Laboratoire de Génétique moléculaire et cellulaire, INRA, CNRS, Institut National Agronomique Paris-Grignon, 78850 Thiverval-Grignon, France
* Author for correspondence (e-mail: boisrame{at}grignon.inra.fr)
Accepted 24 September 2002
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Summary |
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Key words: Translocation, Quality control, Sec61 ß
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Introduction |
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The translocation sites that allow transport of hydrophilic proteins across
a hydrophobic membrane are aqueous channels
(Simon and Blobel, 1991),
formed by the oligomerization of a trimeric complex, the Sec61 complex
(Hanein et al., 1996
). The
first subunit of this complex, Sec61
, was initially identified in
S. cerevisiae as Sec61p (Deshaies
and Schekman, 1987
); it was later discovered in mammalian cells
too and displays strong homology with the Escherichia coli SecY
protein (Görlich et al.,
1992
). The integral Sec61
protein contains several
transmembrane domains that were found in proximity to the nascent chains
during their transfer and were shown to contribute to the hydrophilic
environment reported in translocation pores
(Mothes et al., 1994
).
Sec61ß and Sec61
were co-purified in complex with the Sec61
polypeptide in mammals (Görlich and
Rapoport, 1993
). In S. cerevisiae, the
subunit,
Sss1p, was isolated as a suppressor of the sec61-2
temperature-sensitive mutation (Esnault et
al., 1993
). This single transmembrane domain protein is related to
the SecE subunit of E. coli translocase
(Hartmann et al., 1994
).
Although both polypeptides are encoded by essential genes in the yeast, the
third one, Sbh1p (Sec61ß homolog)
(Panzner et al., 1995
), does
not display an essential function (Finke
et al., 1996
). A second trimeric complex was identified in S.
cerevisiae comprising Ssh1p (a Sec61p
homolog) and Sbh2p (another Sec61ß homolog) proteins together
with Sss1p. In vitro studies indicate that this second Sec61 complex was
specialized in the co-translational translocation pathway
(Finke et al., 1996
). Yeast
Sss1p and mammalian Sec61
proteins are highly conserved and were shown
to be functionally interchangeable
(Esnault et al., 1993
). By
contrast, Sbh1p and Sec61ß show poor homology and are not related to the
third component of E. coli translocation apparatus, SecG. Until now,
no precise function has been attributed to the ß subunit of the Sec61
complex in either yeast or higher eukaryotic cells.
We addressed the function of Sec61 in the yeast Yarrowia
lipolytica. The SEC61 gene had been previously cloned by
reverse genetics (Broughton et al.,
1997) and was shown to complement a null mutation in the S.
cerevisiae SEC61 gene. Antibodies recognizing this translocon component
gave us access to the ribosome-associated membrane protein (RAMP) fraction in
Y. lipolytica and provided biochemical evidence for the predominance
of the co-translational mode of translocation in this yeast
(Boisramé et al., 1998
).
In order to gain insights into the role of the Sec61ß subunit, we decided
to clone and to characterize it. No secretory defect was associated with a
SBH1 gene interruption in Yarrowia, and we obtained for the
first time evidence for an association of Sbh1p with the ER-membrane chaperone
protein calnexin, thus linking this translocon component with folding and/or
quality control of secretory proteins.
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Materials and Methods |
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PINA1326 corresponds to the pFL61 SBH1-complementing vector
(Swennen et al., 1997) cloned
in the S. cerevisiae
sbh1,
sbh2 strain. pINA1328 is a
derivative of the Y. lipolytica integrative URA3 plasmid
pINA300' that contains the 325 base pair SBH1 cDNA amplified
using the two primers: sbh1-1 and sbh1-2
(Table 1) and cloned at the
NcoI and SphI sites.
