©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein O-Glycosylation in Yeast
THE PMT2 GENE SPECIFIES A SECOND PROTEIN O-MANNOSYLTRANSFERASE THAT FUNCTIONS IN ADDITION TO THE PMT1-ENCODED ACTIVITY (*)

(Received for publication, October 20, 1994; and in revised form, November 30, 1994)

Marc Lussier (1)(§) Martina Gentzsch (2) Anne-Marie Sdicu (1) Howard Bussey (1)(¶) Widmar Tanner (2)

From the  (1)Department of Biology, McGill University, Montréal, Québec H3A 1B1, Canada and (2)Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The PMT2 gene from Saccharomyces cerevisiae was identified as FUN25, a transcribed open reading frame on the left arm of chromosome I (Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D. B., and Bussey, H.(1993) Genome 36, 32-42). The product encoded by the PMT2 gene shows significant similarity with the dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase, Pmt1p (EC 2.4.1.109), which is required for initiating the assembly of O-linked oligosaccharides in S. cerevisiae (Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8164-8168). The PMT2 gene encodes a new protein O-D-mannosyltransferase. Yeast cells carrying a PMT2 disruption show a diminished in vitro and in vivo O-mannosylation activity and resemble mutants with a nonfunctional PMT1 gene. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect but retain residual O-mannosylation activity indicating the presence of at least one more protein-O-mannosyltransferase.


INTRODUCTION

Glycosylation of secretory and membrane proteins is the most complex post-translational modification known to occur in eukaryotes. The biosynthetic pathway leading to N-glycosylation has been studied in considerable detail, and the assembly and initial processing of N-linked oligosaccharides in the endoplasmic reticulum are similar from yeast to man, but ensuing phases of glycosylation have been shown to be different in a broad range of organisms (Tanner and Lehle, 1987; Herscovics and Orlean, 1993). In yeast, the N-linked carbohydrate chain is processed by the addition of mannoses from GDPbulletMan to yield either a simple type with 8-14 mannose residues or a more extended class containing up to 200 mannose residues.

Less is known about the structure and biosynthesis of O-linked carbohydrate chains attached to serine and threonine; however, the process of protein O-glycosylation appears to have been less conserved in evolution. The first step in the modification of mammalian O-linked proteins involves the attachment of a GalNAc that has been transferred from UDPbulletGalNAc within the Golgi (Roth, 1984). The carbohydrate chains of mammalian O-linked modified proteins are variable in both length and composition (Elhammer and Kornfeld, 1984; Roussel et al., 1988; Jentoft, 1990; Krijnse Locker et al., 1992). In yeast, it has been demonstrated that O-modified proteins possess a linear carbohydrate chain of up to 5 mannose residues (Tanner and Lehle, 1987; Herscovics and Orlean, 1993). O-glycosylation is initiated in the endoplasmic reticulum, and Dol-P-Man (^1)is the immediate sugar donor for the mannosyl residue transferred to the hydroxy amino acids serine and threonine (Tanner and Lehle, 1987; Strahl-Bolsinger and Tanner 1991). Two genes involved in the elongation of the five mannose residue chain have been isolated. The KRE2/MNT1 gene encodes an alpha-1,2-mannosyltransferase required for the addition of the third mannose residue (Häusler and Robbins, 1992; Häusler et al., 1992; Hill et al., 1992), and the MNN1 gene encodes the terminal alpha-1,3-mannosyltransferase of O-linked chains (Ballou, 1990; Yip et al., 1994). To better functionally define the initial aspects of protein O-glycosylation, the enzyme Dol-P-Man:protein O-D-mannosyltransferase has been purified from Saccharomyces cerevisiae, and the corresponding PMT1 gene has been cloned (Strahl-Bolsinger et al., 1993). Disruption of the gene was not lethal; however, the mutated cells were able to O-glycosylate protein in vivo, although at a reduced rate (Strahl-Bolsinger et al., 1993). The residual enzyme activity remaining in the disruptant has been characterized and shown to differ in a number of properties from the Pmt1 protein (Gentzsch et al., 1994).

