(Received for publication, June 29, 1995; and in revised form, October 16, 1995)
From the
Sodium vanadate is an effective drug for the enrichment of yeast mutants defective in glycosylation reactions that are carried out in the Golgi complex(1) . We have isolated vanadate-resistant, hygromycin B-sensitive mutants that act at very early steps of N-linked glycosylation, occurring in the endoplasmic reticulum. Here we describe the phenotypic characterization of ost4, a vanadate-resistant mutant that is defective in oligosaccharyltransferase (OTase) activity both in vivo and in vitro. The OST4 open reading frame is unusual in that it predicts a protein of only 36 amino acids. We demonstrate that the OST4 gene product is, in fact, an unusually small protein of approximately 3.6 kDa, predicted to lie almost entirely in the hydrophobic environment of the membrane. Strains carrying a disruption of the OST4 gene are viable but grow poorly at 25 °C. The null mutant is inviable at 37 °C, demonstrating that the OST4 gene product is essential for growth at high temperatures. Deletion of the OST4 gene greatly diminishes OTase activity but does not abolish it. These results suggest that the OST4 gene encodes a subunit or accessory component of OTase that is essential at high temperature.
Glycosylation is an important modification that regulates the
structure and function of secreted and membrane proteins. The early
steps of N-linked glycosylation are highly conserved among
eukaryotes and begin in the endoplasmic reticulum. The assembly of a
lipid-linked precursor oligosaccharide occurs through the ordered
addition of sugars onto a dolichol phosphate anchor to generate
GlcMan
GlcNac
-PP-dolichol(2, 3) .
The preassembled oligosaccharide is then transferred as a unit onto an
appropriate asparagine residue in a nascent polypeptide. This transfer
is catalyzed by the enzyme N-oligosaccharyltransferase
(OTase). (
)Recent progress in the characterization of this
enzyme in yeast has been achieved by the convergence of both genetic
and biochemical approaches. In Saccharomyces cerevisiae, five
genes encoding components of OTase have been identified: WBP1(4) , SWP1(5) , OST1(6) , and OST3(7) . WBP1 is required for N-linked glycosylation in vivo and in vitro(4) . SWP1 was isolated as
an allele-specific high copy suppresser of a wbp1 mutant(5) . Both genes encode predicted transmembrane
proteins, which are localized in the endoplasmic reticulum and are
essential for viability. The Wbp1 protein can be cross-linked to the
Swp1 protein in extracts of microsomal membranes, suggesting that these
proteins form a complex in vivo(5) . Yeast OTase
activity co-purifies with a protein complex of six major subunits,
termed
(64 kDa),
(48 kDa),
(34 kDa),
(30 kDa),
(16 kDa), and
(9 kDa)(8) . Among these, the
and
subunits were shown to correspond to the 48-kDa Wbp1 and the
30-kDa Swp1 proteins, demonstrating that Wbp1p and Swp1 are indeed
structural components of OTase(8, 9) . The OST1 gene, a homologue of mammalian ribophorin I, encodes the 64-kDa
subunit gene and is also an essential gene product(6) .
Ost3p corresponds to the 34-kDa
subunit. Unlike the other
subunits, this protein is not required for viability, suggesting that
it functions as an auxiliary component of the OTase
complex(7) . A fifth yeast mutant, m163, has been identified
that is defective in OTase activity in vitro(10) . An
extragenic suppressor of m163 has been identified, but the wild-type
gene defined by this mutant has not been isolated.
We have isolated yeast mutants that are defective in glycosylation, based upon resistance to sodium vanadate(1) . Among these, we found two mutants with defects in steps that affect early N-linked glycosylation. These mutants identify OST4, a novel gene that encodes an unusually small protein required for OTase activity in vivo and in vitro. Our ost4 mutants are allelic to m163.