|
To create the inactivated copy of SBH1, an upstream fragment corresponding to nucleotides 62 to 242 of the SBH1 gene and a downstream fragment from nucleotides 401 to 822 were amplified separately on wild-type genomic DNA using, respectively, the sec61ß-62/sec61ß-R242 and sec61ß-401/sec61ß-R822 primer couples. Sec61ß-62 and sec61ß-R822 contain a 16-bases 5' extension corresponding to a restriction site for AscI; sec61ß-R242 and sec61ß-401 contain, respectively, a EcoRI and BamHI restriction site (Table 1). A second amplification was performed using 50 ng of each purified fragment as template and primers sec61ß-R242 and sec61ß-401. The 600 base pair amplified fragment was digested with EcoRI and BamHI and cloned in pINA300', restricted with the same enzymes. The recombinant plasmid, pINA1325, was linearized using the AscI enzyme before transformation of the Y. lipolytica wild-type strain.
pINA1330, containing the SBH1-coding sequence fused in frame with
the binding domain of Gal4p in pAS2, was constructed for
screening of the Y. lipolytica two-hybrid library
(James et al., 1996
). The
SBH1 open reading frame was excised from pINA1328 by a NcoI
and BamHI digestion and ligated into pAS2
cut with the
same enzymes. The recombinant vector was transformed into the S.
cerevisiae PJ694
strain.
A HA-tagged copy of Sbh1p was constructed by insertion of the HA epitope just upstream of the transmembrane helix, between amino acids 60 and 61, by a PCR strategy. For this purpose, an oligonucleotide called sbh1HA (see Table 1) was designed. Two fragments were amplified separately on the SBH1 cDNA using, respectively, the sbh1-1/sbh1-R209 and sbh1HA/sbh1-2 primer couples. After restriction with SalI, they were ligated and an aliquot of the ligation mixture was used as the template for a new amplification with the sbh1-1 and sbh1-2 primers. The final product was then digested with NcoI and SphI and cloned in pINA300' opened with the same enzymes. The recombinant plasmid, pINA1329, was linearized at the StuI site (upstream from the tag epitope) before integration at the SBH1 locus of the Y. lipolytica wild-type strain. The recombination event leads to a tandem of SBH1 sequences: the first contains the HA epitope under the SBH1 gene transcriptional and translational regulatory elements, and the second is devoid of promoter and translation initiation codon. Expression of a tagged Sbh1p protein was confirmed with anti-HA antibodies for three transformants.
pINA1332 and pINA1331 correspond, respectively, to a pAS2
two-hybrid plasmid containing the ScSBH2 coding sequence fused in
frame with the Gal4p-binding domain and a pINA1269 vector expressing the
ScSBH2 open reading frame under the control of the Y.
lipolytica strong hp4d promoter
(Madzak et al., 2000
). The
SBH2 open reading frame was amplified with two primers: scsbh2-1 and
scsbh2-2 (Table 1) that contain
a BamHI for the first one and a KpnI and XhoI
restriction sites for the second. After digestion of the fragment either by
BamHI and XhoI or BamHI and KpnI, the
digested products were, respectively, ligated with the pAS2
vector cut with BamHI and SalI and pINA1269 opened with
BamHI and KpnI. The recombinant plasmid, pINA1331, was
linearized in the LEU2 gene by an ApaI restriction before
transformation of the
sbh1 strain.
Y. lipolytica strains were grown in YPD complete medium (1% yeast extract, 1% bacto-peptone, 1% glucose) or YNB minimal medium (0.17% yeast nitrogen base without ammonium sulfate and without amino acids, 1% glucose, 0.1% proline); supplements were added to a final concentration of 0.01%. Induction of the alkaline extracellular protease was performed using GPP medium (2% glycerol, 0.17% yeast nitrogen base without ammonium sulfate and without amino acids, 0.3% proteose peptone, 50 mM phosphate buffer, pH 6.8).