During the directed sequencing of chromosome I of S. cerevisiae, a transcribed open reading frame (YAL023/FUN25) was identified (Ouellette et al., 1993) that showed significant similarity with Pmt1p (Strahl-Bolsinger et al., 1993). It is demonstrated here that the FUN25 gene codes for a second Dol-P-Man:protein O-D-mannosyltransferase activity that we now call PMT2. Strains bearing a pmt1 pmt2 double disruption show a severe growth defect, but the double mutant retains residual activity in protein O-glycosylation indicating that S. cerevisiae possesses additional gene products involved in initiating mannosylation of serine and threonine residues.


EXPERIMENTAL PROCEDURES

Yeast Strains, Culture Conditions, and Methods

All yeast manipulations were made in strains SEY6210 (MATa, leu2-3, ura3-52, his3-Delta200, lys2-801, trp1-Delta901, suc2-Delta9) and SEY2101 (MATa, leu2-3-112, ura3-52, ade2-1, suc2-Delta9). Yeast cells were grown under standard conditions, (yeast extract peptone dextrose medium or yeast nitrogen base, buffered with Halvorson medium, when required) as described previously (Boone et al., 1990). Strains were transformed using the LiCl procedure of Ito et al.(1983), using 100 µg of sheared denatured carrier DNA (Schiestl and Geitz, 1989). Transformants were selected on synthetic minimal medium with auxotrophic supplements. Levels of sensitivity to K1 killer toxin was evaluated in SEY6210 by a seeded plate assay using a modified medium consisting of 0.67% YNB, 0.0025% required amino acids, 1.0% Bacto-agar, 0.001% methylene blue, 2% glucose and buffered to pH 4.7 with Halvorson minimal medium (Lussier et al., 1993; Brown et al., 1994).

Computer Analysis

DNA and protein sequence analyses were performed using the GeneWorks (Intelligenetics, Mountain View, CA) and Gene Jockey (Biosoft, Cambridge, UK) software packages. DNA sequence and protein homology searches were conducted on the NCBI mail server using the BLAST program (Altschul et al., 1990).

Gene Disruptions

An deletional disruption of the PMT2 locus was made by the single-step gene replacement procedure (Rothstein, 1991). The 2.94-kb HincII genomic fragment containing PMT2 was subcloned in the EcoRV site of PBSK vector and was subsequently digested by StyI and BglI restriction endonucleases, both of which have sites in the PMT2 coding sequence. The StyI site is located 60 base pairs upstream from the ATG, and the BglI site is found 172 base pairs upstream from the stop codon. This digestion removed a 2.16-kb fragment encompassing 702 amino acids of the PMT2 sequence. A 1.6-kb HpaI-AccI DNA fragment containing the complete LEU2 gene was ligated into the StyI and BglI sites of PMT2. A linear 2.4-kb ApaI-SpeI fragment containing the complete LEU2 gene, as well as the coding plus flanking sequences from the PMT2 gene, was excised and used to disrupt the PMT2 locus into the isogenic diploid SEY6210 cells. Leu transformants were sporulated, and tetrads were analyzed. The construction of the PMT1 gene disruption is described elsewhere (Strahl-Bolsinger et al., 1993). All gene disruptions were confirmed by Southern analysis (data not shown).

Chromosomal Localization and Physical Mapping of PMT1

The PMT1 gene was localized to chromosome IV using a 700-base pair HindIII PMT1-specific random-primed probe (Pharmacia, Montréal, Québec) against a gel wafer of separated S. cerevisiae chromosomes (Clontech, Palo Alto, CA). Physical mapping of PMT1 was performed using the PMT1 probe described above against a set of grids containing a phage library of yeast genomic inserts (Riles et al., 1993).

Preparation of Membranes

Yeast cells were harvested in mid logarithmic growth phase, washed with 50 mM Tris-HCl, pH 7.5, 50 mM MgCl(2) (TM buffer) and broken with glass beads in the same buffer. After centrifugation at 3,000 times g for 1 min, membranes were collected from the supernatant by centrifugation at 48,000 times g for 30 min and resuspended in TM buffer. Protein concentration was determined by the method of Bradford(1976) using bovine serum albumin as a standard.