Figure 4: Complementation analysis defines a minimal DNA sequence required for OST4 activity. The restriction map of the OST4 gene is shown. DNA restriction fragments, depicted by solid lines were tested for the ability to complement the ost4 mutation, as assayed by growth on YPAD plates supplemented with 30 µg/ml hygromycin B (indicated by + or -). The region containing the putative ORF is indicated below the restriction map, as is a region containing a possible frameshift (see text). The arrowhead in pJHC5 denotes the insertion of four base pairs (at the StyI site) in this fragment, introducing a frameshift.
The disruption plasmid, post4::URA3, was constructed by replacing the HindIII/HincII fragment in pOST4RI with a SalI/SmaI fragment containing the entire URA3 gene (filled in with Klenow), thereby replacing all but the last 24 base pairs of the OST4 coding sequence with the URA3 gene.
Spontaneous mutants
resistant to 7 mMortho-vanadate were isolated
essentially as described previously (1) using MCY1093 or
MCY1094 as the starting strains. Resistant colonies arose at a
frequency of about 10 after 3-5 days of
incubation at 30 °C. Two independent spontaneous vanadate-resistant
mutant strains, NDY17.4 and NDY1.4 were back-crossed twice to the
parental strain MCY1093. Tetrad analysis of these back-crossed strains
demonstrated that for both mutants, the glycosylation defect, vanadate
resistance, and hygromycin B sensitivity cosegregated as a single
locus.
Briefly,
cells were grown to logarithmic stage, and spheroplasts were prepared
by digestion with Zymolyase 100T (ICN Radiochemicals, Irvine, CA),
washed and resuspended in glycosylation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MnCl, 1 mM 2-mercaptoethanol). These lysates were incubated for 20 min with
I-bh-Asn-Lys(BzN
)-Thr-NH
, to
allow glycopeptide formation in vitro. After stopping the
reaction by the addition of Nonidet P-40 to 1%, labeled glycopeptide
was separated from unglycosylated peptide by binding to concanavalin
A-agarose beads (Sigma) and quantitated by
scintillation
counting.
Analysis of carboxypeptidase Y (CPY) and the yeast binding protein homologue, Kar2p, was performed by immunoblotting as described previously(16) , except that secondary antibody was conjugated to horseradish peroxidase and detected by chemiluminescence (ECL; Amersham Corp.).
Deglycosylation of CPY was performed by digestion
with peptide N-glycosidase (kindly provided by Robert
Haltiwanger). After acetone precipitation of protein extracts (prepared
as described above), pellets were resuspended in 50 µl of 1% SDS,
1% -mercaptoethanol and boiled 3 min. After cooling, 100 µl of
75 mM Tris (pH 8.6), 15 mM EDTA, 5% Nonidet P-40 was
added. Peptide N-glycosidase was added, and the digestion
carried out for differing lengths of time at room temperature. Samples
were applied directly to 9% SDS-polyacrylamide gels, transferred to
nitrocellulose, and probed with anti-CPY antiserum.
A
1.1-kb EcoRI fragment containing the complementing activity
was sequenced by the dideoxy method(18) . For one strand, a
series of nested deletions was generated using the exonuclease III/S1
nuclease method(19) . A series of synthetic oligonucleotide
primers were subsequently used to sequence the complementary strand.
The 375-bp HindIII/XhoI fragment in pJHC3, which
contains the sequence required for complementing the ost4 mutant, was subjected to more rigorous sequence analysis.
Sequencing reactions, using both dGTP and dITP labeling mixtures
(Sequenase, Version 2.0; U. S. Biochemical) were analyzed by a variety
of gel systems, including 0.5-5 TBE (1
TBE is 90
mM Tris borate, 2 mM EDTA) gradient gels, containing
6% polyacrylamide (5.7:0.3 acrylamide to bisacrylamide), 7 M urea; Long Ranger gels, as described by the manufacturer (AT
Biochem); or standard 6% polyacrylamide gels containing 40% formamide
as described in the Sequenase version 2.0 instruction manual (U. S.
Biochemical) to resolve reaction products.
