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INTRODUCTION |
Genetic and biochemical studies have helped to elucidate major
concepts of cell biology for targeting of proteins into and across
membranes. Typical secreted proteins usually contain amino-terminal signal sequences that ensure their targeting to the translocation machinery. These sequences, called signal peptides, are recognized upon
their emergence from ribosomes by a cytosolic ribonucleoprotein factor,
the signal recognition particle (SRP),1 which in mammalian
cells is composed of one molecule of SRP
RNA and six polypeptides (SRP19p, -54p, -68p, -72p, -9p, and -14p). The
resulting complex is then targeted to the endoplasmic reticulum (ER)
membrane by binding to an integral protein complex called the SRP
receptor. Subsequently the SRP releases the signal sequence to the
translocation channel (1, 2). Several genes involved in protein
translocation into the ER lumen in Saccharomyces cerevisiae have been identified by genetic analysis (SEC61,
SEC62, SEC63, SSS1, SEC71,
and SEC72) (3-10). In this yeast, post-translational translocation has been reproduced in vitro with
reconstituted proteoliposomes containing a heptameric complex
consisting of the trimeric Sec61pC complex (Sec61p, Sss1p, and Sbh1p)
and the tetrameric Sec63pC complex (Sec62p, Sec63p, Sec71p, and Sec72p) (11). Mutational tests have shown that neither SEC71 nor
SEC72 is essential for cell viability. However, cells
lacking Sec71p lack simultaneously Sec72p, grow slowly at elevated
temperature, and depletion of Sec72p leads to the accumulation of
precursor polypeptides (10, 12). The SSS1 gene was found in
genetic screen as a suppressor of a temperature-sensitive sec61
mutation (7), and the Sss1p protein can be co-immunoprecipitated in complex with Sec61p and Sbh1p (11, 12). Sbh1p has never been found in
genetic screens but has been identified during purification of the
Sec61p complex. Sec61p, Sbh1p, and Sss1p are related to the Sec61
,
Sec61
, and Sec61
subunits of the mammalian Sec61p complex,
respectively. The identification of the homologues of SEC61
and SBH1 genes, SSH1 and SBH2,
respectively, in S. cerevisiae have been reported recently.
In contrast to Sec61p and Sss1p, Ssh1p, is not essential for cell
viability but is required for fast growth. Deletion of one of the two
genes SBH1 or SBH2 is not lethal, but deletion of
both genes diminishes the cell growth at elevated temperature and
induces defects in the translocation of Kar2p and of
-factor
precursors (12). With these new components two major trimeric
complexes, Sec61pC and Ssh1pC, are now known. Sss1p is common to both
complexes. The Sec61pC has been shown to be associated with Sec63pC to
form a heptameric complex (11) and thus could be involved either in
post- or co-translational translocation pathways. In contrast, no
interaction of Ssh1pC with Sec63pC has been detected, suggesting that
this second trimeric complex is involved exclusively in
co-translational translocation (13).
In the yeast Yarrowia lipolytica, a thermosensitive mutation
scr2.II-13 was identified in SCR2, one of two
genes encoding the 7 S RNA of SRP (14), and was shown to decrease the
stability of the SRP. Genetic screening for suppressor mutations
identified, among others, the tsr1-1 mutation in the
TSR1 gene (15, 16). This mutation was shown to restore the
stability of an SRP crippled by the scr2.II-13 mutation and
to stabilize its binding to the ribosome at or near the translocation
site (15, 16). The TSR1 gene codes for a serine-rich protein
of 50 kDa spanning the ER membrane. Genetic and biochemical studies
implicated Tsr1p in the stability of SRP in the early steps of the
SRP-dependent translocation pathway of secretory proteins.
Cross-linking studies revealed that Tsr1p interacted with the
ribosome-SRP complex and with BiP (16). Following the sequencing of the
S. cerevisiae genome, we identified four homologues of
TSR1 in this unrelated fungal species, and we suggest here
that they define a new gene family to be called the TSR1
gene family. To assess whether the closest of the TSR1
homologues YHC8 is also critically involved in protein translocation, we constructed a null mutant allele of this gene and
examined the effect of its loss of function in S. cerevisiae.
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EXPERIMENTAL PROCEDURES |
Strains and Plasmids--
Strains and plasmids used in this
study are listed in Table I.