Assay of Enzyme Activity in Vitro

The peptide assay I for measuring Pmt1p dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase activity was performed as described by Strahl-Bolsinger and Tanner(1991) and contained 0.02 µCi of Dol-P-[^14C]Man (specific activity, 303 Ci/mol; enzymatically synthesized following the method of Sharma et al., 1974), 7 mM Tris-HCl, pH 7.5, 7 mM MgCl(2), 0.14% Triton X-100, 3 mM acetyl-YNPTSV-NH(2), and 25 µg of membrane protein in a total volume of 70 µl. Assay II detecting residual mannosyltransferase activity in a pmt1 null mutant contained 0.04 µCi of Dol-P-[^14C]Man, 10 mM KH(2)PO(4)bulletNa(2)HPO(4), pH 6.5, 7 mM MgCl(2), 0.14% Triton X-100, 3.5 mM acetyl-YATAV-NH(2), and 200 µg of membrane protein in a total volume of 140 µl. The in vitro enzyme reactions were stopped by the addition of 2 ml of chloroform/methanol (3:2) followed by 0.5 ml of H(2)O as described by Strahl-Bolsinger and Tanner(1991). After centrifugation, a 0.5-ml aliquot of the aqueous phase was counted in 5 ml of scintillation mixture in a Beckman scintillation counter. The radioactivity was shown by high performance liquid chromatography to be peptide-bound. Control assays were conducted by omission of the blocked acceptor peptide. The radioactvity measured in such controls amounted to less than 2% of that of complete wild-type assays. The blocked peptides were kindly provided by Dr. M. Marriott, Glaxo, UK.

Yeast Cell Extracts and Immunoblotting

Yeast total cell protein extracts were prepared from cultures exponentially growing in Yeast Nitrogen Base selective media by cell lysis with glass beads in the presence of protease inhibitors. Yeast proteins were separated by SDS-polyacrylamide gel electrophoresis and were then transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Blots were treated in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20, 5% nonfat dried milk powder) and subsequently incubated in TBST buffer with affinity-purified anti-Kre9p or anti-chitinase antibodies. The anti-chitinase antibody was directed against the deglycosylated protein obtained after hydrofluoric acid treatment (Mort and Lamport, 1977). After antibody binding, membranes were washed in TBST buffer, and a second antibody directed against rabbit immunoglobulins and conjugated with alkaline phosphatase was then added. The blots were again washed, and proteins were detected using an enhanced chemiluminescence procedure (Amersham Corp.).

Mannose Labeling and beta-Elimination

Yeast cells were grown overnight in minimal medium containing 2% galactose. This overnight culture was then used to inoculate a YP culture (1% yeast extract, 2% Bacto-peptone) supplemented with 2% galactose. 7.5 A units of exponentially growing cells were harvested, washed, and resuspended in 5 ml of YP, 0.5% sucrose. Subsequently, 0.2 mCi of [^3H]mannose (16.5 Ci/mmol; Amersham Corp.) were added, and the cells were incubated for 2 h at 30 °C. 10 A units of labeled cells were washed twice with 50 mM Tris-HCl, pH 7.5, 50 mM MgCl(2), and broken with glass beads by vortexing. The total radioactivity incorporated was determined from an aliquot of the homogenate. The extract was centrifuged for 30 min at 10,000 times g. The pellet containing cell walls and membranes was beta-eliminated. beta-elimination was carried out in 0.1 N NaOH, 1 N NaBH(4) at 29 °C. After 24 h, the sample was adjusted to pH 4 by the addition of 5 N acetic acid and centrifuged, and and the supernatant was deionized on Dowex 50 W-X8 (Serva, Heidelberg). The eluate was lyophilized, washed twice with 1% acetic acid in methanol, and washed three times with methanol to remove boric acid. Half of the beta-eliminated and reduced material was separated by thin layer chromatography on silica gel G/60 (Merck, Darmstadt) and developed twice in acetone/butanol/water, 7:15:15 (v/v/v). Mannitol, maltitol, and maltotriitol were used as size standards and visualized by 0.5% KMnO(4) (w/v) in 1 N NaOH. Radioactive spots on thin layer plates were detected by a TLC scanner (LB 284 from Berthold, Wildbach, Germany). The radioactive peaks corresponding to Man^1-Man^5 were eluted with water and estimated by liquid scintillation spectrometry using 5 ml of scintillation mixture.