SDS hypersensitivity tests
Exponential cultures were harvested and adjusted to an optical density of
1. 5 µl of ten-fold serial dilutions were spotted on YPD containing
increasing amounts of SDS. Plates were incubated at 28°C for 48 hours.
Y. lipolytica two-hybrid library screening
The PJ69-4 strain was transformed with the recombinant plasmid
pINA1330. About 2x109 cells grown in rich medium were mixed
with 1.5-3.5x108 cells of each library
(Kabani et al.,
2000
,Kabani et al.,
2000
), which corresponds to a ratio of 10:1 (bait:prey). Cells
were sedimented by centrifugation and resuspended in 4 ml of YPD for each
pool, which were plated on rich medium and incubated overnight at 28°C for
mating. Cells were harvested in 30 ml of YPD for each pool and plated on
minimal medium plus methionine and uracile for selection of Leu+, Trp+, His+
and Ade+ diploids.
Antibodies
Polyclonal anti-HA antibodies from Santa Cruz Biotechnology and anti-c-myc
antibodies from Upstate Biotechnology were used. For calnexin, Kar2p and
Sec62p, a fusion protein with the glutathione S-transferase was expressed in
E. coli, purified on a gluthatione column and used to immunize
rabbits as previously described
(Boisramé et al., 1996).
The CNX1 open reading frame from nucleotide 840 to nucleotide 1471
(see Fig. 4A) was amplified
using the primers Cnx1-1 and Cnx1-2 (Table
1) and restricted with BglII and EcoRI for
cloning at the BamHI and EcoRI sites of pGEX-2T. Anti-Sec62p
antibodies were raised against a fragment corresponding to amino acids 236 to
381.
|
Cell extract preparation and analysis
Membrane-enriched extracts were prepared as follow: cells from 200 ml of an
overnight culture in YPD were lysed in 2 ml of phosphate saline buffer (PBS)
in the presence of anti-protease and glass beads. After a slow centrifugation
at 2000 g, supernatant was further centrifuged at 18,000
g for 30 minutes. The pellet was then solubilized in 1 ml of
PBS plus anti-protease plus Triton X100 2% at room temperature for 20 minutes,
and a solubilized supernatant, corresponding to proteins from membranous
compartments, was obtained after a new 30 minutes centrifugation at 18,000
g. For immunoprecipitation, 200 µl of this sample was
diluted five times in PBS either with anti-HA, anti-c-myc or anti-Sec62
antibodies, and complexes were recovered with protein-A sepharose beads.
Sepharose beads were washed three times with 500 µl of PBS and precipitates
were eluted in 50 µl of sample buffer (100 mM Tris-HCl pH 6.8, 2% 2
ß-mercaptoethanol, 20% glycerol, 4% SDS, 0.02% Bromophenol blue) for 20
minutes at 65°C. Samples were then applied on a 8% polyacrylamide
denaturing gel, and proteins were transferred onto a nitrocellulose membrane
after migration. Anti-calnexin antibodies were used as primary antibodies,
peroxidase-conjugated anti-IgG antibodies as secondary ones, and detection was
realized using the ECL method (Amersham).
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Results |
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The Y. lipolytica SBH1 gene and the Sbh1p protein
In order to subclone the SBH1 gene in a Y. lipolytica
integrative vector, an amplification was performed on genomic DNA using
primers sbh1-1 and sbh1-2 (Table
1). Instead of the expected 325 base pair fragment, a 850 base
pair amplification product was obtained. Sequencing of this DNA confirmed that
this fragment corresponds to the SBH1 gene (accession number
YLI277554) but revealed that the coding sequence contains an intron of 531
base pairs with typical Y. lipolytica intron features
(Bon et al., 2002). The
5' splicing site GTGAGT is located in the 16th codon, and a TACTAAC box
is present one nucleotide upstream from the 3' splicing site TAG.
Attempts to identify a second gene as in S. cerevisiae using
substringent Southern blot conditions failed.