We
analyzed the steady state levels of the vacuolar protein, CPY, in all
of these mutants by immunoblot analyses using anti-CPY antiserum. From
this analysis, we were surprised to find two mutants that were
defective in early steps of the glycosylation pathway that occur in the
ER. All previously characterized vanadate-resistant were defective in
Golgi-localized glycosylation. Normally, as CPY transits the secretory
pathway, it undergoes a series of modifications. These include a
glycosylated ER form, containing core oligosaccharides (67 kDa) added
at four positions; a Golgi form that is further modified by the
addition of sugars (69 kDa); and a proteolytically processed vacuolar
form (61 kDa)(23) . In wild-type cells at steady state, CPY is
predominantly found as the mature, 61-kDa vacuolar form. Two
independent vanadate-resistant isolates accumulated forms of CPY that
migrated with an increased electrophoretic mobility on
SDS-polyacrylamide gels compared with that of the mature, vacuolar form
normally found in wild-type cells (Fig. 1, compare lane 1 with lanes 2 and 3). In these two allelic
mutants, which we designate ost4-2 and ost4-3 (see below), CPY accumulated as a series of intermediate species,
each differing by about 2 kDa (Fig. 1, lanes 2 and 3). This ladder-like pattern of CPY mobility is strikingly
similar to that described by Aebi and co-workers (4, 5) in their analyses of the yeast
oligosaccharyltransferase mutants wbp1 and swp1.
OTase catalyzes the earliest step in N-linked glycoprotein
addition: the transfer of the core oligosaccharide to an asparagine
residue of nascent proteins in the ER. CPY has four sites for N-linked glycosylation. Leaky mutations in OTase result in the
underglycosylation of CPY, with the accumulation of discrete
intermediates in which zero, one, two, three, or four of these sites
are utilized. Intermediates of CPY that accumulate in mutants with
defects in the synthesis of the core oligosaccharide migrate as a smear
rather that a ladder. ()The increased mobility of CPY in
these ost4 mutants therefore suggested a defect due to a
reduction in the number or size of core oligosaccharides that are added
in the ER.
Figure 1: Glycosylation states of carboxypeptidase Y in different vanadate-resistant mutants. Proteins were extracted from isogenic wild-type cells (MCY1094) (lane 1) or the vanadate-resistant mutants ost4-2 (NDY17.4) (lane 2), or ost4-3 (NDY1.4) (lane 3) and subjected to immunoblot analysis with antiserum against CPY as described in ``Materials and Methods.'' The mobility (m) of the mature, vacuolar form of CPY is indicated with an arrow.
To establish that the CPY intermediates that accumulated in ost4-2 and ost4-3 represent underglycosylated CPY rather than proteolytic products, protein extracts were treated with peptide N-glycosidase prior to fractionating by polyacrylamide gel electrophoresis. This enzyme removes N-linked oligosaccharides from proteins. If the mobility shift in CPY observed in the mutants is due solely to underglycosylation, then enzymatic deglycosylation after peptide N-glycosidase digestion should result in a single nonglycosylated form of CPY that migrates identically in both mutant and wild-type cells. As shown in Fig. 2, this was the case. A time course of peptide N-glycosidase-catalyzed deglycosylation of CPY in extracts from wild-type cells resulted in the progressive cleavage of one, two, three, or four oligosaccharides (Fig. 2, lanes 2-5). These underglycosylated forms of CPY precisely comigrated with those seen in ost4-2 extracts that had not been treated with peptide N-glycosidase (Fig. 2, lane 6, labeled ost4). After 6 h of peptide N-glycosidase digestion, CPY from both ost4-2 and ost4-3 (data not shown) accumulates almost exclusively as a fully deglycosylated form that is identical to the deglycosylated form seen in wild-type cells (Fig. 2, compare lanes 5 and 7). This result demonstrates that the aberrant CPY intermediates that accumulate in ost4-2 and ost4-3 strains are due to underglycosylation rather than proteolysis of the mature CPY protein.
