COLLEY 1989). A hydropathv analy- sis predict5 that Hoclp possesses a single potential transmembrane spanning region near the amino-termi- n u s of the protein (Figure 3B) (KYTE and Doo1.1n-1.E 1982), suggesting that i t too may be a type I1 membrane protein. The sequence homoIo<gy and organizational similarity between Hoclp (Homologous to QCHI) and Och 1 p suggests that HOCl might also encode a glycosyl- transferase.

A synthetic interaction between hoc1 and pkcl- 372: To assess the function of HOCI in viun, a mutant allele was constructed bv the insertion of the LEU2gene into the HOCl coding region. A strain carrying the

640 A. M. Neiman r/ nl.

Rescue of

temperature pkcl-371

- - .. sensitivity plasmid - - - - - S

I I S m m "

SI S

4 I I I I " CDCl1 HOCl

+ p371A

0 - p m 1

--1_____=I7 - p371ABamHlA

I I - p371AHlllA - + pRS425HOC1

FI(;CRI:. 2.-Sucloning of HOCl. The top line shows the extent of the genomic insert in the original HOCl clone. indicate the position of the C,'DCll and HOCl genes and arrows indicate the direction of transcription. The vertical lines above the insert indicate the position of restriction sitrs. RHI, BnmHI; HIII, HindIII. The names of the plasmids used are given at right.

hocl ::LEU2 allele, V M Y Z , was constructed as described in MATERIAI.S AND METHODS. horl mutants are viable, morphologically normal, and display no obvious growth defects. Unlike ochl mutants, which are temperature sensitive

A .

1

21

41

61

81

101

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161

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B.

for growth, horl mutants grew well at both

B L I S L A E E Y B X L E H N S N R T D ~ A C 3 A M A A 7 T T E C A G U ~ A A W A G A ~ ~ A T T A A T ~

A A T A T T C A G A G T I O X A ~ ~ A G I T C A A C A A C A T I T I L Q K I L Q N L P K E I S Q S I N S A N

E N I Q S S V S D L V Q H F E S L A Q E I N 3 C A P T l T 3 A T . U A C A A C G T A A G A -

AI\RArrCARGACI.FGARAUVV\CACCFCCOGAOGCCArrC R H Q Q E V Q A K Q F V K Q R K I L E K

~ A C C A T F X C k T G T C A G T W C C ~ T I T A ~ T K I Q D L K Q T P P E A T L R E R I A H

GAP T F P Y D S H V K F P A F I W Q T W S N

TATAAAXWAXIWZUAKAAGAAXWXK D E G P E R V Q D I K G M W E S K N P G ~ A ~ C C A ~ ~ T ~ C T A G T A C A C C A C T A ~ A C F A H E V L N H D V I N A L V H H Y F Y ~ T ~ T X T ~ " W A T X T M J G A T A G 4 T S I P E I L E T Y E A L P S I I L K I D ~ T A C I T A A T A C I G I T ~ ~ A T Q T C A C A ~ ~ F F K Y L I L L V H G G V Y A V I D T F """ma-

A T P P V Q P I P N W I P E E L S P S D I C L

I V G V E E D A Q R A D W R T K Y I R R CAAGTATATCXkGA

-"ITATA"-E L Q F G T W I I Q A K P G H P V L R E I A T I X T X A A T F A T E ~ A C A P C T ~ T I S R I I E T T L Q R K R D D Q L N V N C T A A G G A A T G A ' I T E A 4 T A T T A ~ ~ C 4 C T A ' T T L R N D L N I H S W T G S G L W T D T T T l T A C G T A " T T C A A l C X l T S A P ACAlWAAhTTA F T Y F N D F M R S G V R E K V T W K L ~ A T A A C C T ~ T C P A ~ ~ A A C T G A ~ ~ F H N L N Q P K L L S D V L V F P K F S T I T A A C P G F C C C 4 C X P X A k 4 T X T A ' l T T C A T F A C T F N C P N Q I D N D D P H K K F Y F I T C A T I T Z C C A ' K A C L 4 ~ T A P H L A S Q F W K N T P K V E Q K .

TAA

~ ~~- " "

200 3w

100 200 300 amino acid

FIGURE 3.-Sequence of the HOCl gene. (A) Nucleotide and predicted amino acid sequence of HOCl. The potential transmembrane domain is underlined. (R) Kvte-Doolittle hv- dropathv plot of the Hoclp amino acid sequence.

high (37") and low (23") temperatures. Thus, HOCI appears not to be required for growth under normal culture conditions.