The cDNA cloned by complementation of the temperature-sensitive growth
phenotype of the S. cerevisiae sbh1,
sbh2 strain
encodes a 91 amino-acid long protein. An alignment between this protein and
the two S. cerevisiae homologues shows that the Y.
lipolytica protein is closer to ScSbh2p than to ScSbh1p
(Fig. 1). Indeed, YlSbh1p and
ScSbh2p share 45 identical amino acids, whereas 34 amino acids only are common
to YlSbh1p and ScSbh1p, giving 51 and 41 percent of identity, respectively.
The hydrophobicity profile obtained using the Antheprot editor shows a
potential transmembrane helix between amino acids 65 and 78. The S.
cerevisiae proteins are predicted to be tail-anchored membrane proteins,
having their soluble N-terminal domain in the cytoplasm and their short
C-terminal domain in the lumen of the ER. YlSbh1p could thus adopt a similar
topology with a predicted ER lumenal domain of 13 amino acids. Functional
equivalence of YlSbh1p and ScSbh2p is further documented below.
|
Interruption of the SBH1 coding sequence
Since the promoter and downstream sequences of the Y. lipolytica
SBH1 gene were not cloned, we used the sticky-end polymerase chain method
(Maftahi et al., 1996) to
construct an interrupted copy of SBH1 (see Materials and Methods).
Integration at the SBH1 locus of Yarrowia was confirmed by
Southern blot analysis for three Ura+ transformants among ten. The Y.
lipolytica
sbh1 strain growth phenotype was then tested
at three temperatures and compared to the wild-type parental strain to detect
a temperature-sensitive phenotype. No difference in the growth rates between
the two strains was observed at 18, 28 or 32°C. The only visible phenotype
was the colonial aspect on solid medium: indeed, after a one-week incubation,
sbh1 colonies appeared smooth in contrast to the rough
colonies formed by the wild-type strain
(Fig. 2A). Since absence of
filamentation is usually correlated to a modification in the cell wall
composition (Richard et al.,
2002
), the SDS sensitivity of the null mutant was assayed. As
shown in Fig. 2B, the
interrupted strain is resistant to a SDS concentration of 0.2%, whereas the
wild-type strain is sensitive to 0.125%.
|
Synthesis and secretion of the Y. lipolytica reporter protein, alkaline extracellular protease, were studied in the null mutant and compared to those observed for the wild-type strain. No difference was detected by western blot analysis of culture supernatants, suggesting that the initial step of the secretion pathway was unaffected in the absence of the Sbh1p protein.
Sbh1 strain is impaired in the unfolded protein response
Since Sbh1p and Cnx1p interact (see below), we supposed that Sbh1p plays a
role in the quality control process that allow retention of misfolded proteins
in the ER either to ensure their normal folding or to target them to a
degradation pathway. First, the two Y. lipolytica wild-type and
sbh1 strains were incubated in the presence of 10 µg/ml of
Tunicamycine for three hours. Growth curves were similar for the two strains
and comparable to the untreated cultures. Intracellular proteins were
extracted from cell pellets and levels of the ER chaperone protein, Kar2p, and
its cofactor Sls1p (Kabani et al., 2001;
Travers et al., 2000
) were
estimated by immunoblotting. As shown in
Fig. 3, although the amount of
Kar2p was induced two to three times in the wild-type strain, its level was
unchanged in the sbh1 null mutant. A similar result was obtained for
Sls1p. Such an observation could indicate that the
sbh1 strain
does not accumulate unglycosylated proteins.
