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INTRODUCTION |
Asparagine-linked glycosylation is one of the most common types of
eukaryotic protein modifications (1-3). N-Glycans are essential for cell viability (4-6) and can have a profound role in the
biological function and physicochemical properties of many secreted and
integral membrane proteins (7). The key step of this pathway is the
en bloc transfer of the core oligosaccharide Glc3Man9GlcNAc2 from dolichyl
pyrophosphate to selected asparagine residues in an
Asn-X-Ser/Thr consensus sequon of nascent polypeptides, where X can be any amino acid except proline (8-13). The
reaction is catalyzed by the
ER1-resident enzyme
N-oligosaccharyltransferase (OST) (for review see Refs. 14
and 15).
Previous attempts to purify and characterize this enzyme have met with
limited success due to its lability upon solubilization (16-18) and
probably also due to the fact that it is a multimeric membrane protein
complex. Meanwhile, however, OST complexes have been purified from
different sources, such as dog pancreas (19, 20), yeast (21-23), hen
oviduct (24), and human (25) and pig (26) liver. The subunit
composition of the various isolated complexes and the protein
sequences, so far obtained, reveal in part a high conservation of the
structural organization of this enzyme throughout evolution.
Independent OST purifications from Saccharomyces cerevisiae
have yielded active complexes consisting of four polypeptides (Ost1p
(64/62 kDa), Wbp1p (47 kDa), Ost3p (34 kDa), and Swp1p (30 kDa)) (21,
23) or of six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p (16 kDa), and
Ost5p (9.5 kDa)) (22). Cloning and functional analysis of
OST1 (23, 27), WBP1 (28), SWP1 (29),
and OST2 (30) have indicated that these genes are essential
for the vegetative growth of the yeast cell and reveal significant
homology to components of the canine complex: to ribophorin I (27),
OST48 (31), to the C-terminal half of ribophorin II (22), and DAD1
(defender against apoptotic cell death) (20), respectively. In
contrast, OST3 (32) and OST5 (33) coding for the
34- and 9.5-kDa subunits, respectively, are not essential, but their
deletion yields glycosylation defects and reduces OST activity in
vitro. In addition to these six proteins, genetic screens have
identified two other loci, OST4 (34) and STT3
(35), that are required for optimal OST function in vivo and
in vitro. Recent evidence indicates now that the derived
proteins are indeed part of the complex (36, 37). OST4
encodes an unusually small, hydrophobic polypeptide of 3.6 kDa; its
deletion leads to underglycosylation of N-glycoproteins and
a temperature-sensitive growth phenotype. STT3 encodes a
78-kDa transmembrane protein with the highest conservation among the
proteins associated with OST function. The essential Stt3p was found to
be necessary for stability or assembly of the complex, and its lack
affects the substrate specificity for the lipid-linked oligosaccharide
donor (35).
So far, the specific function of the various enzyme subunits is obscure
and remains to be defined. In addition, the question must be answered
which subunit is a bona fide constituent of the complex or
serves only an auxiliary function. In this report, we describe the
isolation and functional characterization of a new, not essential gene,
designated OST6, that has sequence homology and in
particular a very similar membrane topology to OST3.
Disruption of OST6 causes only a minor defect in
N-glycosylation, both in vivo and in
vitro. However, a
ost6
ost3 double mutant exhibits a synthetic phenotype with a strong underglycosylation of soluble and
membrane-bound glycoproteins as well as a defect in complex formation.
By blue native electrophoresis, we demonstrate that the OST complex has
a molecular mass of about 240 kDa and consists of all hitherto defined
OST subunits (except for the small 3.6-kDa Ost4p, for which no antibody
is available for detection).
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Genetic Methods--
Yeast strains were SS330
(MATa ade2-101 ura3-52 his3
200
tyr1), MA7-B (MATa ade2-101 ura3-52
his3
200 lys2-801 wbp1-1), YG191 (MAT
ade2-101 ura3-52 his3
200
ost3::HIS3), YG176 (MATa
ade2-101 ade3 leu2 his3
200 tyr1 stt3-3),
RKY325 (MATa ade2-101 ura3-52 his3
200
tyr1
ost6::URA3), RKY326
(MAT
ade2-101 ura3-52 his3
200
ost3::HIS3
ost6::URA3),
RKY327 (MAT
ade2-101 ura3-52
his3
200
ost3::HIS3
ost6), RKY346 (MAT
ade2-101 ura3-52
his3
200
ost3::HIS3 YEp352-OST6), and RKY348 (MAT
ade2-101
ura3-52 his3
200
ost3::HIS3
ost6 YEp352-OST6). Strain SS330 is the
isogenic wild-type. Plasmids encoding recombinant CPY forms (38) were
transformed into yeast using standard techniques. Cells were grown in
standard yeast media (39).
Cloning and Disruption of OST6--
The OST6 coding
sequence was originally reported as an ORF of unknown function on
chromosome XIII by the yeast genome sequencing project (ORF YML019W). A
genomic clone of OST6 was isolated from a yeast gene bank
with 4-kilobase pair Sau3A-DNA fragments in the multicopy
vector YEp352 (40). As a probe for colony hybridization, a
PCR-amplified, digoxygenin-labeled genomic OST6 fragment was used. A genomic clone containing the OST6 open reading frame
together with 5'- and 3'-flanking sequences was isolated from ~20.000
Escherichia coli colonies.
