Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica

Anita Boisramé*, Marion Chasles, Anna Babour, Jean-Marie Beckerich and Claude Gaillardin

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


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The core component of the translocation apparatus, Sec61p or {alpha}, was previously cloned in Yarrowia lipolytica. Using anti-Sec61p antibodies, we showed that most of the translocation sites are devoted to co-translational translocation in this yeast, which is similar to the situation in mammalian cells but in contrast to the situation in Saccharomyces cerevisiae, where post-translational translocation is predominant. In order to characterize further the minimal translocation apparatus in Y. lipolytica, the ß Sec61 complex subunit, Sbh1p, was cloned by functional complementation of a {Delta}sbh1, {Delta}sbh2 S. cerevisiae mutant. The secretion of the reporter protein is not impaired in the Y. lipolytica sbh1 inactivated strain. We screened the Y. lipolytica two-hybrid library to look for partners of this translocon component. The ER-membrane chaperone protein, calnexin, was identified as an interacting protein. By a co-immunoprecipitation approach, we confirmed this association in Yarrowia and then showed that the S. cerevisiae Sbh2p protein was a functional homologue of YlSbh1p. The interaction of Sbh1p with calnexin was shown to occur between the lumenal domain of both proteins. These results suggest that the ß subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation.

Key words: Translocation, Quality control, Sec61 ß


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In eukaryotic cells, the protein secretory pathway initiates with transport across the endoplasmic reticulum (ER) membrane. Two major pathways have been described. In the first one, the preprotein is not released in the cytoplasm but is directly transferred into the ER lumen in a process called co-translational translocation. The ribosome-nascent chain complex is recognized and targeted to the ER membrane by the signal recognition particle (Walter and Blobel, 1980Go) and docks at the translocation site. Translation then provides the energy for precursor progression through the ER membrane (Nicchitta and Blobel, 1993Go). In the second pathway, which has been well studied in the yeast Saccharomyces cerevisiae, proteins are translocated post-translationally in an unfolded state (Rothblatt and Meyer, 1986Go). In this mode, cytosolic heat shock proteins (HSP) belonging to the HSP70 family interact with the nascent protein to prevent its folding (Deshaies et al., 1988Go), and the translocation machinery specifically recognises the secretory protein at the ER membrane and actively pulls the precursor into the lumen of the ER (Panzner et al., 1995Go; Matlack et al., 1999Go).

The translocation sites that allow transport of hydrophilic proteins across a hydrophobic membrane are aqueous channels (Simon and Blobel, 1991Go), formed by the oligomerization of a trimeric complex, the Sec61 complex (Hanein et al., 1996Go). The first subunit of this complex, Sec61{alpha}, was initially identified in S. cerevisiae as Sec61p (Deshaies and Schekman, 1987Go); it was later discovered in mammalian cells too and displays strong homology with the Escherichia coli SecY protein (Görlich et al., 1992Go). The integral Sec61{alpha} 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., 1994Go). Sec61ß and Sec61{gamma} were co-purified in complex with the Sec61{alpha} polypeptide in mammals (Görlich and Rapoport, 1993Go). In S. cerevisiae, the {gamma} subunit, Sss1p, was isolated as a suppressor of the sec61-2 temperature-sensitive mutation (Esnault et al., 1993Go). This single transmembrane domain protein is related to the SecE subunit of E. coli translocase (Hartmann et al., 1994Go). Although both polypeptides are encoded by essential genes in the yeast, the third one, Sbh1p (Sec61ß homolog) (Panzner et al., 1995Go), does not display an essential function (Finke et al., 1996Go). 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., 1996Go). Yeast Sss1p and mammalian Sec61{gamma} proteins are highly conserved and were shown to be functionally interchangeable (Esnault et al., 1993Go). 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., 1997Go) 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., 1998Go). 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.


    Materials and Methods
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 Summary
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 Materials and Methods
 Results
 Discussion
 References
 
Strains, plasmids and media
The S. cerevisiae YKF16 strain mat{alpha}, {Delta}sbh1::HIS3, {Delta}sbh2::ADE2, his3-11, -15, leu2-3, -112, trp1-1, ura3-1, ade2-1, can1-100 was used for cloning of the Y. lipolytica SBH1 cDNA (Finke et al., 1996Go). Interruption of the Y. lipolytica SBH1 gene was done in the 136463 strain MatB, scr1::ADE1, SCR2, his-1, leu2, ura3. The S. cerevisiae strain PJ694{alpha} MAT{alpha}, trp1-901, leu2-3,112, ura3-52, his3-200, gal4{Delta}, gal80{Delta}, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ (James et al., 1996Go) was chosen for screening of the Y. lipolytica two-hybrid library (Kabani et al., 2000Go,Kabani et al., 2000Go).

