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.
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 GDP
Man 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 UDP
GalNAc 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 (
)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
-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
-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-
200, lys2-801,
trp1-
901, suc2-
9) and SEY2101 (MATa,
leu2-3-112, ura3-52, ade2-1, suc2-
9). 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
(TM buffer) and
broken with glass beads in the same buffer. After centrifugation at
3,000
g for 1 min, membranes were collected from the
supernatant by centrifugation at 48,000
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-[
C]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
, 0.14% Triton X-100, 3 mM acetyl-YNPTSV-NH
, 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-[
C]Man, 10 mM KH
PO
Na
HPO
,
pH 6.5, 7 mM MgCl
, 0.14% Triton X-100, 3.5 mM acetyl-YATAV-NH
, 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
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
-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 [
H]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
, 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
g. The pellet containing cell walls and membranes was
-eliminated.
-elimination was carried out in 0.1 N NaOH, 1 N NaBH
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
-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
(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
-Man
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
[
C]mannose residues from
Dol-P-[
C]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 (
)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
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
10
/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 [
H]mannose-labeled yeast cells by
-elimination and resolved by thin-layer chromatography (Fig. 6). The wild-type strain showed the normal five
oligosaccharide peaks (Man
-Man
) 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:
-elimination profiles. Thin-layer
chromatograms of manno-oligosaccharides released by
-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.
-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
-Man
) 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. (
)
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
-Man
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.