Increased dosage of HOC1 can rescue the tempera- ture-sensitive phenotype of strains harboring pkcl-371 (Figure 1). We therefore wished to examine if loss of HOCl might exacerbate the defect of a plzcl-371 strain. A diploid heterozygous for both hoc1 ::IXU2 and pkc l - 371 was constructed by mating strain VMY2 to 371-5G 4GlD. Upon sporulation, this diploid gave rise to four viable progeny at 23" indicating that the hocl ::LEU2 pkcl-371 double mutants are viable. However, when we tested growth at a range of temperatures between 23" and 3 7 , we found that the restrictive temperature of the hocl pizcl-371 double mutant (identified as tempera-

HOClp Ochlp sur:p Ybr:Blp spaCi'9P.l!p

FIGURE 4.-Sequence alignments. (A) Alignment of the Hoclp and Ochlp sequences. Identical residues are shaded. (R) Alignment of the homology domain found in Hoclp, Ochlp, Surlp,YbrlGlp, and Spacl7g8.11~. Consensus is four out of five identical.

ture-sensitive, Leu+ progeny) had decreased relative to the parental @cl-371 strain from 37" to 34" (Figure 5 ) .

hocl mutants are sensitive to CaIcofluor White and hygromycin B: The genetic interactions with f)lrrl-371 suggest a role for H O C 1 i n cell wall synthesis. To test this possibility, we asked whether a hoc1 mutant strain was sensitive to Calcofluor White (CFW). CF" is a fluo- rescent dye that binds to chitin and disrupts cell wall assembly. Mutants defcctive in various aspects of cell wall synthesis have been isolated on the basis of sensitiv- i ty to CF" (Rw Pt nl. 1994). As shown in Figure 6, inclusion of CF" in the medium completely inhibited the growth of a llorl strain, though the isogenic wild- type strain was unaffected. Introduction of the wild-type HOCI gene on a plasmid rescued the sensitivity of the mutant. This result provides a second indirect piece of evidence that HOCl is important for proper synthesis o f thc cell w a l l .

Given the homology of Hoclp to Ochlp, it seemed likely that Hoclp might function in cell wall synthesis as a glycos!lltransferase. Mutants defective in most

classes of Nlinked glycosylation are sensitive to hygro- mycin B (BAI.ILW pt nl. 1991; Dl;.xs 199.5). We therefore examined the effect o f this drug on a hocl mutant strain. At a concentratioc of 50 pg/ml, hygromycin B com- pletely inhibited the growth of a hoc1 strain, though the isogenic wild-type strain was unaffected (Figure 6 ) . As a wide variety ofglycosylation-defective mutants are sen- sitive to this concentration of hygromycin R (DE.\N 1995), this result does not indicate a specific function for Hoclp in glycosylation. It does suggest, however, that hocl mutants are defective in some aspect of pro- tein glycosylation.

hocl mutants display no detectable defect in protein glycosylation: To explore more directlv the role of NOCI in protein glvcosylation, we examined a variety of glycosylated proteins in wild-type and hocl mutant cells. Invertase, a secreted protein, is subject to exten- sive ,Winked glycosylation during transit through the secretory pathway (REDDY pt nl. 1988). Immunoblot analysis of a r-my-tagged version of invertase revealed no reproducible change in the mobility of invertase from hoc1 cells (Figure 7, lane 2) relative to that from HOCl cells (Figure 7, lane 1). Using a similar approach we were unable to detect any alteration in the N-linked glycosylation of the vacuolar protease carboxypeptidase Y (data not shown). We have also examined Oglvcosyla- tion by studying the mobility of secreted chitinase, a protein exclusively decorated with Olinked sugars (KURANDA and ROBRINS 1991). Again, disruption of HOCl did not detectably alter the mobility of the pro- tein (data not shown). To date, we have been able to find no gross changes in protein glycosylation in the horl mutant.