Media--
YPG medium contained 1% yeast extract, 1%
Bacto-Peptone, and 2% glucose. YPinv medium contained 1% yeast
extract, 1% Bacto-Peptone, and 0.1% glucose. The selective minimal
medium contained 1% glucose, 0.17% yeast nitrogen base without
ammonium sulfate (Difco), and 0.1% proline as nitrogen source and was
supplemented with appropriate nutrients.
DNA Techniques--
All enzyme reactions and DNA preparations
were performed as described by Maniatis et al. (17).
Oligonucleotides used for PCR disruption of YHC8 gene were
as follows: oligonucleotide 1, CCC CGG CGC GCC CCC CAT CGA
ACG GTT GCT ACT G; oligonucleotide 2, GCA TAT AAC GCT ACA TAC TAG
CC; oligonucleotide 3, TCC AGG AGG GTT CTG CG; oligonucleotide 4, GGG GGG CGC GCC GGG GTT ATA GAC GGT GAC TCT TAT G. Bold
characters indicate the sticky end 16-base extension used to construct
the AscI site (18).
PCR reactions were developed as described by Maftahi and colleagues
(18). The first PCR reactions utilized primers 1 and 2 or 3 and 4. A
second PCR using the products of the first PCR as a template was
performed using oligonucleotides 2 and 3. The 1-kilobase pair amplified
fragment was tested for the presence of the newly synthesized
AscI site and blunt-ended using the T4 DNA polymerase. The
plasmid pSC10 was obtained by integration of the blunt-ended fragment
obtained from the second PCR at the PvuII site of pINA-KAN
(18).
Protein Immunoblotting--
Yeast cultures were grown overnight
to early log phase and 2 A600 × ml were
collected. The cells were washed in 10 mM NaN3 and resuspended in 100 µl of 2× SDS-polyacrylamide gel
electrophoresis sample buffer containing 1 mM
phenylmethylsulfonyl fluoride. Cells were lysed by vortexing with glass
beads (0.5 mm diameter), heated for 10 min at 95 °C, separated on
10% SDS-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose filters. Preincubation, antibody incubations, and washes
were conducted in TBST buffer (10 mM Tris-Cl, pH 8, 150 mM sodium chloride, 0.05% Tween 20) and 5% of skim milk. A chemiluminescence kit (ECL, Amersham Pharmacia Biotech, France) followed by autoradiography was used to detect the protein of interest.
Immunofluoresence Experiment--
Yhc8 open reading frame was
amplified using two oligonucleotides carrying flanking BamHI
and NotI restriction sites (CGG GAT CCG CAA AAA CGC ATG CAG
ACG and CCC GCG GCC GCC GTT CAT TAG, respectively) and cloned into
pYEF2 designed by C. Cullin (19). This construction put yhc8
under the control of the GAL10 promoter with the HA tag at
the carboxyl-terminal end. In order to exchange the HA tag for the
protein A tag, a NotI-Bsu36I fragment was
amplified from the pE9 plasmid and inserted in the corresponding
NotI-Bsu36I. Expression of the tagged Yhc8p under
the control of GAL1 promoter was monitored by growing cells
to A600 = 1 in synthetic media containing 2%
raffinose. Galactose was added to a final concentration of 2%. Cells
were collected at 0, 30, 60, and 90 min (20). Fixation and antibody
decoration procedures were adapted from Pringle and collaborators (21).
The primary antibody was an anti-protein A raised in rabbit (Sigma
reference number P-3775) used at a dilution 1:300 and the fluorescent
secondary antibody was a Cy3-conjugated anti-rabbit IgG from donkey
(reference number 711-165-152, Jackson ImmunoResearch, West Grove, PA)
at a dilution 1:300. Observations were performed on a Leitz Laborlux S microscope.
Transformation of S. cerevisiae--
Yeast cells were
transformed with linear DNA fragments using the lithium acetate method
(22). Cells were grown at 30 °C in YPD for 4-5 h and spread on YPD
plates containing 200 mg/liter G418. Resistant clones were verified by
Southern or PCR analyses for disruption of the YHC8 locus.
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RESULTS |
TSR1 Gene Family--
We have previously described the in
vivo evidence for the role of the ER membrane protein, Tsr1p, in
the translocation pathway of the yeast Y. lipolytica (15). A
first homology search had identified two homologues, YHC8
from S. cerevisiae and YLU2 from Hansenula
polymorpha (23) of the TSR1 gene. A new search through the entire S. cerevisiae genome sequence data base led to
the identification, in addition to YHC8, of three other
homologues called Hre556, Scynl283, and
UNF378. All of these genes encode putative proteins which,
like Tsr1p and Ylu2p, contain an amino-terminal signal sequence and
share a highly conserved distribution of 5 domains as follows:
cysteine-rich (Cys-rich), serine/threonine-rich (Ser/Thr-rich),
intermediary, transmembrane, and cytoplasmic (Fig. 1, A and B) (15).