Figure 2:
Peptide N-glycosidase digestion
of carboxypeptidase Y in OST4 and ost4-2 mutant
extracts. Total cellular proteins were extracted from OST4 (strain MCY1094) (lanes 1-5) or ost4-2
cells (strain NDY17.4) (lanes 6 and 7) and treated
with buffer alone (lanes 1 and 6) or digested with
buffer containing peptide N-glycosidase for the times
indicated (in hours) above each lane. Proteins were then fractionated
on SDS-polyacrylamide gels and subjected to immunoblot analysis with
antiserum against CPY as described under ``Materials and
Methods.'' The mobility of the fully glycosylated vacuolar form of
CPY (CPY) is indicated with an arrow.
Genetic crosses between the OTase mutant, m163(19) , and either ost4-2 or ost4-3 resulted in diploid strains that failed to grow on medium supplemented with hygromycin B, suggesting that ost4-2 and ost4-3 are allelic to the OTase mutant previously identified by Roos et al.(10) . Because this allele was previously mentioned in the literature (10) but had not been given a name, we have designated m163 as ost4-1. Tetrad analysis of 12 sporulated diploids produced by crossing ost4-1 with either ost4-2 or ost4-3 exhibited complete linkage of hygromycin B sensitivity and confirmed that these mutants are indeed allelic (data not shown). Extracts prepared from strain m163 indicate that ost4-1 is defective in peptide glycosylation activity in vitro ( (10) and see Table 1). ost4-1 is also vanadate-resistant and hygromycin B-sensitive. Unlike ost4-2 and ost4-3, ost4-1 is temperature-sensitive for growth and even at the permissive temperature, grows much more slowly than ost4-2 or ost4-3. Gel filtration analysis of purified oligosaccharide isolated from the oligosaccharyl pyrophosphoryl dolichol synthesized in vivo demonstrated that a full-length core is synthesized by m163, indicating that the ost4-1 mutation does not substantially affect the synthesis of the core oligosaccharide(10) . While this assay is not sufficiently sensitive to detect a loss of one or two sugars, these results strongly suggest that the primary defect in ost4-2 and ost4-3 is in the transfer of oligosaccharide. We propose to name the wild-type gene defined by these mutants OST4 (oligo saccharyl transferase 4).
Figure 3: Complementation of hygromycin B sensitivity of the ost4-2 mutation by pOST4. Isogenic OST4 (MCY1094) and ost4-2 mutant strains (NDY17-4), harboring either control parental plasmid, or a plasmid containing the 1.1-kb EcoRI complementing fragment were streaked onto either YPAD (lower panel) or YPAD plates supplemented with 30 µg/ml hygromycin B (upper panel).
To confirm that the cloned fragment
indeed contained the OST4 locus rather than an extragenic
suppressor, the 1.1-kb fragment containing OST4 was cloned
into the integrative plasmid pRS306(13) , which carries URA3 as a selectable marker. The plasmid was linearized at a
unique HincII site within the putative OST4 gene to
allow homologous recombination at the ost4 locus in a ost4
ura3 strain. Ura transformants were crossed to an OST4 ura3 strain, to produce diploids that were sporulated and
dissected. This analysis demonstrated a 2:2 segregation pattern for
Ura
:Ura
and a 4:0 pattern for
Ost4
:Ost4
(as assayed by hygromycin
B sensitivity) in the 14 tetrads analyzed. These results confirm that
the cloned DNA is very tightly linked to the ost4 mutation and
suggest that this DNA fragment does indeed contain the OST4 gene.
Figure 5: A, DNA and predicted protein sequence of the OST4 gene. The predicted protein sequence of the Ost4 protein is underlined with a thick solid line, with asterisks indicating stop codons. The region suspected to result in a larger ORF due to a frameshift is underlined with a thin line (see text). B, hydropathy profile of the small 36-amino acid Ost4 protein. Hydropathy was calculated according to the algorithm of Kyte and Doolittle(33) . C, open reading frame map of the OST4 gene. Shown are the two relevant reading frames, with the shaded frame 2 containing the predicted ORF. Full vertical lines denote the position of stop codons, while half vertical lines denote the position of methionine codons. The six-base pair region suspected to contain a frameshift is indicated by a horizontal bar above the map.