For disruption of the OST6 locus by homologous
recombination, DNA fragments of 0.9 and 1.2 kilobase pairs from the 5'-
and 3'-untranslated regions of OST6 were amplified from
genomic wild-type DNA by PCR and ligated to an URA3
cassette. The 5' fragment covered
840 to +58 base pairs (relative to
the start ATG of the OST6 locus), the 3' fragment 41 base
pairs upstream of the stop codon and 1159 base pairs of the
3'-untranslated region. Successful replacement of the OST6
locus was confirmed by PCR using genomic DNA from uracil prototrophic transformants.
Isolation of Microsomal Membranes and Preparation of Solubilized
Enzyme Extract--
Rough microsomal membranes were isolated as
described (21). The membranes were resuspended in membrane storage
buffer (30 mM Tris-HCl (pH 7.5), 3 mM
MgCl2, 1 mM DTT, 35% glycerol, v/v) at a
concentration of 12 mg/ml protein. For solubilization, membranes were
adjusted to 15 mM Tris-HCl (pH 7.5), 1.5 mM
MgCl2, 0.5 M KCl, 0.5 mM DTT, and
17% glycerol. After adding 1% Nikkol (final concentration), the
reaction was incubated 20 min on ice. The solubilized extract was then
separated from insoluble material by centrifugation at 150,000 × g for 40 min. Unless otherwise stated, all steps were
carried out at 4 °C.
Determination of Oligosaccharyltransferase
Activity--
Oligosaccharyltransferase activity was determined as
described (16, 35).
In Vivo Labeling with [35S]Methionine and
Immunoprecipitation--
Yeast cells were grown to mid-log phase in
yeast complete medium. 1.5 × 108 cells were collected
at 1,200 × g for 3 min, washed once, and resuspended
in 10 ml of minimal medium (0.67% yeast nitrogen base, 2% glucose,
and the appropriate amino acids). After a 30-min adaptation at
30 °C, or 25 °C in case of temperature-sensitive strains, 75 µCi of [35S]methionine was added and cells were labeled
for 30-45 min. In case of invertase, cells were derepressed for 20 min
in yeast minimal medium with 0.1% glucose prior to labeling.
Incubation was stopped with 10 mM NaN3 and any
35S label not incorporated was removed by washing the cells
once with 10 mM NaN3. Cells were resuspended in
300 µl of buffer (50 mM Tris-HCl (pH 7.5), 1 mM EtSH, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine) and broken with glass beads for 10 min on a Vibrax shaker at 4 °C. After collecting the supernatant, the glass beads were washed once with 300 µl of buffer. The extract was centrifuged for 30 min at 48,000 × g, and the
supernatant was incubated with 1% SDS for 5 min at 95 °C. SDS was
then reduced to < 0.2% by addition of TNET (50 mM
Tris-HCl (pH 7.5), 3 mM EDTA, 150 mM NaCl, 1%
Triton X-100), the appropriate antiserum was added, and the mixture
incubated at 4 °C over night. For precipitation of the antigen/IgG
complexes, 5 mg of Protein A-Sepharose equilibrated in TNET buffer were
incubated with the solubilized extract by rolling end over end for
2 h at 4 °C. The Sepharose beads were centrifuged for 1 min at
14,000 × g, washed four times with 1 ml of TNET buffer
and once with 1 ml of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, resuspended in 50 µl of SDS-PAGE sample buffer,
and incubated at 95 °C for 5 min. After another centrifugation, the supernatant was analyzed by SDS-PAGE on 8% polyacrylamide gels followed by autoradiography.
Blue Native (BN-PAGE) and Denaturing Gel
Electrophoresis--
BN-PAGE was carried out according to Refs. 41 and
42. Microsomal membranes (12 mg of protein/ml) in storage buffer were solubilized in buffer containing 15 mM Tris-HCl (pH 7.5),
1% Nikkol, and 500 mM 6-aminocaproic acid.
Unsolubilized material was removed by centrifugation for 40 min at
128,000 × g. After addition of 1/20 volume of sample
buffer (5% Serva blue G, 500 mM 6-aminocaproic acid), the
supernatant was analyzed on a 6-13% polyacrylamide gradient gel with
a 4% stacking gel in 50 mM Bistris-HCl (pH 7.0) 500 mM 6-aminocaproic acid. Marker proteins were: bovine serum albumin (monomeric and dimeric form), 66 and 132 kDa, respectively;
-amylase, 230 kDa; apoferritin, 443 kDa.
Denaturing SDS-polyacrylamide gel electrophoresis was carried out
according to Refs. 43 or 44, respectively, and visualized by silver
staining (45). For Western blotting, proteins were transferred to
nitrocellulose or Immobilon (in case of BN-PAGE) membranes using the
semidry blotting technique (46).