PINA1326 corresponds to the pFL61 SBH1-complementing vector (Swennen et al., 1997Go) cloned in the S. cerevisiae {Delta}sbh1, {Delta}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.


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Table 1. List of the different oligonucleotides used in this study

 

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{Delta}{Delta}, was constructed for screening of the Y. lipolytica two-hybrid library (James et al., 1996Go). The SBH1 open reading frame was excised from pINA1328 by a NcoI and BamHI digestion and ligated into pAS2{Delta}{Delta} cut with the same enzymes. The recombinant vector was transformed into the S. cerevisiae PJ694{alpha} 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{Delta}{Delta} 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., 2000Go). 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{Delta}{Delta} 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 {Delta}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{alpha} 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., 2000Go,Kabani et al., 2000Go), 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., 1996Go). 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.



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Fig. 4. Intracellular level of precursor forms of the alkaline extracellular protease in Y. lipolytica wild-type and {Delta}sbh1 strains. Total intracellular protein was extracted from wild-type cells (lane 1), wild-type cells transformed with the mutated copy of the XPR2 gene (lanes 2 and 3), {Delta}sbh1 cells (lane 4) and {Delta}sbh1 cells expressing the mutant protease (lanes 5 and 6). Proteins were analyzed by SDS-PAGE and blotted with anti-AEP antibodies.

 

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).


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cloning of the SBH1 cDNA from Y. lipolytica
The temperature-sensitive S. cerevisiae strain {Delta}sbh1 {Delta}sbh2 was transformed with pools B and C of the Y. lipolytica cDNA expression library (Swennen et al., 1997Go). Ura+ transformants were first selected on minimal medium at permissive temperature and then replica-plated on rich medium and incubated at 38°C. Among 100,000 clones, eight were able to grow at this temperature. To test if the temperature resistance was due to the resident plasmid, Ura— segregants were obtained on rich medium at 20°C and then checked for reappearance of the thermosensitive phenotype. Six transformants displayed a temperature-sensitive growth when the pFL61 recombinant plasmid was lost. Plasmids from these S. cerevisiae clones were extracted and used to retransform the initial {Delta}sbh1 {Delta}sbh2 mutant. All plasmids rescued the growth defect of this strain. Restriction analysis of the different plasmids using the NotI enzyme revealed two types of insert of either 350 or 450 base pairs. cDNA inserts were sequenced using primers that hybridize in the PGK promoter and terminator of pFL61. Four different ORF sequences were obtained and compared to databases. The first one, found into two clones, had no homologue in the databases; the second one, present into two clones, corresponded to a vacuolar ATPase subunit; the third one was in inverse orientation relative to the PGK promoter and was not studied further; and the last one was the Y. lipolytica SBH1 cDNA.

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., 2002Go). 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 {Delta}sbh1, {Delta}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.



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Fig. 1. Alignment of the Y. lipolytica Sbh1p protein and the two Sbh2p and Sbh1p proteins from S. cerevisiae. Identical amino acids in the three proteins are in black. Identical amino acids in YlSbh1p and ScSbh2p or YlSbh1p and ScSbh1p are in red and blue.

 

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., 1996Go) 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 {Delta}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, {Delta}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., 2002Go), 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%.



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Fig. 2. (A) Comparison at 28°C on rich medium of the growth and colonies morphology of the Y. lipolytica wild-type and {Delta}sbh1 strains. (B) Comparison of the SDS resistance of the Y. lipolytica wild-type and {Delta}sbh1 strains.

 

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.

{Delta}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 {Delta}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., 2000Go) 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 {Delta}sbh1 strain does not accumulate unglycosylated proteins.



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Fig. 3. Levels of Kar2p in Y. lipolytica wild-type and {Delta}sbh1 strains treated with tunicamycine. Exponential cultures were treated for 3 hours with 10 µg/ml tunicamycine before intracellular protein extraction. Same amounts of total protein were separated by SDS-PAGE, transferred onto nitrocellulose and blotted using anti-kar2p antibodies.

 

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., 1991Go). 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 {Delta}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 {Delta}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, 1989Go). The PJ69-4{alpha} 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., 1996Go). 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.



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Fig. 5. (A) Schematic representation of the different genomic fragments encoding the Y. lipolytica calnexin protein selected in the two-hybrid library screen using the Gal4BD-Sbh1p fusion protein as the bait. The position of the fusion point, relative to the initiator codon of YlCNX1, with the Gal4p activating domain is indicated. (B) Schematic representation of the different deletions of Cnx1p tested for their interaction with Sbh1p in the two-hybrid assay.

 

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.



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Fig. 6. (A) Nucleotidic sequence of the CNX1 gene from Y. lipolytica and amino acid translation. The start and stop codons are in bold and the SacI (positions 1207 and 1743), SalI (position 1396) and XhoI sites (positions 406 and 1432) are both in italic and underlined. The signal sequence (amino acids 1 to 18) and the transmembrane helix (amino acids 495 to 524) are in bold. The different fusion points found in the two-hybrid library are both in bold and underlined.