Upon examination of the predicted open reading frame map (Fig. 5C), we noted that two reading frames (underlined in Fig. 5A) could be joined by the addition of a single base, resulting in an ORF of 276 bp that could encode a 10-kDa protein. As the purified yeast OTase complex includes a polypeptide of about 9 kDa(8) , it was assumed that a sequencing error had occurred, despite the clarity in this region. The sequence in the area surrounding the putative frameshift region, AAGAAC, does not conform to GC-rich regions normally associated with artifactual compressions (see Fig. 5A). Moreover, the sequence in this region was unambiguous with respect to both the resolution and spacing of bases on both strands (data not shown). After exhaustive sequencing efforts, in which a number of different gel and reaction conditions were used, no sequencing errors in this region could be identified (data not shown).
Given the small size of the complementing fragment, we first wished to confirm that this DNA encoded a protein-encoding RNA. If this were the case, the introduction of a simple frameshift mutation in the ORF, by insertion or deletion of one or two bases, should result in loss of function. Alternatively, if this DNA encoded an untranslated RNA, a frameshift mutation might have a minimal effect. To test this idea, we took advantage of a unique StyI restriction site at the 5`-end of the putative open reading frame. Digestion with StyI results in 5` overhangs of four bases. After digestion with StyI, the ends were filled in with Klenow, thus introducing four additional base pairs to create a frameshift mutation in the plasmid pJHC5. This mutated form of the OST4 gene failed to rescue the ost4-2 mutant (Fig. 4). In addition, RNase treatment of purified microsomes had no affect on OTase activity in vitro (data not shown), suggesting that the functional molecule is comprised of protein and not RNA. Based upon these results, we inferred that this DNA does encode a protein and that the StyI site is within the ORF.
A search of nucleotide sequence data bases using the BLAST algorithm (26) provided further evidence to support the notion that this small gene encodes a protein. The S. cerevisiae OST4 cDNA sequence was identified in the Expressed Sequence Tags data base (dbest, accession number T36335). This data base contains cDNA sequences derived from mRNA. A comparison the two OST4 sequences demonstrated these to be identical where the overlap occurred, except for two ambiguities (N) in the deposited sequence and an inversion of two bases. None of these changes could result in the formation of another or extended ORF. The deposited cDNA sequence extended from nucleotide position 85 (as shown in Fig. 5) to approximately 80 bases 3` of the XhoI site.
Figure 6:
In vitro translation of Ost4p
in the presence of either [S]cysteine or
[
S]methionine. pJHC3, containing the HindIII/XhoI fragment was used as template for the
synthesis of OST4 RNA in vitro. In vitro translation
reactions containing either [
S]methionine (lanes 1 and 3) or [
S]cysteine (lanes 2 and 4) were performed. Lanes 1 and 2 contain protein derived from translation of a control RNA
that encodes a protein that containing both methionines and cysteines
residues, demonstrating the fidelity of
[
S]cysteine-containing reactions. Lanes 3 and 4 contain protein derived from OST4 RNA.
Figure 7:
The OST4 gene is essential for
growth at 37 °C. Panel A is a schematic representations of
the restriction map of the region surrounding the OST4 gene. Panel B depicts the strategy used to create the ost4:URA3 disruption plasmid. Panel C, shows tetrad analysis of
diploid strains heterozygous for the ost4-disrupted allele.
Tetrads obtained from the sporulation of strain JCY11 were dissected on
YPAD plates. The spores were incubated for 5 days at 30 °C. Each
column is labeled numerically, and each spore is labeled
alphabetically. Two large spores and two small spores from each tetrad
are evident. All large spores were Ura and all small
spores were Ura
. Panel D, colonies from four
spores of one tetrad obtained from the diploid strain heterozygous for
the ost4 deletion (shown on panel C) were streaked
onto YPAD plates and incubated at 25 or 37 °C for 4
days.