Immunodetection was performed according to standard procedures and was
visualized by the ECL method (Amersham Pharmacia Biotech). Polyclonal
antisera against Wbp1p and Swp1p were raised in rabbits using synthetic
peptides corresponding to the 16 C-terminal amino acids coupled to
bovine serum albumin with glutaraldehyde (47) as described (21). The
polyclonal anti-Ost3p and Ost6p antisera were raised against amino
acids 26-350 and amino acids 25-192, respectively, fused to the
maltose-binding protein from E. coli. The fusion protein was
isolated by amylose column chromatography using the protein fusion and
purification system from New England Biolabs and used for immunization
of rabbits.
Quantification of Glycosylation--
The average number of
oligosaccharides for the various glycoproteins was determined by
scanning of ECL images using a Bio-Rad MultiImager, or by radioanalytic
scanning of fluorographs using a Packard PhosphoImager. The ratio
between the average number of oligosaccharides on a given glycoprotein
in the different mutants and the average number of glycan chains for
that glycoprotein in the wild-type strain was expressed as the
percentage of glycosylation relative to wild-type.
In Vivo Labeling of Lipid-linked Oligosaccharides with
[3H]Mannose--
Yeast cells were grown to mid-log phase
in complete medium. 4 × 108 cells were collected at
1,200 × g for 3 min, washed once with complete medium
lacking glucose, resuspended in 200 µl of complete medium without
glucose, and labeled with 100 µCi of [3H]mannose for 15 min at 30 °C. The reaction was stopped with 4 ml of
chloroform/methanol (C/M) 3:2 (v/v), and the cells were collected at
1,200 × g for 5 min. The supernatant was removed and
lipid-linked oligosaccharides were extracted by washing the cells three
times with 3 ml of C/M 3:2 (v/v), twice with 3 ml of
chloroform/methanol/H2O (C/M/H) 3:48:47 (v/v/v) upper phase containing 4 mM MgCl2, two times with 3 ml of
upper phase without MgCl2 and finally three times with 3 ml
of C/M/H 10:10:3 (v/v/v).
The composition of the lipid-linked oligosaccharides was determined
after cleavage of the carbohydrate from the lipid component by mild
acid hydrolysis. For this purpose the C/M/H extracts were dried under
nitrogen, resuspended in 0.02 M HCl in 1-propanol, and
incubated for 30 min at 100 °C. The hydrolysate was then analyzed by
HPLC on a Supelco Sil LC-NH2 column running a gradient from acetonitrile/H2O 70:30 (v/v) to
acetonitrile/H2O 50:50 (v/v) over 75 min, 5 min at 50:50
(v/v), and returning to 70:30 (v/v) over 5 min. The eluate was mixed
continuously with scintillation fluid Flow 302 (Zinsser) at a ratio of
1:2, and the radioactivity in the eluate fractions was quantified with
a flow-through scintillation counter (Berthold).
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RESULTS |
Isolation and Sequence Analysis of the OST6 Gene--
In the
process of cloning and characterizing the OST3 gene encoding
the 34-kDa subunit (our accession number X79596, GeneEMBL data base) a
report appeared dealing with the same gene (32). Our nucleotide
sequence analysis is in agreement with the published data. However, the
N terminus of the mature Ost3 protein, isolated by us and determined by
gas phase sequencing, was found to start three amino acids further
downstream at residue 26 with asparagine (Fig.
1A). We do not know whether
this difference is strain-specific or due to proteolytic degradation. A
search of protein sequence data bases using the BLASTP protein sequence
comparison algorithm (48) revealed a homology between Ost3p and an open
reading frame of unknown function on chromosome XIII of S. cerevisiae (YML019W). The sequence identity (20%) and similarity
(46%) between OST3 and the yeast ORF, which we named
OST6 on account of its function in
N-oligosaccharide transfer (see below), are rather modest
(Fig. 1A). However, the two proteins have a strikingly
similar membrane topology (Fig. 1B) with a typical
N-terminal cleavable signal sequence (49) and a C-terminal arrangement
of four transmembrane domains using the algorithm of Kyte and Doolittle
(50). Searches of protein sequence data bases have revealed that
OST3 and OST6, respectively, have homology to
three other ORFs: to an ORF of Caenorhabditis elegans on
chromosome 3 (identity 21%, similarity 46%) encoding a 37.7-kDa
protein, to a candidate human tumor suppressor gene on chromosome band
8p22, named N33 (51), and to the mammalian protein
TRAP, a component
involved in protein translocation into the ER (identity 23%,
similarity 48%; in both cases) (52). Whereas the first two proteins
have a similar hydropathy profile, and thus may be structural
homologues to Ost3p/Ost6p,
TRAP is a type I membrane protein with
only one transmembrane domain and probably different function.
OST6 encodes a protein of 332 amino acid residues with a
calculated molecular mass of 37.9 kDa, but runs on SDS gels as 32 kDa,
somewhat faster than Ost3p (34 kDa) and hardly to resolve from Swp1p
(see below). The presumed signal sequence cleavage site (Fig.
1A) is in agreement with the predictive method of von Heijne
(53). A consensus site for N-glycosylation is located at
Asn-175, but not used, since endo H treatment or the N-glycosylation inhibitor tunicamycin do not alter the
mobility of Ost6p (data not shown). Thus OST6, like
OST3, is not a glycoprotein. A genomic clone of
OST6 was obtained by PCR techniques amplifying a fragment
that comprises the sequence from 233449 through 234455 from chromosome
XIII (YML019W).