The topology of this last domain was previously established for Tsr1p
(16). Fig. 1 summarizes the features of this gene family that we called
the TSR1 family. In addition to the structural conservation (Fig.
1A), the similarity of the sequences of the four predicted
proteins with Tsr1p increases significantly toward the
NH2-terminal cysteine-rich domain (results not shown). Comparison of the putative cytosolic domain of the six proteins shows
high conservation between Scynl283p and Hre556p, and Tsr1p and Yhc8p
(Fig. 1, C and D). Based on the fact that the
Tsr1p was more similar to Yhc8p than to any of the other homologues (Fig. 1, C and D), we focused our study on the
YHC8 gene of S. cerevisiae, and we tested its
possible involvement in the early steps of the secretory pathway.

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Fig. 1.
The TSR1 gene family.
A, structural organization of the five putative homologous
proteins encoded by the genes of the TSR1 gene family. The
percentages of identity between Tsr1p and the other five proteins of
the TSR1 gene family for the cysteine-rich,
serine/threonine-rich, and cytoplasmic domains are indicated.
Tm, transmembrane; Cyt, cytoplasmic;
S.c., S. cerevisiae; H.p., H. polymorpha; Y.l., Y. lipolytica;
aa, amino acid. Tsr1p (15), Yhc8p (SwissProt accession
number, P38739), Scynl238p (EMBL accession number, Z71559), Hre556 (PIR
accession number, S51892), Ylu2p (23) and Unf378p
(GenBankTM accession number, U39481). B,
comparison of the pattern of hydrophobicity of the different members of
the TSR1 gene family. C, a tree of sequence
similarities showing the Tsr1p with its homologues. The tree was
generated using the Pileup program from GCG. D, alignment of
the cytosolic domains of the five homologous proteins using the PILEUP
program from the GCG software package with the scoring matrix of Risler
et al. (40). Identities between Scynl238p, Hre556p, Ylu2p,
Tsr1p, and Yhc8p are presented in bold.
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The nucleotide sequence of the YHC8 gene revealed an open
reading frame of 1815 base pairs. RNA hybridization experiments confirmed that this gene was expressed and produced a single transcript of approximately 2 kilobase pairs (data not shown). The YHC8
gene is predicted to encode a protein of 605 amino acids residues with a molecular mass of approximately 64 kDa. This protein shows an amino-terminal signal peptide and a predicted signal cleavage site
between amino acids 26 and 27 (Fig. 1, A and B).
The last cytoplasmic domain (170 amino acids) is strongly conserved
between Yhc8p and Tsr1p and was predicted to play an important role in Tsr1p interaction with SRP and/or ribosomes (16).
To assess the YHC8 null phenotype, a sticky-end polymerase
(SEP) disruption strategy was adopted (18). The promoter and terminator
regions of the YHC8 gene were amplified separately and
served to construct the disrupting plasmid pSC10 carrying the kanamycin
resistance cassette. The linearized plasmid was then targeted for
integration into the YHC8 genomic locus of the diploid
strain FY1679, and Kanr clones were selected. Disruption of
one of the YHC8 alleles was confirmed by Southern blot on
several transformants (results not shown). One of the heterozygous
disrupted diploids was sporulated, and tetrads were dissected (Fig.
2A). Each of the 20 dissected tetrads yielded four viable spores, two of which were Kanr
implying that they were disrupted for YHC8, which was
confirmed by PCR (data not shown). We tested the growth of
yhc8 at 28 and 38 °C and compared it with the wild
type and with the sec61-3-thermosensitive mutant. The growth of
yhc8 was slightly affected compared with the wild type
strain but not blocked as is the case for Sec61-3 at 38 °C (Fig.
2B). This result shows that the YHC8 gene is not essential in S. cerevisiae under standard laboratory
conditions.

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Fig. 2.
A,
Yhc8::kanr disrupted strains are
viable. Tetrad dissection of the parental diploid strain FY1679
(lane 1) and six isolated
YHC8+/yhc8::Kanr-disrupted
diploids (lanes 2-7) was developed. The four spores were
isolated and grown on YPD or YPD supplemented with 200 µg/ml
geniticin (G418). B, mild defect of
yhc8::Kanr strain at 38 °C.