Heterozygous diploids were sporulated,
and dissected tetrads were analyzed for cell viability at 25 °C (Fig. 7C). This experiment demonstrated that the OST4 gene encodes a protein that is not essential for viability at 25
°C but that is important for normal growth. Colonies from spores
carrying the null allele (i.e. Ura) could not
be detected until 5 days after the wild-type colonies arose (Fig. 7C). Cells carrying the null mutation grew more
slowly than those carrying either the ost4-1 or ost4-2 alleles. The growth of four spores from a single
tetrad was also compared at 25 and 37 °C. No growth was observed in ost4::URA3-disrupted colonies grown at 37 °C (Fig. 7D), even on plates containing osmotic
stabilizers (data not shown). We therefore conclude that the OST4 gene is necessary for normal growth at room temperature and is
essential for growth at high temperatures.
In S. cerevisiae, genes encoding four components of OTase have been identified: WBP1(4) , SWP1(5) , OST1(6) , and OST3(7) . WBP1, SWP1, and OST1 encode proteins that are essential for viability. As OST4 is only required for growth at higher temperatures, we wished to determine what effect the loss of OST4 function had on OTase activity at the nonrestrictive temperature of 30 °C. Protein extracts were prepared from the disruption strains derived from each of the four sister spores shown in Fig. 7and subjected to immunoblot analysis to detect any changes in the level of CPY glycosylation (Fig. 8a). This experiment demonstrated that the loss of OST4 function results in a marked decrease in OTase activity in vivo. In this experiment, the ratio of underglycosylated CPY to fully glycosylated CPY was increased in cells containing the null allele compared with the mutant, ost4-2 (Fig. 8a, compare lanes 4 and 5 with lane 6). We have observed some experiment variability in the ratio of the different intermediates that accumulate in all of the ost4 mutant strains (compare, for example, Fig. 1, lane 2, and Fig. 8a, lane 6). This may result from the growth stage at which cells are harvested, which may in turn affect the rate of CPY transport from the ER, allowing more time with which oligosaccharides may be transferred. In the absence of OST4 gene product, there remained a low, but detectable, amount of fully glycosylated CPY that comigrated with the mature form seen in wild-type cells (Fig. 8, compare lanes 4 and 5 with lane 1).
Figure 8: Glycosylation states of CPY in wild-type, ost4-2, and the ost4::URA3 mutant. Protein was extracted from yeast and subjected to immunoblot analysis using antiserum against carboxypeptidase Y, as described under ``Materials and Methods.'' Panel A, proteins were prepared from OST4 cells (MCY1094) (lane 1), NDY17.4, containing the ost4-2 allele (lane 6), or from strains derived from the four spores of one tetrad derived from the ost4::URA3 diploid, JCY11 (see Fig. 7C) containing the wild-type OST4 allele (lanes 2 and 3) or a deletion of the OST4 gene (lanes 4 and 5). The position of the mature vacuolar form of CPY is indicated with an arrow; the position of the underglycosylated forms are indicated by the bracket. Panel B, proteins were extracted from OST4 cells (MCY1094) (lanes 3 and 4), NDY17.4, containing the ost4-2 allele (lanes 5 and 6), or JCY12, containing ost4:URA3 null allele (lanes 1 and 2) and treated with buffer alone (lanes 1, 3, and 5) or digested with buffer containing peptide N-glycosidase for 8 h (lanes 2, 4, and 6). Proteins were then fractionated on SDS-polyacrylamide gels and subjected to immunoblot analysis with antiserum against CPY as described under ``Materials and Methods.''
The
unglycosylated precursor form of CPY migrates with a molecular mass of
59 kDa, similar to that of the fully glycosylated mature form (61
kDa)(23) . To confirm that the form observed in the
ost4 strains is fully glycosylated, protein extracts from
these strains were treated with peptide N-glycosidase prior to
immunoblotting. As seen in Fig. 8b, upon peptide N-glycosidase treatment, all forms of CPY that accumulate in
the ost4:URA3 mutant are sensitive to digestion with peptide N-glycosidase, demonstrating that no forms correspond to
unglycosylated CPY. Since the ost4 null mutant retains some
ability to express fully glycosylated CPY, the Ost4 protein is not an
essential component of OTase at the permissive temperature, but rather
greatly augments its activity.