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Fig. 1.
A, comparison of the amino acid
sequences of Ost3p and Ost6p. Solid lines
represent identical amino acids; single and
double points indicate conservative amino acid
replacements. Ost3p, the sequence of the N terminus of the
mature protein (starting with Asn) and of an internal tryptic peptide
fragment, determined by direct amino acid sequencing, are
underlined. Ost6p, the arrow indicates
the potential signal sequence cleavage site. B, Hydropathy
analysis of Ost3p and Ost6p according to the algorithm of Kyte and
Doolittle (50) using a window of 17 amino acids. Note the nearly
identical arrangement of four hydrophobic regions in the C-terminal
half of both proteins.
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OST3 and OST6 Are Required for N-Glycosylation in Vivo, and Their
Disruption Leads to a Synthetic Phenotype--
To analyze the function
of OST6 and OST3 and, in particular, to disclose
a possible distinction between the two genes, we examined the
glycosylation pattern of well characterized soluble and membrane
glycoproteins in single and double mutant strains. Either cell extracts
from metabolically labeled cells followed by immunoprecipitation and
gel electrophoresis (in case of carboxypeptidase, invertase, dipeptidyl
aminopeptidase B), or protein immunoblots (in case of Wbp1p) were
analyzed. As depicted in Fig. 2 and Table I, soluble vacuolar carboxypeptidase Y
(CPY) shows in single mutants only a slight reduction in glycosylation
indicated by the appearance of a minor band of CPY lacking one
N-linked chain; the defect in
ost3 is
consistently more pronounced than in
ost6. Mature CPY
contains four N-linked core type oligosaccharide chains and
has a molecular mass of 61 kDa (54). In striking contrast, the double
mutant
ost3
ost6 severely underglycosylates CPY and lacks one, two, and, to some extent, also three and four chains. Such a
reduction in glycosylation can also be observed in OST mutants
defective in the essential genes WBP1 (28) or
OST1 (27). All underglycosylated bands were endo H-sensitive
and shifted to a 53-kDa band, the predicted size of unglycosylated
mature CPY, indicating that the bands differ in oligosaccharide content rather than in polypeptide mass (data not shown). A similar
underglycosylation pattern in the order
ost6 <
ost3 <
ost3
ost6 was demonstrated also for the soluble, extracellular glycoprotein invertase (only the
core glycosylated ER form has been considered, since the high molecular
weight cell wall form does not allow a reliable quantification of the
glycosylation defect), for the vacuolar membrane protein dipeptidyl
aminopeptidase B (DPAP B) and for the OST subunit Wbp1p (Fig. 2; Table
I). The glycoproteins vary to some extent in the degree of
underglycosylation. Our results, however, do not allow to postulate a
bias for underglycosylation of membrane glycoproteins, as has been
previously discussed for the case of ost3 (32).

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Fig. 2.
In vivo
N-glycosylation defects in ost3,
ost6, and ost3 ost6
disruptants. Analysis of glycosylation of CPY, invertase,
and DPAP B was performed by metabolic labeling of yeast cells with
[35S]methionine, followed by immunoprecipitation, PAGE,
and autoradiography. The position of the mature form of CPY
(mCPY) and of the underglycosylated forms lacking one to
four oligosaccharide chains (CPY 1 to
4) are indicated; mDPAP B, mature
DPAP B. Glycosylation of Wbp1p was analyzed by Western blot technique.
50 µg of membrane protein were loaded per lane. Mature Wbp1p
(mWbp1p) and the two underglycosylated forms ( 1, 2) are
indicated. A presumably proteolytic fragment of Wbp1p running somewhat
faster than the unglycosylated form ( 2) is marked by an
asterisk (*).
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Table I
Reduced in vivo glycosylation activity in ost3, ost6, and
ost3/ ost6
The underglycosylation of 35S-labeled glycoproteins was
quantified by densitometric scanning of fluorographs or phosphor
imaging as described under "Experimental Procedures." In the case
of Wbp1p, an ECL image obtained from Western analysis was scanned. The
values in column 3 represent the number of N-glycosylation
sites of each protein. The numbers in parentheses indicate the average
number of glycosylation sites used in wild-type yeast. Quantification
of the glycosylation is described under "Experimental Procedures."
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Since OST3 and OST6 show a synthetic phenotype,
we asked whether an overexpression of OST6 in a multicopy
vector can functionally replace OST3 and thus cure
underglycosylation of CPY in an
ost3 single and an
ost3
ost6 double mutant, respectively. Indeed, this is
the case as shown in Fig. 3, lanes
1 and 4. No suppression of CPY underglycosylation was
observed, when OST1, WBP1, or SWP1 were overexpressed (data not shown). We did not examine overexpression of OST3 in an
ost6 mutant, since the
underglycosylation defect in this strain is rather mild.

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Fig. 3.