RESULTS

Identification of the PMT2 Gene

DNA sequencing of chromosome I of S. cerevisiae has identified an open reading frame similar to the Pmt1p dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase (EC 2.4.1.109) responsible for the transfer of mannose from Dol-P-Man to serine/threonine residues of O-linked modified yeast proteins. This open reading frame called YAL023 was also identified as the 2.5-kb FUN25 transcript (Ouellette et al., 1993; Barton and Kaback, 1994) and PMT2 based on the studies presented below. PMT2 encodes a 758-amino acid protein (Pmt2p) that shares 31% identity with Pmt1p (Fig. 1). The hydropathy profiles of both proteins are similar (Fig. 2). Like Pmt1p, Pmt2p is predicted to be an integral membrane protein with multiple transmembrane domains. The N and C termini of the proteins are lipophilic, whereas their central parts are hydrophilic. Both Pmt2p and Pmt1p possess 3 putative N-glycosylation sites, although none are conserved between the two proteins ( Fig. 1and Fig. 2).


Figure 1: Sequence comparison of the PMT2- and PMT1-encoded proteins Pmt2p and Pmt1p. Predicted amino acid sequences of the PMT2 and PMT1 genes are represented in the single-letter amino acid code. The names of the proteins are shown on the left, with the amino acid residue numbers on the right. Identities between the two proteins are boxed. Gaps represented by dashes have been introduced to improve alignment. Putative asparagine-linked glycosylation sites found in both proteins are marked by arrowheads. The GenBank accession number containing PMT2 as YAL023 is # L50146.




Figure 2: Hydropathy profiles of Pmt2p and Pmt1p. Hydropathy was compiled according to Kyte and Doolittle(1982) with a window of 16 amino acids. Arrowheads indicate potential N-glycosylation sites. Positive values correspond to hydrophobic regions, whereas negative values correspond to hydrophilic segments.



The PMT2 and PMT1 Genes Are Not Physically Linked

To test if PMT2 and PMT1 were linked and arose from a gene duplication event, the chromosomal localization of PMT1 was determined. PMT2 lies directly centromere proximal to the LTE1 gene on the left arm of chromosome I (Ouellette et al., 1993). The PMT1 gene was localized to chromosome IV using a PMT1-specific probe against separated Saccharomyces cerevisiae chromosomes. Precise physical mapping of PMT1 was achieved using the PMT1 probe against a library of ordered yeast genomic inserts (Riles et al., 1993). PMT1 mapped to overlapping prime clones 6605 and 4114, which contain DNA from the region close to the PHO2 gene in the PHO2 SIR2 interval on the left arm of chromosome IV.

Functional Analysis of PMT2

The possible role of the PMT2 gene product as a protein mannosyltransferase was analyzed. First, a one-step gene replacement of PMT2 was carried out using the LEU2 gene (see ''Experimental Procedures``). Tetrad analysis of spore progeny derived from SEY6210 pmt2::LEU2 heterozygotes showed that PMT2 was not essential for cell viability but was required for normal vegetative cell growth. Haploid pmt2::LEU2 cells grew at a slightly slower rate than the wild-type strain (Fig. 3). To assess whether a strain carrying a pmt1::HIS3 pmt2::LEU2 double disruption possesses a more severe phenotype, cells carrying pmt1 or pmt2 single deletions were crossed, the resulting diploid strains were sporulated, and individual meiotic tetrads were dissected and grown at 30 °C. As can be seen in Fig. 3, a strain harboring a pmt1::HIS3 null mutation grows at a significantly slower rate than a pmt2::LEU2 strain or the wild-type. Double pmt1 pmt2 null strains have a severe growth phenotype and grew more slowly than the single null mutants, indicating a genetic interaction between the two genes.


Figure 3: Growth phenotypes of wild-type, pmt2, pmt1, and pmt1 pmt2 double null mutants. SEY6210 cells carrying pmt1 or pmt2 single deletions were crossed, the resulting heterozygous pmt1::HIS3 pmt2::LEU2 diploid strains were sporulated, meiotic spore tetrads were dissected, and spore progeny were grown at 30 °C. An enlargement of the spore progeny from such a dissected tetratype tetrad is shown displaying all four possible outcomes of the cross.