A large body of data suggests that correct glycosylation is required for the proper folding of many glycoproteins(28) . Therefore, it was of interest to ascertain whether or not the temperature-sensitive lethality of the ost4 null mutant may reflect a temperature-dependent protein folding requirement for glycosylation. To test this, we assayed the level of Kar2p (BiP), whose synthesis is normally induced in response to unfolded proteins in mutant and wild-type cells grown at 25 or 37 °C(7) . Isogenic wild-type and ost4::URA3 null mutants were grown to mid log phase at 25 °C, after which aliquots of cells were removed and shifted to growth at 37 °C. After 12 additional h of growth, equal numbers of cells were centrifuged, and both the intracellular and extracellular levels of Kar2p was assayed by immunoblot analysis. In wild-type cells, the amount of intracellular Kar2p increased by 5-fold at 37 °C compared with 25 °C (Fig. 9, compare lanes 1 and 3), and none was secreted at either temperature (Fig. 9, lanes 2 and 4). This was expected since the synthesis of Kar2p, a member of the heat shock family of proteins, is known to be induced as a result of high temperature. Even after induction, its level was apparently below that required to saturate the normal ER retention system, and it was not secreted into the culture supernatant. In the ost4 null mutant, at 25 °C, Kar2p was at levels similar to that seen in wild-type cells grown at 37 °C, suggesting that the protein unfolding response was induced 5-fold even at the permissive temperature. Kar2p synthesis did not significantly increase when ost4::URA3 cells were grown at 37 °C (Fig. 9, compare lanes 5 and 7). There was, at most, a 1.5-fold increase. No Kar2p was secreted into the culture media at either temperature (Fig. 9, lanes 6 and 8). These results suggest that this mutation leads to an accumulation of misfolded proteins. The lack of further induction at high temperature in the null mutant may reflect the possibility that Kar2p induction was already at a maximal levels at the permissive temperature.
Figure 9: Immunoblot analysis of Kar2p in wild-type and the null ost4::URA3 mutant. Equal aliquots of wild-type (W303) (lanes 1-4) or mutant cells (JCY12) (lanes 5-8) were grown at 25 °C (lanes 1, 2, 5, and 6) or at 25 °C and then shifted to growth at 37 °C for 16 h (lanes 3, 4, 7, and 8.) Protein extracts were prepared from the cell pellet (labeled I for intracellular), and the culture supernatant (labeled S for secreted) as described under ``Materials and Methods.'' Proteins (15 µg/lane) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis, as described under ``Materials and Methods,'' using anti-HDEL antiserum to detect Kar2p.
We have described the isolation of vanadate-resistant mutants that are defective in early steps of N-linked glycosylation. We demonstrate that the ost4-2 and ost4-3 mutants are defective in OTase activity both in vivo and in vitro and are allelic to a previously identified OTase mutant, m163 (designated ost4-1). In addition to the two isolates carrying the ost4 mutation, we also identified another, as yet uncharacterized vanadate-resistant mutant with defects in early glycosylation (data not shown). While the basis for resistance to sodium vanadate is unknown, it has been demonstrated to be a useful agent for the enrichment of mutants defective in steps that affect glycosylation in the Golgi complex(1) . The isolation of early glycosylation mutants described here suggests that it may be useful for the selection of early glycosylation mutants as well.
The nucleotide sequence of the DNA fragment that complements the ost4 mutant identified a small ORF, predicted to encode a 3.9-kDa protein. While many proteins of this size exist, most are proteolytic products of preproteins. The small, almost unprecedented, predicted size of this protein led us to initially suspect that an error had occurred in determining the DNA sequence, despite rigorous sequencing efforts. Also, complementation analysis with pJHC4, which contains a fragment that spans this region (Fig. 4), suggested that the small ORF was insufficient to complement the mutant phenotype. Examination of the DNA sequence indicated that a potential frameshift could result in a larger 10-kDa translation product, which is coincidentally the approximate molecular mass of a known OTase subunit (8) . Therefore, a number of different experiments were performed to firmly establish that the predicted ORF encoded a small protein of approximately 3.9 kDa.