High copy number suppression of the
ost3 glycosylation defect by
OST6. OST6 was expressed in the
multicopy vector YEp352 (40) in the ost3 single mutant
(lane 1) and the ost3 ost6 double
mutant (lane 4). CPY was analyzed in the transformants by
metabolic labeling with [35S]methionine, followed by
immunoprecipitation, PAGE, and autoradiography. The vector control is
shown in lanes 2 and 3. The positions of mature
CPY (mCPY) and of glycoforms lacking one to four
oligosaccharide chains are designated accordingly ( 1 to 4).
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Ost3p and Ost6p Influence OST Activity in Vitro, but Not Formation
of Lipid-linked Oligosaccharide--
The phenotype of
underglycosylation of proteins could be attributed to a defect in the
assembly of lipid-linked oligosaccharide (alg mutants)
(55-57) or to a reduced OST activity (27, 28, 30). To clarify this, we
analyzed the dolichol-linked oligosaccharides from isogenic wild-type,
ost3,
ost6, and
ost3
ost6 cells
labeled with [3H]mannose. In all strains synthesis of
fully assembled core oligosaccharide was observed (Fig.
4). The oligosaccharide pattern in the
double mutant was slightly shifted toward
Glc3Man9GlcNAc2, which is in accord
with the observed underglycosylation in this strain. We conclude that
the glycosylation defect is not due to a specific defect in the
biosynthesis of the lipid-oligosaccharide precursor, as is the case in
different alg mutants.

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Fig. 4.
Analysis of lipid-linked oligosaccharides in
wild-type cells (A), ost3
(B), ost6
(C), and
ost3 ost6
(D) mutant cells. Lipid-linked
oligosaccharides were labeled by incorporation of
[3H]mannose, and the oligosaccharides released by
mild acid hydrolysis were analyzed by HPLC.
GN2M5,
GlcNAc2Man5;
GN2M8,
GlcNAc2Man8;
GN2M9G3,
GlcNAc2Man9Glc3.
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Next, we tested whether OST activity was affected in vitro
in these strains. Since we had previously observed that the
stt3-3 mutation results in a sensitivity of OST toward only
a suboptimal, non-full-length substrate (35), two different glycosyl
donor substrates (lipid-linked chitobiose and lipid-linked
Glc3Man9GlcNAc2) were used; the
synthetic hexapeptide Tyr-Asn-Leu-Thr-Ser-Val served as the
oligosaccharide acceptor (16). As shown in Table
II, the measured OST activity reflects
the underglycosylation observed in vivo. Whereas the single
mutations display only a small decrease in activity, the
ost3
ost6 double mutant is severely affected, in a
similar fashion as, e.g., a wbp1 mutant (28). OST
activity in
ost6 using the chitobiose donor was
consistently somewhat lower than in
ost3, opposite to the
situation in vivo; however, glycosyl transfer using the
full-length substrate is in accord with the in vivo data.
The data also indicate that OST6 may influence the glycosyl
donor recognition, but compared with stt3-3 only to a very
minor extent.
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Table II
In vitro OST activity of wild-type and mutant strains with different
lipid-linked oligosaccharides as substrates
In vitro OST activity was determined by measuring the
transfer of a radiolabeled oligosaccharide from DolPP-GlcNAc2
or DolPP-GlcNAc2Man9Glc3 as glycosyl donor to
the synthetic hexapeptide YNLTSV as glycosylacceptor (cf. 16).
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We have also determined various enzyme activities involved in
biosynthesis of lipid-linked oligosaccharide such as DolP-Man, DolP-Glc, and DolPP-GlcNAc2, respectively, and found no
differences between the mutants and wild-type (data not shown).
Similarly, biosynthesis of O-linked glycans, which is
initiated in yeast in the ER (3, 58), is not affected. Chitinase, an
exclusively O-mannosylated glycoprotein, is not altered in
its mobility when analyzed by Western technique and, furthermore, no
defect in O-mannosylation activity is detectable in
vitro (data not shown).
Ost3p and Ost6p Are Not Involved in Peptide Substrate Recognition
but Are Needed for Optimal Oligosaccharyltransfer--
In search of a
specific function for OST3 and OST6, we
considered among other possibilities that these genes could be involved in peptide substrate recognition. To address this point, we expressed simultaneously with wild-type CPY a recombinant form (in single copy as
is the case for the wild-type form) containing only one of the four
carbohydrate chains, either A, B, C, or D (Fig.
5A). The corresponding
carboxypeptidase species are called CPY-A, CPY-B, CPY-C, and CPY-D,
respectively. As shown in Fig. 5B, both in the
ost3 single and in the
ost3
ost6 double
null mutant, all four recombinant CPY forms are glycosylated. This
indicates that Ost3p and Ost6p, respectively, are not involved in the
recognition of a particular glycosylation site of the nascent
polypeptide chain. The slight increase in mobility of CPY-C mutant
(Fig. 5B, lane 3) is an inherent feature of the
recombinant form and not due to altered glycosylation (38);
it is also observed when expressed in a wild-type strain (Fig.
5B, upper panel).

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Fig. 5.
ost3 ost6 mutant is
affected in the glycosylation capacity of the OST complex.
A, schematic representation of the CPY precursor showing the
position of the four N-linked glycosylation sites A-D.