Overexpression of HOCl cannot rescue an ochl mu- tant: One possible explanation for the modest pheno- type of the hocl mutant is redundancy between Hoclp and the related Ochlp. We attempted to assess this possibility by comparing the phenotype of an ochl hocl double mutant strain to isogenic ochl and hoc1 single mutants. However, in the two strain backgrounds we examined (A364A and RSY255) the ochl single mutant was either dead or extremely slow growing, making analysis of the double mutant problematic. Therefore, a s an alternative way to assess redundancy of OCHI and HOCI, we tested the ability of overexpression of HOCl to rescue the growth defects in the original ochl mutant strain (NAKAYAMA et nl. 1992). Strains deleted for OCHI are blocked in extension of Nlinked oligosaccharide chains beyond the core glycosylation added to the pro- tein in the ER. OCHI is not essential in this background, but deletion strains are temperature-sensitive and sensi- tive to hygromycin B. Introduction of HOCZ on a 2y plasmid had no discernible effect on growth of the orill mutant at high temperature or on plates containing hygromycin B (data not shown).

As a further test for overlap in HOCI and OCHI func- tion, we tried the reciprocal experiment of overex- pressing OCHI in a hocl mutant. The hygromvcin B

642 A . M. Neiman c/ nl.

Y PAD YPAD + hygromycin B YPAD + Calcofluor White

sensitivity of strain VMY2 (hocl::IEU2) was not s u p pressed by introduction of a plasmid that expresses OCHI from the TPI promoter (Figure 8). Thus, overex- pression of OCHI cannot suppress hoc1 and vice versa. These results suggest that Hoclp function is distinct from that of Och 1 p.

Hoclp is localized in the Golgi: To localize the Hocl protein, we constructed a fusion of a sequence encod- ing the c-fnyr epitope (E\:\N p t nl. 1985) to the 3' end of the HOCl gene. This HOCI-my- fusion was expressed constitutively from the TIY promoter and could be de- tected by immunohlotting in total cell protein extracts (Figure 8E). Expression of this fusion protein in the Irorl::1.1.X12strain VMY2 rescued its hvgromycin Rsensi- tive phenotype, indicating that the Hocl-myc fusion protein was functional in vi710 (data not shown).

M'e next performed indirect immunofluorescence ex- periments using the Hocl-mvc fusion protein. M'e also

200 -

1 invertase

97 - 66 -

46 -

FIGURE 7.--Glycosylation of invertase in hoc1 mutants. Pro- tein extracts of isogenic strains RSY25.5 (I-IOCI) and V"2 (hoc1 ::IJW2) carrying a plasmid expressing c-ny-tagged in- vertase were Western blotted and probed with anti-myc anti- bodies. Positions of molecular size standards are indicated on the right.

stained cells with antibodies to Pdilp (LA\I;\STIA and LESSAKZ 1993) and a c-rny-tagged Mntlp (DEAS and POSTER 1996) as standards for ER and Golgi localiza- tion, respectively. Cells expressing HOCI-myc showed a punctate staining pattern (Figure 8R) with an average of three to seven bright dots per cell. These bright dots are not visible in the same strain without the HOCI- my- fusion (Figure SA). The Hocl-my firsion protein staining pattern is distinct from the ER staining of Pdil p (Figure XD) and ven similar to the pattern of'the r-?ny- tagged Mntlp control (Figure 8C) and other Golgi- localized proteins (FRASZLXOFF PI nl. 1991; COOPER and BUSSEY 1992). Additionally, Hocl p cofractionated with a different Golgi marker, Mnnlp (LCSSIEK PI 01. 199.5) in differential centriftlgation experiments (Figure 10). Hoclp fractionation was distinct from that of the ER marker Karlp. M'e conclude that Hoclp is probably localized to the Golgi apparatus in yeast.

DlSCL'SSlON

M'e report here the isolation of HOC1 as a dosage suppressor of a temperature-sensititve pltcl mutant. The

YPAD + hygromycin B

FIG~.KI;. X.-O\c.t.c.sl~r('ssioll 01. O ( : / l / tloc*s n o t suppress the hygromycin R sensitivity of a lrorl mutant. Stlain \'MY2 was transformed with pWp3.52 :: HOCl (pHOCI ), pTiOCH I-HA (pTiOCHI), or pRS316 (vector) as indicated and streaked out on a WAD plate containing 50 pg/mI hygromycin R.