O-Mannosylation Activity of Pmt2p and Pmt1p

Analysis of the enzymatic activity of Pmt2p and Pmt1p in O-linked carbohydrate chain elaboration was performed using two in vitro assays measuring the transfer of [^14C]mannose residues from Dol-P-[^14C]Man to an acceptor peptide (Table 1). Assay I was developed to detect Pmt1p O-mannosyltransferase activity (Strahl-Bolsinger et al., 1993), and assay II was subsequently devised to detect the activity remaining in a pmt1 null mutant (Gentzsch et al., 1994). Assay II is more permissive than assay I as it allows detection of an expanded range of O-mannosyltransferase activities. As previously found, a pmt1 disruptant has considerably less activity than the wild-type in both assays (Strahl-Bolsinger et al., 1993; Gentzsch et al., 1994). A strain carrying a pmt2::LEU2 null mutation also shows a diminished enzymatic activity in both assays. Double pmt1 pmt2 null strains possess no activity relative to wild-type in assay I and less than 20% of wild-type activity in assay II, and both assays show a cumulative reduction in mannosyltransferase activity in the double null strain over the single mutants.



Overexpression of PMT2 or PMT1 in a Double pmt1 pmt2 Null Strain Restores Normal Elaboration of O-linked Mannoprotein Oligosaccharides

The functional capacity of Pmt2p and Pmt1p was also assessed in vivo by using a killer toxin sensitivity assay (Fig. 4). K1 killer yeast strains secrete a small pore-forming toxin that requires a cell wall receptor for function (Bussey, 1991). PMT1 null mutations lead to a decrease of mannose chains on O-linked modified cell wall mannoproteins disrupting the toxin receptor and leading to partial resistance (Fig. 4; Strahl-Bolsinger et al., 1993). Yeast strains harboring single as well as a double null mutations were assayed for killer toxin sensitivity, and the results are shown in Fig. 4. Compared with the wild-type toxin sensitive SEY6210 strain, pmt1 or pmt2 single disruptions are partially resistant to the killer toxin, pmt1 being more resistant. Yeast cells bearing a double pmt1 pmt2 null disruption are totally toxin resistant and show no killing zone in the assay. The killer phenotype of the double null mutants allowed a test of possible suppression of the loss of both genes by one homologous counterpart. Overexpression of PMT2 in a strain carrying a double pmt1 pmt2 null mutation enhanced the vegetative growth (^2)and completely suppressed the killer resistance phenotype (Fig. 4) of this mutant, indicating that Pmt2p can function in vivo as a protein O-mannosyltransferase. Reciprocally, overexpression of PMT1 in a strain carrying a double null mutation partially restored cellular growth^2 and fully suppressed the killer phenotype (Fig. 4), providing evidence for functional homology between these two proteins.


Figure 4: Killer toxin sensitivity phenotypes of wild-type, pmt2, pmt1, and pmt1 pmt2 double null mutants. Concentrated K1 killer toxin was spotted on a lawn of approximately 1 times 10^6/ml cells from a fresh culture of each strain (see ''Experimental Procedures``). After subsequent incubation, toxin-sensitive cells are killed, and a killing zone was detected in the growth lawn. Toxin-resistant cells grow in the presence of the toxin and show no killing zone.



Pmt2 Mutants Are Defective in O-Glycosylation and Exacerbate the O-Mannosylation Defect of a pmt1 Mutant

The extent of O-glycosylation in yeast strains bearing different pmt mutations was analyzed by measuring the mobility of two yeast O-glycoproteins, chitinase (Kuranda and Robbins, 1991) and Kre9p (Brown and Bussey, 1993). Neither protein receives N-linked modifications. Chitinase possesses a predicted size of 60 kDa. As can be seen in Fig. 5, chitinase produced by single null disruptants migrates more rapidly in SDS-polyacrylamide gel electrophoresis than the fully glycosylated wild-type protein (130 kDa), consistent with reduced O-mannosylation. Chitinase synthesized in a double pmt1 pmt2 null strain migrates at an apparent molecular mass of 100 kDa and is clearly smaller than when produced in either single mutant strain. Kre9p was also found to show a mobility shift consistent with underglycosylation in the different mutant strains. The molecular mass of Kre9p is predicted to be 30 kDa, but when synthesized in a wild-type strain it migrates at an apparent mass of 60 kDa. Kre9p produced in strains containing pmt1 or pmt2 single disruptions migrates with an apparent mass of 50 kDa (Fig. 5). Kre9p synthesized in yeast cells bearing a double pmt1 pmt2 null disruption migrates at an apparent mass of 45 kDa. The fact that chitinase and Kre9p are smallest when produced in a double null mutant indicates a cumulative effect of both mutations on the proportion of O-linked chains received by these proteins.