|
In order to test this hypothesis, a mutated copy of the gene encoding the
reporter protein, alkaline extracellular protein (AEP), was integrated at the
XPR2 locus of the two strains using pINA317. This copy encodes a
protease with a mutation in its glycosylation site that leads to a partial
intracellular retention of a precursor form devoid of its signal sequence but
containing the unglycosylated pro-region
(Fabre et al., 1991). Two
transformants for each strain were then cultivated in inducing medium for
three days at 28°C, and intracellular and extracellular AEP were detected
by western blot analysis. Although no precursor was revealed in the total
protein extract of the parental strains
(Fig. 4, lanes 1 and 4) and in
the two transformants derived from the
sbh1 strain (lanes 5
and 6), the two others (lanes 2 and 3) accumulated intracellular precursors as
already described. Only the mature form was detected in the supernatant of all
the tested strains (data not shown). This observation is in accordance with an
absence of accumulation of unfolded or misfolded precursors in the ER of the
sbh1 strain.
Screening of the two-hybrid library for partners of Sbh1p
In order to elucidate the Sbh1p function, we chose to look for partners
interacting with this Sec61 complex subunit using the two-hybrid system
(Fields and Songs, 1989). The
PJ69-4
strain, transformed with plasmid pINA1330 encoding the fusion
protein Gal4BD-Sbh1p, was mated with aliquots of the three pools of the
library constructed in pGAD-C1 to C3 plasmids in PJ69-4A
(James et al., 1996
). The
number of diploids obtained was comparable for the three pools (about
15x106 each), and was sufficient to ensure a good
representation of the 4x106 PJ694A clones present in each
pool. Diploid cells were directly plated on minimal medium devoid of leucine,
tryptophane, histidine and adenine and incubated at 30°C. After seven
days, 80, 9 and 35 Ade+, His+ clones were isolated, respectively, for pools 1,
2 and 3. The next day, 78, 23 and 38 new clones were picked, and three days
later, 329, 61 and 183 were retained. The total number of candidates was thus
836. Yeast colonies were purified on minimal medium before amplification of
the inserted genomic fragments by PCR using two primers flanking the cloning
site and sequencing. About two hundred sequences, representative of each
subgroup, were analyzed using the GCG package (University of Wisconsin,
Madison, WI). Redundant and overlapping fragments were only found for one open
reading frame (see Fig. 5A),
which matches calnexins and thus was identified as the Y. lipolytica
CNX1 gene (accession number YLI277589). No other candiate protein was
repeatedly obtained in this screening.
|
The open reading frame and its protein product in Y. lipolytica are presented in Fig. 6A. YlCnx1p displays a potential sequence signal between amino acids 1 and 18, a large lumenal domain that is highly conserved (see Fig. 6B), a potential transmembrane domain lying between amino acids 495 and 524 and a short cytoplasmic domain containing many acidic residues.
|
|
Analysis of the Sbh1p-Cnx1p interaction
In a second step, a deletion analysis was performed to map the interacting
domain in each of the partners. A truncated Gal4BD-Sbh1p protein that
eliminates the transmembrane helix and the 13 terminal amino-acid lumenal
residues was constructed using the SalI restriction site. Expression
of the fusion protein in S. cerevisiae was controlled for by western
blot analysis and was similar to the full-length hybrid protein (data not
shown). Unlike the entire protein, Gal4BD-Sbh1pC was unable to interact
with full-length Gal4AD-Cnx1p in the two-hybrid assay (compare sectors 1 and 3
in Fig. 7), suggesting that the
C-terminal tail of Sbh1p is required for interaction.
|
For calnexin, we knew from the screening of the two-hybrid library that the
interaction domain was located downstream from amino acid 339. Three deletions
were made by digestion: the first one, called SacI, eliminates
amino acids 397 to 576 in the protein; the second one,
SalI,
starts at amino acid 460 and continues to the end; and the third one,
XhoI, fuses in frame amino acid 130 to amino acid 473
(Fig. 5B). None of these
Gal4AD-Cnx1p-deleted proteins was able to reconstitute an active Gal4p
activator when co-expressed with Gal4BD-Sbh1p (data not shown). This indicates
that amino acids 460 to 473, at least are required for the interaction with
Sbh1p, whereas the transmembrane domain and the C-terminal tail of Cnx1p
present in the last deletion are not sufficient. Considering these results,
Sbh1p and Cnx1p are thought to interact through their lumenal domains.