We first demonstrated that this gene encodes
a protein, rather than an untranslated RNA. An insertion mutation,
introduced just downstream the initiating methionine resulted in the
loss of OTase function. Furthermore, treatment of microsomes with RNase
had no effect on oligosaccharide transferase activity in vitro (data not shown). A search of nucleotide sequence data bases
provided further evidence to support the notion that this small gene
encodes a protein. The OST4 sequence was identified in the
dbest data base, which contains cDNA sequences derived from mRNA.
Finally, this DNA contains the information for the synthesis of protein in vitro. Differential labeling with
[S]cysteine versus [
S]methionine clearly demonstrated that the in vitro translation product is a protein that migrates with a
molecular mass of 3.6 kDa, containing no cysteine residues. If one
invoked a frameshift in the DNA sequence, the 10-kDa translation
product would contain two cysteines. These results, taken together,
demonstrate that the OST4 gene encodes an unusual protein,
merely 36 amino acids in length, predicted by hydropathy analysis to
lie almost entirely in a membrane.
The complete absence of the OST4 gene product resulted in a severely compromised growth rate at normal temperature and temperature-sensitive lethality. One explanation for such a phenomenon is that lethality is an indirect effect of the glycosylation defect that is manifested only at high temperatures. An obvious candidate for such a temperature-sensitive process is protein folding. Oligosaccharide addition is necessary for the proper folding or assembly of many glycoproteins(28) . A number of glycosylation mutants exhibit temperature-sensitive growth phenotypes, both in yeast (2, 29, 30) and mammalian cell lines(31) ; and inhibition of glycosylation is known to affect the folding of many proteins(28, 32) . At the higher temperature, the folding of some glycoprotein(s) required for viability may be impaired in ost4 mutants. We tested this by monitoring the synthesis of Kar2p, whose induction occurs in response to misfolded proteins(7) . We found high levels of Kar2p induction in the null mutant even at the permissive temperature, suggesting a defect in protein folding.
An alternative explanation is that Ost4p facilitates or maintains the assembly of the OTase polypeptide complex, whose activity is required for viability. In the absence of Ost4p at the permissive temperature, complex formation may be impaired but can be tolerated. At higher temperatures, the complex is unstable, resulting in an inactive enzyme. Consistent with this notion is the correlation between the severity of the glycosylation defect and the growth defect observed in ost4 mutants.
What is the function of Ost4p? A 3.6-kDa protein was not observed as a component of the purified yeast protein complex(8, 9) . However, the identification of polypeptides in this size range would not be expected by conventional SDS-polyacrylamide gel electrophoresis. Consequently, it is quite possible that Ost4p is a structural component of oligosaccharytransferase. Ost4p is predicted to be a very hydrophobic membrane protein, containing a single membrane-spanning domain. Indeed, little of the protein sequence is predicted to be anywhere but buried in a membrane, raising questions concerning what portion of the protein may be available for interaction with other proteins outside of the membrane. No dolichol-binding consensus sequence is obvious in the membrane-spanning portion of Ost4p. Experiments designed to map the mutations in each of the different ost4 alleles may help to address this issue. Based upon its predicted membrane localization, it seems likely that this protein is a structural component of OTase, or a proximal accessory component that regulates OTase activity or assembly. The observation that mature, fully glycosylated CPY accumulates in the complete absence of Ost4p suggests that it is not an essential component. Despite this, the dramatic effect that the loss of OST4 function has on the normal growth rate of cells and on the efficiency of glycosylation demonstrates its importance for normal OTase activity. The identification of OST4 and the other genes that encode subunits or regulators of OTase will enable us to define how this multifunctional enzyme functions in glycosylation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42519[GenBank].