Figure 5: Immunological detection of chitinase and Kre9p synthesized in wild-type, pmt2, pmt1, and pmt1 pmt2 double null mutants. Yeast total-protein extracts were immunoblotted with affinity-purified anti-chitinase or anti-Kre9p polyclonal antibodies (see ''Experimental Procedures``). The molecular mass standards are shown in kilodaltons.



PMT1 and PMT2 Are Not the Only Genes Involved in Initiating the Assembly of O-Linked Oligosaccharide Structures

Pmt2p and Pmt1p are responsible for the direct elaboration of O-linked carbohydrate chains. However, based on their respective electrophoretic mobilities, chitinase and Kre9p produced in a double null mutant are still apparently glycosylated. To determine whether some of the residual glycosylation is indeed O-linked, the carbohydrate chains present on the glycoproteins of strains lacking functional PMT1 and/or PMT2 genes were analyzed. Total O-linked carbohydrate chains were specifically released from the glycoprotein fraction of in vivo [^3H]mannose-labeled yeast cells by beta-elimination and resolved by thin-layer chromatography (Fig. 6). The wild-type strain showed the normal five oligosaccharide peaks (Man^1-Man^5) as was also the case with the pmt1 and pmt2 single null disruptants. Some O-linked oligosaccharides were still being assembled in a strain carrying a double pmt1 pmt2 null mutation since it showed a pattern of five oligosaccharide peaks. S. cerevisiae hence possesses, in addition to PMT1 and PMT2, other gene products involved in initiating the mannosylation of serine and threonine residues of O-linked modified proteins.


Figure 6: beta-elimination profiles. Thin-layer chromatograms of manno-oligosaccharides released by beta-elimination from bulk yeast glycoproteins of wild-type cells (A) and the same strain where PMT1 (B), PMT2 (C), or both genes were disrupted (D) are shown. Aliquots of extracts corresponding to equal amounts of cells were run on thin-layer plates (see ''Experimental Procedures``). M1-M5 represent carbohydrate chains bearing one to five mannoses.




DISCUSSION

Pmt1p was purified as an enzyme catalyzing the initial reaction of protein O-glycosylation in yeast (Strahl-Bolsinger and Tanner, 1991). In vivo, O-mannosylation in a pmt1 null mutant was 50% of that of a wild-type strain, arguing for the existence of at least one additional transferase (Strahl-Bolsinger et al., 1993). Evidence has been presented showing that Pmt2p is also involved in the O-mannosylation of serine and threonine residues of proteins as a pmt2 null mutation caused phenotypes similar to those seen in strains carrying a PMT1 disruption.

The influence of a PMT2 disruption on protein glycosylation was analyzed by in vitro enzymatic assays. In the two different assays used, an effect of a pmt2 single disruption was clearly seen and was almost as marked as that seen with a pmt1 single disruption. Compared with the single mutants, enzymatic activity was reduced in a mutant strain carrying a double pmt1 pmt2 disruption, consistent with both proteins having similar functions. However, in the double mutant, assay II clearly indicated the presence of residual Dol-P-Man-dependent protein O-mannosyltransferase activity.

A strain carrying a nonfunctional copy of PMT2 is partially K1 killer toxin resistant, suggesting, as with PMT1, that PMT2 null mutations lead to a reduced number of O-linked mannose chains on cell wall mannoproteins perturbing the cell surface toxin receptor and leading to resistance. Apparent functional homology between the two genes is implied by the demonstration that multiple copies of either the PMT2 or PMT1 genes are able to individually suppress the killer resistance phenotype of a double null mutant. Each of the proteins thus has the capacity to substitute in vivo for the absence of the other. Interestingly, overexpression of either gene in the double null mutant partially restored vegetative growth, suggesting a relationship between normal cell division and O-glycosylation.