Interaction between Sbh1p and Cnx1p in Y. lipolytica
To validate the Sbh1p-Cnx1p interaction observed in an heterologous
context, we performed a co-immunoprecipitation experiment on Y.
lipolytica protein extracts. A polyclonal serum was obtained against the
Cnx1p protein that recognized a 80 kDa product in a protein extract from a
wild-type strain extract (Fig.
8A, lane 1). Although calnexin is a 582 amino-acid long
polypeptide, such an aberrant migration was already described
(Degen and Williams, 1991). A
solubilized supernatant of a membrane-enriched fraction was prepared from a
Y. lipolytica strain expressing the HA-tagged Sbh1p protein (see
Materials and Methods). The sample was diluted in phosphate buffer with salt
and incubated with anti-HA antibodies in the presence of protein-A sepharose
for 2 hours at 4°C. The immunoprecipitate was further analysed by
SDS-PAGE, blotted with anti-Cnx1p antibodies and compared with crude extracts.
As shown in Fig. 8A lane 2,
calnexin was detected in the anti-HA precipitate when the tagged version of
Sbh1p was present, but no Cnx1p was observed if immunoprecipitation was
performed on a wild-type extract (Fig.
8A, lane 3).
|
In order to show that the Sbh1p-calnexin association detected using this approach is specific, we performed the same experiment for two other ER membrane components: Sec61p and Sec62p. A solubilized fraction of a membrane-enriched extract was prepared from a SEC61-c-myc-tagged strain, and the two proteins were independently immunoprecipitated using anti-c-myc and anti-Sec62p antibodies. Western blot analysis of immunoprecipitates using anti-calnexin antibodies allowed detection of Cnx1p in anti-c-myc precipitate (Fig. 8B, lane 2) but not in anti-Sec62p precipitate (Fig. 8B, lane 3). This result confirms that a pool of calnexin is located closer to the minimal translocation apparatus formed by the Sec61 complex.
ScSbh2p is a functional homologue of YlSbh1p
To determine if ScSbh2p was able to interact with YlCnx1p, as shown for
YlSbh1p, the S. cerevisiae SBH2 coding sequence was cloned into the
pAS2 vector. The S. cerevisiae strain containing the
pGADCNX1 two-hybrid vector was transformed with the recombinant
plasmid, pINA1332. Co-expression of the Gal4BD-ScSbh2p and Gal4AD-Cnx1p fusion
proteins allowed growth on minimal medium devoid of leucine, tryptophane,
histidine and adenine, indicating that the two proteins interact to
reconstitute a functional Gal4p activator (data not shown). The control strain
expressing the Gal4BD-ScSbh2p hybrid protein with the Gal4p-activating domain
alone did not grow on the same medium.
Considering this positive result, we expressed the S. cerevisiae
SBH2 coding sequence in Y. lipolytica under the control of the
hp4d promoter (Madzak et al.,
2000). We confirmed by immunoblotting that the heterologous
protein was detectable in a total protein extract from one of the Leu+
transformants. This transformed strain recovered the ability to form rough
colonies on rich solid medium. We first showed that the Sbh2p protein
co-fractionated with calnexin in a membrane-enriched extract of the Y.
lipolytica recombinant strain (data not shown). An immunoprecipitation
experiment was then performed using a solubilized supernatant of a
membrane-enriched fraction and anti-Sbh2p antibodies. As shown in
Fig. 8C, YlCnx1p was
co-precipitated with ScSbh2p (Fig.
8C, lane 2). The reverse experiment was performed, and ScSbh2p was
revealed in an anti-Cnx1p precipitate (data not shown). These results suggest
that the S. cerevisiae protein behaves similarly to Sbh1p in Y.
lipolytica.