Wild type, sec61-3, yhc8::kanr or a
sec61-3+yhc8::kanr strains were tested
for growth of YPD at 28 and 38 °C.
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Deletion of YHC8 Induces Defects in the Translocation of Soluble
Secretory Proteins--
To determine whether YHC8 is
important for protein translocation, we examined the fate of several
well characterized secreted proteins in a
yhc8::Kanr strain by immunoblotting of
whole cell extracts.
Carboxypeptidase Y (CPY) is a vacuolar protease that is synthesized as
a 59-kDa inactive precursor (prepro-CPY). Removal of its amino-terminal
signal sequence in the ER lumen gives rise to the 57-kDa pro-CPY. ER
glycosylation of pro-CPY yields the 67-kDa p1 form (24), which is then
transported to the Golgi apparatus where it is glycosylated to generate
the 69-kDa p2 form. Upon arrival in the vacuole, an amino-terminal
sequence of approximately 8 kDa is removed from the p2 form, yielding
the 61-kDa mature CPY, active form (m-CPY). This maturation of p2
requires the PEP4 gene product (Ref. 25; see Fig.
3A). Fig.
3A shows the fate of CPY in
yhc8, wild type (SEC+), and sec
mutant cells. To discriminate between the prepro and m-CPY forms all
the strains used in the CPY immunoblotting experiments carried the
pep4-3 mutation so that no maturation of the p2 could occur
in the vacuole. In addition, we used the ER-to-Golgi blocked sec18-1 mutant (Fig. 3A, 6th lane) to
detect the ER p1 form. Results in Fig. 3 show that for sec61,
-62, and -63 mutants primarily prepro-CPY and p2 forms
were detected and for
yhc8 mutants primarily prepro-CPY
and pro-CPY forms were detected.

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Fig. 3.
A, translocation of carboxypeptidase Y
in a yhc8-deleted strain. Yeast whole cell extracts were prepared
from yhc8::Kanr and wild type strains
growing under permissive conditions and from sec mutant
cells after a 2-h shift to 38 °C. The extracts were electrophoresed
through 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and
probed with anti-CPY serum. Bound antibodies were visualized by
enhanced chemiluminescence (Amersham Pharmacia Biotech). B,
translocation of invertase in yhc8-deleted strain. Cells were grown
on YPD until A600 0.2 and then transferred to
YPinv medium. Yeast whole cell extracts were prepared from
yhc8::kanr and wild type strains
growing under permissive conditions and from sec mutant
cells after a shift to restrictive conditions. The extracts were
electrophoresed through 7.5% SDS-polyacrylamide gel, blotted to
nitrocellulose, and probed with anti-invertase serum. Bound antibodies
were visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech). C, translocation of -factor in yhc8-deleted strain. Cells were grown on YPD
until A600 0.2. Yeast whole cell extracts were
prepared from yhc8::kanr and wild type
strains growing under permissive conditions and from sec
mutant cells after a shift to restrictive conditions. The extracts were
electrophoresed through 12.5% SDS-polyacrylamide gel, blotted to
nitrocellulose, and probed with anti- -factor serum. Bound antibodies
were visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech). D, translocation of dipeptidyl-aminopeptidase B in
yhc8-deleted strain. Yeast whole cell extracts were
prepared from yhc8::kanr growing under
permissive conditions and from sec mutant cells after a
shift to restrictive conditions. The extracts were electrophoresed
through 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and
probed with anti-DPAP B serum. Bound antibodies were visualized by
enhanced chemiluminescence (Amersham Pharmacia Biotech).
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Fig. 4.
Yhc8p is an ER membrane protein.
Localization of Yhc8p by cells expressing a tagged fusion of
YHC8 with a protein A tag. Cells were grown overnight on
complete medium containing 2% raffinose and then transferred to a
pre-warmed complete medium containing 1% raffinose and 2% galactose.
Samples were taken at 0, 30, and 60 min after shift. The cells were
fixed with 5% formaldehyde and, after processing with anti-protein A
antibodies, were decorated with Cy3-conjugate anti-rabbit antibodies.
Nuclear DNA was visualized with 4,6-diamidino-2-phenylindole
(dapi).