The O-glycosylated proteins chitinase and Kre9p are both similarly underglycosylated in a pmt1 or pmt2 null mutant compared with a wild-type strain. Both proteins receive even less carbohydrate chains when synthesized in a double null strain but are still modified in some way since their migration patterns do not coincide with those expected for their unglycosylated forms. beta-elimination experiments confirmed that some O-glycosylation still occurs in strains where both PMT1 and PMT2 have been rendered inactive. No qualitative differences were seen in the distribution of counts from the released oligosaccharides (Man^1-Man^5) from either wild-type, single, or double null, whereas the total amount of counts corresponding to individual peaks was reduced in the different mutant strains, consistent with their containing reduced amounts of O-linked oligosaccharides. A direct correlation between the diminished height of the mannose peaks and reduction in number of carbohydrate chains was difficult to demonstrate since the mutant strains incorporate labeled mannose to a lesser extent than wild-type cells. (^3)

Taken together, our results clearly indicate that O-modified proteins synthesized in a pmt1 pmt2 double disruptant are still O-glycosylated but that they receive a reduced number of O-linked mannose chains. The phenotypic severity of the double mutant strongly suggests that the missing carbohydrate chains are required for normal cellular growth. The remaining Man^1-Man^5 chains must be initiated by at least one more dolichyl phosphate-D-mannose:protein O-D-mannosyltransferase that remains to be identified.

PMT1 and PMT2 represent a diverged pair of functionally homologous genes. It appears likely that there is at least one further functional member of this PMT family in S. cerevisiae. The reason for these multiple protein O-mannosyltransferases is unclear. However, the differing specificities of the Pmt1p and Pmt2p proteins in our two in vitro assays suggest that the transferases may initiate glycosylation on different acceptor proteins or on specific serine and threonine residues in a variety of sequence contexts, and may collectively permit a broader range of protein O-mannosylation.

The question of whether O-glycosylation is an essential process still remains and will be resolved only when all other genes coding for the remaining protein O-mannosyltransferase are identified and disrupted. However, the demonstration of growth phenotypes in the single mutants that are cumulative in the pmt1 pmt2 double mutants already indicates that the Pmt proteins and O-glycosylation play important roles in the vegetative growth of yeast.


FOOTNOTES

*
This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Genome Analysis and Technology Program (to H. B.) and from the Deutsche Forschungsgemeinschaft (SFB 43) and by the Fonds der Chemischen Industrie (to W. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L50146[GenBank].

§
Recipient of a postdoctoral fellowship from the Medical Research Council of Canada.

To whom correspondence should be addressed: Dept. of Biology, McGill University, 1205 Dr. Penfield Ave., Montréal, Québec H3A 1B1, Canada. Tel.: 514-398-6439; Fax: 514-398-2595; hbussey{at}monod.biol.mcgill.ca.

(^1)
The abbreviations used are: Dol-P-Man, dolichyl phosphate-D-mannose; kb, kilobase pair(s).

(^2)
M. Lussier, A.-M. Sdicu, and H. Bussey, unpublished observations.

(^3)
M. Gentzsch and W. Tanner, unpublished observations.


ACKNOWLEDGEMENTS

We thank Mark Goebl and Dahn Vo for bringing the sequence similarities of PMT2 and PMT1 to our attention, Jeff Brown for providing the anti-Kre9p Ab, Thomas Immervoll for providing the antibody against the protein moiety of chitinase, the members of the Bussey laboratory for helpful comments and suggestions, and Carole Smith and Guy l'Heureux for photographic work.