B, analysis of wild-type and recombinant forms of CPY in
wild-type (upper panel), ost3 (middle
panel), and ost3 ost6 (lower panel)
strains. Cells were metabolically labeled with
[35S]methionine, and CPY was analyzed by
immunoprecipitation followed by PAGE and autoradiography.
Wild-type CPY indicates CPY with four
N-glycosylation sites encoded by the chromosomal
PRC1 locus. rCPY designates the recombinant CPY
form, which contains only one of the four sequons.
Underglycosylated forms of wild-type CPY are marked 1 to 4;
underglycosylated recombinant CPY is marked rCPY-1.
Quantification of the glycosylation defect is described under
"Experimental Procedures" and in the text.
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In order to estimate the degree of glycosylation of the wild-type and
recombinant CPY forms in the
ost3
ost6 double mutant, the following calculation was made. Since the recombinant glycosylated and unglycosylated CPY forms comigrate with the
3 and
4 wild-type forms, respectively, the amount of radioactivity of the
3/
4 bands
of the control (lane 5) was subtracted from the
corresponding bands in lanes 1-4 to give the amount of
radioactivity in the recombinant forms (assuming that
underglycosylation of wild-type CPY is the same, when both species are
expressed). We calculate that in
ost3
ost6 cells
wild-type CPY contains on average 1.8 instead of four
N-linked saccharide chains, corresponding to 45% glycosylation compared with wild-type cells (Table I). In the recombinant forms, having only one glycosylation site, 0.58 chains of
CPY-A, 0.75 of CPY-B, 0.71 of CPY-C, and 0.65 of CPY-D are glycosylated, corresponding on average to 67% glycosylation. Thus, a
nascent polypeptide with multiple glycosylation sites is somewhat more
affected, indicating that OST activity is limiting in the double mutant.
In order to exclude a defect in the protein translocation machinery
that could also account for the underglycosylation, we investigated by
Western analysis whether the amount of components of the translocon,
such as Sec61p, Sec62p, and Sec63p, was altered in the
ost3
ost6 double mutant. However, no difference was
observed as compared with wild-type cells (data not shown).
Growth Phenotypes of OST3 and OST6 Disruptants--
Both
OST3 and OST6 are not essential for yeast growth,
and also the double knock-out mutant does not exhibit an altered growth rate or morphology as compared with wild-type when tested up to 37 °C in complete medium (data not shown). Mutations in other OST
subunits, like WBP1, OST1, or OST2,
cause temperature-sensitive growth defects (27, 28, 30). However, we
noticed that ost3 and ost6 null mutants display
at 30 °C differential phenotypes, when stressed by agents that
interfere with cell wall biogenesis, such as Calcofluor White,
caffeine, or SDS. Cells with weakened cell walls are sensitive against
Calcofluor White (CFW) (59) at concentrations that do not impair growth
of wild-type cells. As shown in Fig.
6A, growth of the
ost6 disruptant is completely abolished in the presence
of 5 µg/ml CFW, whereas in
ost3 no inhibition occurs. A
similar effect is also exhibited by the phosphodiesterase inhibitor
caffeine (Fig. 6B). Evidence has been obtained (60-62) that
a protein kinase C is a central element in the regulation of cell
integrity via regulating cell wall formation. Likewise,
ost6 but not
ost3 cells are sensitive to
caffeine and SDS. In the case of
ost3, it was found that
the mutant is even somewhat more resistant against SDS, compared with
the isogenic wild-type strain (Fig. 6C). Various mutants
with a defective cell wall biosynthesis or N-glycosylation
were reported to reveal an SDS sensitivity (63, 64). The different
behavior of
ost6 and
ost3 null mutants toward these compounds could mean that both genes may in part be
involved in different functions important for cell wall biogenesis and/or stability.

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Fig. 6.
OST3 and OST6
interfere with cell wall biosynthesis and cell wall
stability. 3 µl of a serial 1:10 dilution of the indicated
strains in liquid medium were plated on YEPD plates containing 5 µg/ml CFW (A), 7.5 mM caffeine (B),
and 0.005% SDS (C), starting with 3 × 104
cells (left). Plates were incubated at 30 °C for 72 h.
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Subunit Composition of the OST Complex by Blue Native and
Denaturing Electrophoresis--
Our affinity purification procedure
using an anti-Wbp1p affinity column (21) led to the isolation of a
tetrameric, enzymatically active OST complex consisting of Ost1p,
Wbp1p, Ost3p, and Swp1p, whereas the complex purified by Kelleher and
Gilmore (22) contained in addition Ost2p and Ost5p. In none of the two
procedures the essential and genetically identified subunits Stt3p and
Ost4p, or the newly discovered Ost6p were present in detectable amounts by silver or Coomassie staining. Using the more sensitive method of
Western analysis, we show now that the tetrameric OST preparation from
wild-type cells contains also Stt3p, Ost2p, and Ost5p, as well as Ost6p
(no antibodies are available for Ost4p; Fig.
7B). We find that Stt3p, which
has a predicted molecular mass of 78 kDa, migrates on SDS gel as a
diffuse band with an anomalous mobility of ~60 kDa that is not
resolved from Ost1p (lanes 1 and 3). Proof for
the correct identification of the Stt3p signal comes from the
observation that it disappears, when Stt3p depleted membranes were
analyzed (data not shown). Ost6p turns out to have a mobility almost
identical to that of Swp1p and, therefore, may have escaped detection
so far.