643

sequence of HOCI predicts a protein with significant similarity to the a-1 ,~mannosvltransferase, Ochlp. Both the homology to Ochlp and thc predicted topol-

Hoclp +

r Karlp +

FI(;I:RE IO.-Hoclp cofractionatcs w i t h t l w (hlgi protein Mnn 1 p. Extracts of strain SEW210 expressing I-IA-tagged Mnnlp (LAXII:.R rt nl. 1995) and c-Pqc-tagged Hoclp were suh jccted t o differential centriflqation. Samples from the 1000 x .g (PI) 13,000 X .g (PIS) and 100,000 X .g (PIOO) pellets as well as the 100,000 X .g supernatant (S100) were analyzed on a Western blot using anti-HA, anti-r-ny, or anti-HDEL (HARD- WKK rt rrl. 1990) antibodies. b r l p is recognized by the anti- HDEL, antibodies and serves a s a markcr [or the ER.

1

FIGL-RI:. 9.-Hoclp l o - calizes to a Golgi-like compartment. Strain SEY(Zl0 carrying no plasmid ( X and D), c- I n w a g g e d HOCl (R) , o r c-ny-taggrtl M\Tl (C) was stained for indirect immrlnoflrlorescence with anti-myc antihotlies (A-C) or anti-PDI anti- bodies (D) . (E) Protein extracts of strain SEY6210 caTing a plasmid ex- pressing either c-my- tagged HOCl or c-rny- tagged MAT1 were M'est- ern blotted and probed with anti-ny- antibodies. Positions of molecular size standards are indi- cated on the right.

ogy of Hoclp are suggestive that the Hoclp might itself be a glycosyltransferase.

Apart from the synthetic interaction with pkcl-371, the hoc1 mutant displays no obvious cell growth pheno- types. It is, however, sensitive to the inclusion of CFM' or hygromycin R in the growth medium (Figure 6 ) . Mutants defective in cell wall synthesis are sensitive to CF" (RAM PI 01. 1994) while mutants defective in N linked glycosylation are commonly sensitive to amino- glycosides such as hygromvcin R (DEW 1995).

Rased on these observations, an economical hypothe- sis is that HOCZ is a glycosyltransferase required for proper glycosylation of cell wall proteins. To test this possibility we examined two Nglycosylated (invertase and CPY) proteins and one Oglycosylated (chitinase) protein in the horl mutant. None of these proteins show any glycosylation defect in the hoc1 mutant (Figure 7; data not shown). Although we cannot exclude the possi- bility that HOCI has a severe effect on glycosylation of a specific protein or set of proteins that we have not assayed, our current data suggest at best a minor role for Hoclp in Nlinked or Olinked glycosylation.

Hoclp has a distinct role from Ochlp: The OCHI and HOCI gene products are similar in primary se- quence, and both Hoclp and Ochlp are localized to the Golgi. These results raise the possibilty that Hoclp and Ochlp have overlapping functions. Certainly there are numerous examples in yeast of genes with com- pletely or partially redundant function (see e . 6 , TI-~OMAS 1993). To assess this possibility we overex-

644 A. M. Neiman et al.

pressed each gene in a strain mutant for the other. Neither overexpression of HOCl in an ochl mutant nor overexpression of OCHl in a hocl mutant (Figure 8) suppressed the mutant phenotypes. These results and the disparate phenotypes of ochl and hocl mutants sug- gest that Ochlp and Hoclp play distinct roles in the cell.

It is perhaps not surprising to find related glycosyl- transferases with distinct functions. S. cerevisae, for ex- ample, contains a family of at least six putative glycosyl- transferases related to the a-l,2-mannosyltransferase encoded by KRE2/MNTl (LUSSIER et al. 1993; A. M. NEIMAN, unpublished data). Mutation of KRE2 results in a defect in Olinked glycosylation. Specifically, O linked oligosaccharide chains are truncated after the second mannose residue. Mutation of one of these KRE2-related genes, KTR2, results in partial resistance to killer toxin, suggestive of a perturbation in cell wall structure (LUSSIER et al. 1993). However, reminiscent of our results with HOCl , ktr2 mutants display no detect- able alteration in protein glycosylation.

If HOCl has a distinct role from OCHl in protein glycosylation, why is the mutant phenotype so modest? A sequence search of S. cmevisiae protein sequences available in the database using known or suspected gly- cosyltransferases as probes (e.g., Mnnlp, MnnlOp, Kre2p) reveals at least 20 putative glycosyltransferases falling into at least five distinct sequence families (A. M. NEIMAN, unpublished data). This large number of potential glycosyltransferases suggests that there may be extensive redundancy among enzymes involved in glycosylation. Alternatively, it may be that certain glyco- syltransferases are specific for particular proteins. In either event, and the two possibilities are not mutually exclusive, it may be difficult to detect gross abnormali- ties of protein glycosylation in single mutants.