REFERENCES

  1. Atlschul, S. F., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ballou, C. E., (1990) Methods Enzymol. 185, 441-472
  3. Barton, A. B., and Kaback, D. B. (1994) J. Bacteriol. 176, 1872-1880 [Abstract]
  4. Boone, C., Sommer, S. S., Hensel, A., and Bussey, H. (1990) J. Cell Biol. 110, 1833-1843 [Abstract]
  5. Bradford, M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  6. Brown, J. L., and Bussey, H. (1993) Mol. Cell. Biol. 13, 6346-6356 [Abstract]
  7. Brown, J. L., Roemer T., Lussier, M., Sdicu, A.-M., and Bussey, H. (1994) in Molecular Genetics of Yeast : A Practical Approach (Johnston, J. R., ed) pp: 217-231, IRL Press, Oxford University Press, Oxford, United Kingdom
  8. Bussey, H. (1991) Mol. Microbiol. 5, 2339-2343 [Medline] [Order article via Infotrieve]
  9. Elhammer, A., and Kornfeld, S. (1984) J. Cell Biol. 98, 327-331
  10. Gentzsch, M., Strahl-Bolsinger, S., and Tanner, W. (1995) Glycobiology , in press
  11. Haselbeck, A., and Tanner, W. (1983). FEBS Lett. 158, 335-338 [CrossRef][Medline] [Order article via Infotrieve]
  12. Häusler, A., and Robbins, P. W. (1992) Glycobiology 2, 77-84 [Abstract]
  13. Häusler, A., Ballou, L., Ballou, C. E., and and Robbins, P. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6846-6850 [Abstract]
  14. Herscovics, A., and Orlean, P. (1993) FASEB J. 7, 540-550 [Abstract/Free Full Text]
  15. Hill, K., Boone, C., Goebl, M., Puccia, R., Sdicu, A.-M., and Bussey, H. (1992) Genetics 130, 273-283 [Abstract/Free Full Text]
  16. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  17. Jentoft, N. (1990) Trends Biochem. Sci. 15, 291-294 [CrossRef][Medline] [Order article via Infotrieve]
  18. Kuranda, M. J., and Robbins, P. W. (1991) J. Biol. Chem. 266, 19758-19767 [Abstract/Free Full Text]
  19. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 [Medline] [Order article via Infotrieve]
  20. Locker, J. K., Griffiths, G., Horzinek, M. C., and Rottier P. J. (1992) J. Biol. Chem. 267, 14094-14101 [Abstract/Free Full Text]
  21. Lussier, M., Camirand, A., Sdicu, A.-M., and Bussey, H. (1993) Yeast 9, 1057-1063 [Medline] [Order article via Infotrieve]
  22. Mort, A. J., and Lamport, D. T. A., (1977) Anal. Biochem. 82, 289-309 [Medline] [Order article via Infotrieve]
  23. Ouellette, B. F. F., Clark, M. W. C., Keng, T., Storms, R. G., Zhong, W., Zeng, B., Fortin, N., Delaney, S., Barton, A., Kaback, D. B., and Bussey, H. (1993) Genome 36, 32-42 [Medline] [Order article via Infotrieve]
  24. Riles, L., Dutchik, J. E., Baktha, A., McCauley, B. K., Thayer, E. C., Leckie, M. P., Braden, V. V., Depke, J. E., and Olson, M. V. (1993) Genetics 134, 81-150 [Abstract/Free Full Text]
  25. Roth, J. (1984) J. Cell Biol. 98, 399-406 [Abstract]
  26. Rothstein, R. (1991) Methods Enzymol. 194, 281-301 [Medline] [Order article via Infotrieve]
  27. Roussel, P., Lamblin, G., Lhermitte, M., Houdret, N., Lafitte J.-J., Perini, J.-M., Klein, A., and Scharfman, A. (1988) Biochimie (Paris) 70, 1471-1482 [CrossRef][Medline] [Order article via Infotrieve]
  28. Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346 [Medline] [Order article via Infotrieve]
  29. Sharma, C. B., Babczinski, P., Lehle, L., and Tanner, W. (1974) Eur. J. Biochem. 46, 35-41 [Medline] [Order article via Infotrieve]
  30. Strahl-Bolsinger, S., and Tanner, W. (1991) Eur. J. Biochem. 196, 185-190 [Abstract]
  31. Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R., and Tanner, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8164-8168 [Abstract/Free Full Text]
  32. Tanner, W., and Lehle, L. (1987) Biochim. Biophys. Acta 906, 81-89 [Medline] [Order article via Infotrieve]
  33. Yip, C. L., Welch, S. K., Klebl, F., Gilbert, T., Seidel, P., Grant, F. J., O'Hara, P. J., and MacKay, V. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2723-2727 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.