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Fig. 7.
Subunit composition of the enzymatically
active, anti-Wbp1p immunoaffinity purified OST complex from wild-type
and ost3 ost6 cells. A, silver
stain. 30 µg of protein from anti-Wbp1p immunoaffinity-purified
complex (21) were analyzed on a 12.5% SDS gel (43). B,
Western analysis. 15 µg of protein were loaded on a 10%
SDS-polyacrylamide gel (lanes 1-8) or a 10% Tris-Tricine
gel (lanes 9-16), respectively, and a Western blot analysis
was performed with antibodies to Stt3p, Ost1p, Wbp1p, Ost3p, Ost6p,
Swp1p, Ost2p, and Ost5p as indicated and visualized by ECL detection.
In the left lanes within each antibody column, OST from
wild-type is loaded; right lanes contain OST from
ost3 ost6 .
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Affinity purification and silver staining (Fig. 7A) of OST
from
ost3
ost6 cells reveals a reduction of Ost1p and,
as expected, a lack of Ost3p (and Ost6p by Western blot, see Fig.
7B). Ost1p and Wbp1p, respectively, the two glycosylated
subunits of the complex are partly underglycosylated (Fig. 7,
A, lane 2; B, lanes 4 and
6). By Western analysis, the presence of Stt3p, Ost2p, and Ost5p could also be demonstrated. Furthermore, a comparison of the
composition of the complexes from wild-type strain and
ost3
ost6 double mutant (Fig. 7B) indicates
that the amount of Stt3p, Ost1p, and Ost5p is reduced. Thus, the
structural organization of OST is severely affected and leads, as shown
above (Table II), to a defect in enzyme activity, observed so far for
cells lacking an essential OST protein.
In order to estimate the size of the native complex and identify
possible subcomplexes (see below), we applied the method of blue
native-polyacrylamide electrophoresis that was reported to be a
powerful tool for the separation and size determination of native
membrane bound complexes (41, 42, 65). By this technique, in
combination with Western analysis, we can identify in wild-type
membranes a complex of ~240 kDa (Fig.
8), in which all OST subunits are
assembled. In the case of Wbp1p, we also observe in low amounts an
additional band (larger than the expected mass of Wbp1p) that is
recognized by the antibody; its origin remains obscure, however. The
differences in signal intensities do not reflect different amounts or
the stoichiometry of the subunits in the complex, but are rather due to
differences in the antibody titer.

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Fig. 8.
Blue native PAGE of solubilized OST complex
from wild-type cells. Solubilized complex was prepared as
described under "Experimental Procedures," and equal amounts of
protein (260 µg) were loaded in each lane on a 6-13% polyacrylamide
gel. Proteins were transferred to a polyvinylidene difluoride membrane
and probed with antibodies to Stt3p, Ost1p, Wbp1p, Ost3p, Ost6p, Swp1p,
Ost2p, and Ost5p as indicated and visualized by ECL detection.
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DISCUSSION |
We have described the identification and functional
characterization of OST6, a novel gene encoding a subunit of
the OST from yeast. Ost6p is a 32-kDa transmembrane protein not
disclosed in any of the previously purified OST complexes. It is
homolog to Ost3p, the 34-kDa
-subunit of yeast OST (32). The
identity between both proteins with 21% is rather low, but they
display a strikingly similar membrane topology containing four
predicted C-terminal membrane-spanning domains. Such a hydropathy
profile and the same degree of identity (23%) is also found for a
Caenorhabditis elegans ORF of unknown function and a human
candidate tumor suppressor. The relationship of OST6 and
OST3 to a neoplastic phenotype is obscure, but altered
glycosylation of cell surface proteins is a well known feature of tumor
cell lines (66, 67).
Disruption of OST3 was reported (32), and is confirmed in
the present study, to cause an only moderate underglycosylation of
proteins in vivo and a small decrease in OST activity
measured in vitro. In the case of disruption of
OST6 the underglycosylation defect is even less. However, an
ost3
ost6 double mutation leads to a synthetic
phenotype with a severe underglycosylation, both in vivo and
in vitro (Fig. 2; Table I), as is the case for ts mutations of essential OST genes (27, 28, 30). Surprisingly, however,
growth is not impaired at temperatures up to 37 °C. Overexpression of Ost6p not only complements the
ost6 knockout, but also
rescues the
ost3 underglycosylation effect. Although we
have not performed the reverse experiment (due to the very mild
ost6 defect), it seems that both genes have in part a
redundant function and are able to partially replace each other. On the
other hand, they also reveal specific effects (cf. Fig. 6).
We observed that upon stressing the cells by compounds interfering with
cell wall biogenesis, like Calcofluor White, caffeine, or SDS, an
ost6 mutant behaves differently compared with an
ost3. Even though no distinct target reaction can be
given for these findings, they may eventually help to unravel the
complexity of formation and function of N-linked saccharide chains.