HOCl interactions with PKCl: HOCl displays two types of genetic interactions with pkcl-371. First, overex- pression of HOCl, even from a CEN plasmid, allows growth of pkcl-371 strains at high temperature (Figure 1). Second, mutation of HOCl in a pkcl-371 mutant background lowers the restrictive temperature from 37" to 34" (Figure 5). Thus, the phenotype of a pkcl-371 mutant can be suppressed or enhanced depending on the dosage of HOCl. An effect on pkcl mutants similar to that which we

describe here for HOCl has previously been described for the KRE6 gene (ROEMER et al. 1994). KRE6 encodes a Golgi-localized protein required for the synthesis of P- 1,Gglucan. Overexpression of KRE6, even from a CEN plasmid, can rescue the lysis defect of a p k c l a mutant, whereas kre6pkcl mutants are dead even on osmotically stabilized media. In the same study, a variety of other KRE genes were tested for suppression and synthetic lethality with pkcl. While pkcl showed synthetic lethality with a number of kre mutants, only KREGwhen overex- pressed could rescue a pkcl mutant.

Though overexpression of KRE6 rescues a p k c l a mu-

tant, it does not rescue the lethality of mutations in kinases downstream of PKCl, such as MPKl (ROEMER et al. 1994). This observation suggests that overex- pression of KRE6 is suppressing a requirement for a function of PKCl that is distinct from regulation of the MAP kinase cascade. We have found that overex- pression of HOCl will not rescue the temperature-sensi- tive growth phenotype of bckl or mpkl mutants (N. DEAN, unpublished data), further strengthening the parallel between KRE6 and HOCl .

What is the function of HOClp?: Though there is some reduction in the mannan content, the weakened cell wall in pkcl mutants appears to result primarily from defects in the inner, P-glucan layer of the cell wall (LEVIN and BARTLETFHUEBUSCH 1992; PARAVICINI et al. 1992; ROEMER et al. 1994). This may be due in part to the upregulation of a P-glucanase encoded by BGL2 in pkcl mutants (SHIMIZU et al. 1994). A primary defect in the P-glucan layer is also consistent with the genetic interac- tions seen between pkcl and the kre genes (ROEMER et al. 1994) that are involved in P-1,6-glucan synthesis.

By contrast, the homology of Hoclp to Ochlp sug- gests that Hoclp is a mannosyltransferase. We expect, therefore, that hocl should influence the structure of the mannan layer of the cell wall by altering Nlinked or Olinked glycosylation. If so, then how might overex- pression of HOCl suppress a cell lysis phenotype caused by defects in the 0-glucan layer of the cell wall? We suggest two possibilities. First, Hoclp might be required for the proper modification (and therefore function) of some protein involved in P-glucan synthesis. Consistent with this possibility, a number of the proteins required for P-glucan assembly, including KreGp, are known to be glycoproteins (BROWN and BUSSEY 1993; ROEMER et al. 1994).

A second possibility is suggested by the description (MONTIJN et al. 1994) of a third class of oligosaccharide chains in some S. cerevisiae cell wall proteins. These side chains, which are distinct from Nlinked and Olinked sugars, have a mixed composition containing both P- 1,Glinked glucose residues and a-1,Glinked mannose residues. The proteins attached to these oligosaccha- rides can be released from the cell wall by digestion with the p-l,3-glucanase laminarase (MONTIJN et nl. 1994) suggesting that they may reside in the P-glucan layer of the cell wall. We speculate that Hoclp might be in- volved in the addition of a-1,Gmannose to this class of oligosaccharide. It has also been suggested (ROEMER et al. 1994) that Kre6p might be required for incorpora- tion of P-l,Gglucose into these glycans. If both HOC1 and K R E G were required for production of this type of side chain, this could explain the similar interactions that these genes display with pkcl.

The authors thank J. KONOPKA for plasmids and N. HOI.I.IN(:S WORTH, B. HAI TIWANGER, and R. STERNCIANZ for comments on the manuscript. A.M.N. thanks R. STERNCIANZ for encouragement and material support during the course of this project. This work was funded by National Institutes of Health (NIH) grant GM-48467 to

HOCI, a Suppressor of p k c l 645

N.D., NIH grant GM-28220 to R. S T E R N G I ~ Z , a n d a Council for Tobacco Research grant to N.D. A.M.N. was supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation, DRG-1276.

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Communicating editor: F. WINSTON