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Discussion |
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More recent studies brought some insights into Sec61ß function. First,
a function was suggested for Sec61ß in a post-targeting step to
facilitate the polypeptide insertion into the translocation pore
(Kalies et al., 1998). Second,
cross-links between a multiple-spanning membrane protein, Sec61ß, and
other partners were observed using a membrane-permeable heterobifunctional
reagent (Laird and High,
1997
), suggesting that the ß subunit favors the exit of
transmembrane domains from the translocation site. Third, two studies revealed
that Sec61ß could be co-immunoprecipitated with secretory proteins that
failed to translocate across or to integrate into the ER membrane and that
were finally targeted to the proteasome for degradation
(Chen et al., 1998
;
Bebök et al., 1998
). This
suggested a possible function of Sec61ß in selecting or escorting the
polypeptide to the cytosol.
Using two complementary approaches, we have shown for the first time that
the ß subunit of the Sec61 complex directly associates with the ER
chaperone, calnexin. The two proteins were described as membrane proteins with
a single membrane-spanning domain: although Sec61ß exposes only a short
C-tail to the lumen of the ER, calnexin displays a large N-terminal lumenal
domain (Kalies et al., 1998).
The Sbh1p protein newly identified in Y. lipolytica shows a better
homology with the Sbh2p component of the yeast S. cerevisiae and
displays 42% identity to the human Sec61ß protein. YlCnx1p is also well
conserved when compared to other calnexin sequences and displays 45% identity
to human calnexin. Since the overall primary structure of the polypeptides was
conserved and a transmembrane domain was predicted for each one, we may assume
topology conservation for these two proteins in Yarrowia.
Deletion analysis of the interacting domain between these two partners
strongly suggests that the association involves regions localized in the ER
lumen. These results could be put together with the work done on the S.
pombe Cnx1p protein that mapped the essential region of the protein to
the terminal 52 amino-acid residues of the lumenal domain
(Jannatipour and Rokeach,
1995; Elagöz et al.,
1999
). This domain was also identified as sufficient for the
formation of a complex including the chaperone protein BiP, and the authors
speculate that the essential function of Cnx1p could reside in its ability to
associate with proteins involved in protein folding
(Elagöz et al., 1999
).
Our results reveal the existence of a new type of interaction for calnexin
that involves the translocation pore.
Calnexin was the unique Sbh1p-interacting protein identified during our
study; this does not exclude the existence of other partner(s) undetectable
using the two-hybrid method. For example, an association of Sec61ß with a
subunit of the signal peptidase (Kalies et
al., 1998) was described using a cross-linking experiment, which
does not imply a direct interaction between the two partners. No interaction
of Sbh1p with the Spc2p subunit of the S. cerevisiae signal peptidase
complex was detected using the two-hybrid assay. Similarly, previous assays to
map the Sec61ß-interacting domain in Sec61
using this method did
not allow us to show any association (A.B., C.M., A.B., J.-M.B. et al.,
unpublished).
Our work and others converge upon the idea that numerous membrane proteins
surround the translocation site: either to facilitate translocation of soluble
proteins or membrane insertion of membrane proteins, like TRAM
(Görlich and Rapoport,
1993), to modify these proteins, such as signal peptidase
(Kalies et al., 1998
) or to
ensure proper folding and/or to target misfolded proteins for proteasomal
degradation, like the membrane-bound chaperone protein calnexin. The role of
Sec61ß in the quality control of secretory proteins could thus consist of
maintaining the chaperone calnexin in the vicinity of the translocation pore.
Such a proximity allows calnexin to interact with some nascent chains as soon
as they emerge in the lumen of the ER. The absence of the docking protein
Sbh1p would lead to an uncoupling of translocation and quality control
process, and misfolded polypeptides would no longer be retained in the ER
compartment as suggested by the preliminary results obtained in the null
strain. This will be further studied in Y. lipolytica.
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