Previous biochemical investigations of the yeast OST suggested that the
enzyme consists of four (Ost1p, Wbp1p, Ost3p, Swp1p) (21, 23), five
(68), or six subunits (Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p, Ost5p) (22).
Genetic screens have identified in addition STT3 (35) and
OST4 (34). In particular the lack of the essential and
highly conserved Stt3p in all OST preparations was puzzling, and it was
hypothesized that Stt3p could be a substoichiometric assembly factor
(35). We have shown now by more sensitive probing with antibodies that
the "tetrameric," enzymatically active complex isolated by us (21)
also contains Stt3p, Ost2p, Ost5p, and the newly discovered Ost6p. The
present study, and also recent experiments employing affinity
purification of tagged Stt3 protein, or analysis of
co-immunoprecipitates of in vivo radiolabeled subunits
clearly identify Stt3p (36, 37) and also Ost4p (37) as components of
the complex. Moreover, an estimation of the amount of radioactivity incorporated into the subunits of the co-immunoprecipitates (37) is
compatible with the notion that the eight subunits identified therein
(Stt3p, Ost1p, Wbp1p, Ost3p, Swp1p, Ost2p, Ost5p, and Ost4p) may be
present in equimolar amounts. Thus, the underrepresentation of some
subunits in the various complex preparations seems to be due to their
depletion during isolation rather than to a real substoichiometric
participation in the in vivo complex. This interpretation is
also in agreement with our analysis of the native complex composition using the method of blue native electrophoresis. A complex in the range
of ~240 kDa was found, which agrees with a calculated molecular mass
assuming all 9 subunits are present in equimolar amounts. Nevertheless,
the isolation of an active "tetrameric" complex may indicate that
for the actual catalysis of the glycosylation reaction less subunits
are sufficient, and the additional components may be essential or
important only for in vivo function.
OST3 and OST6 are not essential for OST activity,
but their simultaneous lack drastically decreases glycosylation
in vitro and in vivo. Therefore, one could
envisage that the products of these genes are needed for optimal OST
activity either directly by interacting with and regulating the
catalytic subunit, or indirectly by affecting the assembly of an
optimal complex, or being involved in proper positioning the OST to the
polypeptide at the translocation site or to the site of formation of
the lipid-linked oligosaccharide precursor. Analysis of the subunit
composition of the affinity-purified complex in the
ost3
ost6 double mutant by Western analysis clearly indicates a severe defect in the structural organization of the complex
(Fig. 7).
Studies demonstrating genetic interactions among different OST genes,
either by using a high copy number suppression approach or by
constructing double mutants with a synthetic phenotype, have led to the
suggestion that OST subunits can be sorted into three groups: I,
Ost1p-Ost5p; II, Wbp1p-Swp1p-Ost2p; and III, Stt3p-Ost4p-Ost3p (15, 36,
37). The structural similarity of Ost3p and Ost6p, their synthetic
interaction, as well as the suppression of
ost3 by
OST6 overexpression suggests a grouping of Ost6p to (III).
Complementary biochemical evidence indicates a direct physical
interaction between Wbp1p and Swp1p (29) and Ost2p,2 as well as between
Stt3p, Ost3p, and Ost4p (37), supporting the idea that these groups may
represent discrete subcomplexes. In this context, it is interesting to
note that upon simultaneous disruption of the OST3 and
OST6 genes, Ost5p and Ost1p (subcomplex I) and Stt3p
(subcomplex III) are decreased, whereas Wbp1p, Swp1p, and Ost2p
(subcomplex II) are not affected in their amount (Fig. 7). Since
glycosylation still occurs in the
ost3
ost6 strain, albeit reduced, one could speculate that Wbp1p, Swp1p, and Ost2p represent the catalytic core unit of the complex.
At present it is not clear, however, whether the proposed subcomplexes
comprise intermediates in the assembly of a fully functional complex,
or whether they are autonomous in vivo pools that may assemble into OST complexes with slightly different composition and
function. Recent findings indicate the occurrence of multiple pathways
of protein translocation into the ER (69-72), one dependent on the
signal-recognition particle and the other independent. The identified
translocation complexes share in part subunit components. The fact that
no distinct OST subcomplexes can be detected in wild-type cells by blue
native polyacrylamide gel electrophoresis makes it less likely that
OSTs of different composition are associated with the respective
translocation machineries. In agreement with this idea is also our
observation that in the
ost3
ost6 mutant proteins are
underglycosylated irrespective whether they use the SRP-dependent (e.g. invertase, DPAP B) or the
SRP-independent (e.g. CPY) sorting pathway. Nevertheless,
this view does not exclude specific interactions of particular OST
subunits and/or not yet identified auxiliary proteins with the
different targeting pathways.
The combination of biochemistry and powerful yeast genetic methods has
advanced tremendously our knowledge of the N-glycosylation of proteins. Now that the composition of one of the most complex enzymes in nature as well as some of the interactions between its nine
subunits has been defined, further work needs to concentrate on a
number of intriguing issues. These include the specific functions of
the various subunits of the complex, the regulation of the enzyme and
the coupling of OST to protein translocation and protein folding.
Finally, the results obtained in yeast may also lead to the isolation
of the homologous mammalian proteins, not yet identified in respective complexes.