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The secretory invertase is synthesized as a precursor of 61 kDa with a
19-residue hydrophobic signal sequence (absent from the cytosolic form)
which ensures its targeting to the ER membrane. Upon translocation to
the ER, this precursor undergoes signal peptide cleavage and core
glycosylation (26, 27). Transported to the Golgi apparatus, it is
subjected to further mannosylations before reaching its periplasmic
location (28). To test the effect of the absence of the YHC8
product on the translocation and transit of the invertase precursors,
we compared the secretory phenotype of the
yhc8 mutant
with that of wild type and sec mutant cells (Fig.
3B). In the wild type cells primarily highly glycosylated forms were detected (5th lane). For sec mutants
primarily pre-invertase and the highly glycosylated forms
(1st to 3rd lanes) were detected;
yhc8 mutant cells, however, accumulated preinvertase
(4th lane), and only a small level of highly
glycosylated forms can be detected.
The
-factor mating pheromone is a 13-amino acid peptide that is
secreted into the culture medium by MAT
cells (29). It is
synthesized as a precursor polypeptide of 21 kDa (pp-
F) that contains a prepro-leader sequence of 83 amino acids. Cleavage of the
signal sequence after translocation into the ER gives rise to the
pro-
-factor. This is then decorated with three core oligosaccharides during its translocation across the ER membrane, yielding a 26-kDa ER
form (30-32). Directed to the Golgi apparatus, this form undergoes outer chain glycosylation and proteolytic maturation (33). The processing is then completed within the secretory vesicles by Kex2p and
dipeptidyl-aminopeptidase A (DPAP A) (34). For
yhc8 and
like sec mutants (Fig. 3C, 1st to 3rd
lanes) the cells accumulate the prepro-
-factor (4th
lane), which indicates a significant defect in the
translocation of this molecule into the ER.
Deletion of YHC8 Induces Defects in the Assembly of Membrane
Proteins--
To test the effect of YHC8 deletion on the
insertion of membrane proteins into the ER membrane, we used the
dipeptidyl-aminopeptidase B (DPAP B) as a reporter protein and compared
its kinetics of insertion in
yhc8 and sec
mutant strains. DPAP B is an integral membrane glycoprotein with a
carboxyl-terminal domain localized in the lumen of the ER (35). The
unglycosylated pre-DPAP B can be observed at 96 kDa, and the mature
vacuolar form migrates as a 120-kDa species. Contrary to the wild type
or sec61-2 mutant strains where no accumulation or only a
small accumulation of pre-DPAP B was detected (Fig. 3D, 1st
and 5th lanes) (4), immunoblotting from
yhc8
showed accumulation of pre-DPAP B (4th lane).
However, the amount is not as great as that observed in
sec62-1 and sec63-1 mutants (2nd and
3rd lanes). These results clearly demonstrate a
partial defect in the assembly of this integral membrane protein in the
yhc8 null mutant cells.
Yhc8p Is an ER Membrane Protein--
In order to localize Yhc8p
inside the cell, it was tagged at its carboxyl-terminal end using the
vectors developed by Cullin and Minvielle-Sebastia (19) which fused the
HA tag and placed the open reading frame under the control of the
GAL1 promoter. When grown on 2% galactose as carbon source,
cells decorated with anti-HA antibodies displayed a strong accumulation
of fluorescent material in an intracellular organelle away from the
nucleus (data not shown) which was probably the vacuole. This
localization could result from a mistargeting due to the HA tag and/or
be a consequence of its overexpression in conditions of GAL1
induction. The HA tag was exchanged for a protein A tag, and the
induction conditions were changed. The cells were grown on 2%
raffinose, a non-repressible carbon source (20). At time
t = 0, the cells were transferred into a pre-warmed
medium containing 2% galactose, and aliquots were fixed and decorated
at 0, 30, and 60 min. Under these conditions, only few cells were
decorated by the anti-protein A antibodies, and this proportion did not
increase with the incubation time on galactose. Results in Fig. 4 show
that the cells were primarily labeled at the periphery of the nucleus
and the plasma membrane. This pattern is characteristic of the
endoplasmic reticulum location. At 60 min, the cells appeared to be
more heavily decorated, and the labeling around the nucleus appeared to
be more diffuse. We concluded that Yhc8p was first directed to the
endoplasmic reticulum membrane as suggested by the structural features
of its sequence. Upon its accumulation in this compartment, the
overproduced polypeptides were then transferred to the vacuolar compartment.
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DISCUSSION |
We have identified four coding sequences, YHC8,
Hre556, Scynl283, and UNF378, in the
genome of S. cerevisiae, as homologues (43, 33.5, 34.5, and
32.5% amino acid sequence identity, respectively) of the
TSR1 gene of Y. lipolytica. Our study on Tsr1p
suggested that it is localized in the ER membrane and is an important
component of the SRP-dependent translocation pathway (15,
16). The proteins encoded by these TSR1 homologues share
high homology in both the amino-terminal and cytosolic domains; these
two domains were demonstrated to be involved in the interaction of
Tsr1p with BiP and with the SRP-ribosome complex, respectively (16). We
called this new family of genes, TSR1 gene family. Homology
of the members of this family with Tsr1p and mutational test on
YHC8 gene suggest that they may be involved in the
SRP-dependent translocation pathway.
Here we focused on YHC8 because its putative product, Yhc8p,
was most closely matched with Tsr1p. By using immunofluorescence experiments, we showed that Yhc8p is localized in the ER. The presence
of an amino-terminal signal sequence and of a membrane-spanning domain
suggested that Yhc8p, like its homologue Tsr1p, is a component of the
ER membrane. We have demonstrated that deletion of one member of this
family, YHC8 gene, although without effect on viability, induces large defects in the translocation of secretory soluble proteins, resulting in the accumulation of preinvertase, pre-CPY, prepro-
-factor. Only a slight defect was observed on the
translocation of pre-DPAP B.
Previous studies have shown that mutations in sec61,
sec62, and sec63 lead to a large accumulation of
precursors of several secretory and soluble vacuolar proteins, such as
-factor precursor, CPY, and acid phosphatase (5, 36) (see Fig. 3,
A and C, 1st to 3rd lanes).
However, these mutations have only marginal defects on the insertion of
the integral membrane protein dipeptidyl-aminopeptidase B (DPAP B) (4)
(see Figs. 3D, 1st to 3rd lanes).
Other genetic screenings permitted identification of new mutants in the
same genes that were defective in the insertion of integral membrane proteins (4). More recently, Pilon and colleagues (37) have characterized strains of S. cerevisiae expressing
cold-sensitive alleles of SEC61 and show that these mutants
exhibit a large cytoplasmic accumulation of co- and
post-translationally translocated precursors. All together these data
pointed to a model where Sec61p acts as the core of the translocon,
controlling both the docking step onto the receptor site and
insertion/translocation, whereas Sec63p and Sec62p were implicated
specifically in the SRP-independent translocation pathway (1, 4,
11).
Our results are consistent with those obtained for the
sec61, sec62, and sec63 mutants where
the level of accumulation of precursors was dependent on the allele
involved and the reporter protein used (4) (see Fig. 3A,
1st lane, and Fig. 3C, 1st
lane). Only a small accumulation of ER forms was detected in
the cases of invertase, CPY, and
-factor compared with that observed
in sec18-1 mutant. The sec18-1 mutant has been
isolated as a thermosensitive mutant that exhibits a block in protein
transport from the ER to the Golgi apparatus (38, 39), resulting from
impaired targeting of the vesicles to an early Golgi compartment (40).
The results obtained with
yhc8 suggest that Yhc8p
controls primarily the translocation step and has only little effect on
ER glycosylation. This could explain why the translocation defect and
the accumulation of ER intermediates were more or less pronounced and
dependent on the reporter protein used.
Our data show that the secretory defect in
yhc8 mutant is
pleiotropic. Why is the null phenotype of the YHC8 gene not
lethal? The fact that four homologues of the TSR1 gene have
been identified in this yeast suggests that the products of these
remaining three genes cooperate to allow partial suppression of the
yhc8 null. Our results with the
yhc8 mutant
are reminiscent of those with SEC71 and SEC72
mutants, which show pleiotropic defects in protein trafficking across
the ER membrane but do not affect cell viability (10, 12). The Tsr1p
gene product which was studied in more detail in Y. lipolytica was shown to interact with the SRP-ribosome complex on
the cytoplasmic side and with BiP in the ER lumen in the predominant
SRP-dependent translocation pathway in this yeast (16). If
Yhc8p is involved in the same process, it is difficult to understand
why the prepro-
-factor and CPY, which were determined to be
post-translationally translocated (41), are affected in the
yhc8 mutant cells. One explanation is that the loss of
Yhc8p could induce a large decrease in the number of sites accessible for post-translational translocation. The fact that Tsr1p interacts with the SRP ribosome complex and deletion of YHC8 gene
induces a large translocation defect suggests that Yhc8p may be an
intermediary between the docking site on the SRP-receptor and the SEC61
complex, allowing it to play a general role in co- and
post-translational translocation.