 


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Fig. 6. (B) Amino acid alignment of YlCnx1p and calnexins from Aspergillus nidulans (ANCNX), Schizosaccharomyces pombe (SPCNX), Homo sapiens (HSCNX) and Saccharomyces cerevisiae (SCCNX).

 

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-Sbh1p{Delta}C 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.



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Fig. 7. Analysis of the interacting domain of Sbh1p with Cnx1p using the two-hybrid system. All diploids are able to grow on medium A, and only interaction between Sbh1p and Cnx1p allows growth on medium B (expression of two reporter genes is required). 1 and 2, pAS2{Delta}{Delta}SBH1 + pGADCNX1 (entire protein); 3, pAS2{Delta}{Delta}SBH1{Delta}SalI (deletion of the C-terminal domain) + pGADCNX1; 4, pAS2{Delta}{Delta} + pGADCNX1.

 

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 {Delta}SacI, eliminates amino acids 397 to 576 in the protein; the second one, {Delta}SalI, starts at amino acid 460 and continues to the end; and the third one, {Delta}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, 1991Go). 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).



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Fig. 8. (A) Co-precipitation of Cnx1p with Sbh1p. A membrane-enriched fraction of the strain expressing the HA-tagged Sbh1p or wildtype was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-HA immunoprecipitate (corresponding to a 100 µl aliquot of the solubilized fraction) from the epitope-tagged strain (lane 2) and from the wild-type strain (lane 3). A western blot analysis was then done using anti-Cnx1p antibodies. (B) Precipitation of Cnx1p with Sec61p or Sec62p. A membrane-enriched fraction of the strain expressing the c-myc-tagged Sec61p was prepared by centrifugation of a total protein extract for 30 minutes at 18,000 g. For solubilization, the pellet was resuspended in phosphate buffer saline and 2% Triton X-100 was added. The sample was further clarified by a centrifugation at 18,000 g. 5 µl of the solubilized supernatant was directly resolved by SDS-PAGE (lane 1) and compared to an anti-c-myc (lane 2) or an anti-Sec62p immunoprecipitate (lane 3) corresponding to a 100 µl aliquot of the solubilized fraction. A western blot analysis was then done using anti-Cnx1p antibodies. (C) Co-precipitation of YlCnx1p with ScSbh2p. A membrane-enriched extract of the strain expressing the ScSbh2p was solubilized in 2% Triton X-100 and centrifuged at 18,000 g for 30 minutes. Proteins in the supernatant were either directly analyzed by SDS-PAGE (lane 1) or first immunoprecipitated in the presence of anti-ScSbh2p antibodies (lane 2) before western blotting using anti-Cnx1p antibodies.

 

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{Delta}{Delta} 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., 2000Go). 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.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Until recently the precise function of Sec61ß was unclear. Previous results suggested that this subunit was the least essential one for the function of the trimeric Sec61 complex involved in protein translocation. Indeed, in the yeast S. cerevisiae, mutants lacking either the {alpha} (Sec61p) or {gamma} subunit (Sss1p) are not viable (Deshaies and Schekman, 1987Go; Esnault et al., 1993Go), whereas deletion of the two genes (SBH1 and SBH2) encoding the ß subunit conferred a growth defect only at high temperature (Finke et al., 1996Go). The growth phenotype displayed by the Y. lipolytica sbh1 null mutant is not very different from the wild-type strain (as shown in this study), suggesting that in this yeast too the ß subunit does not have an essential function although we can not totally exclude the existence of a second gene. Moreover, whereas Sec61{alpha} was identified as the major component adjacent to the polypeptide during its transfer across the ER membrane by a photocross-linking approach (Mothes et al., 1994Go) and as the major ribosome-binding site in reconstitution experiments (Kalies et al., 1994Go), Sec61ß-depleted proteoliposomes showed the same affinity for ribosome binding as proteoliposomes containing the intact Sec61 complex.

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., 1998Go). Second, cross-links between a multiple-spanning membrane protein, Sec61ß, and other partners were observed using a membrane-permeable heterobifunctional reagent (Laird and High, 1997Go), 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., 1998Go; Bebök et al., 1998Go). 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., 1998Go). 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, 1995Go; Elagöz et al., 1999Go). 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., 1999Go). 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., 1998Go) 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{alpha} 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, 1993Go), to modify these proteins, such as signal peptidase (Kalies et al., 1998Go) 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.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Bebök, Z., Mazzochi, C., King, S. A., Hong, J. S. and Sorscher, E. J. (1998). The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61ß and a cytosolic, deglycosylated intermediary. J. Biol. Chem. 273,29873 -29878.[Abstract/Free Full Text]

Boisramé, A., Beckerich, J.-M. and Gaillardin, C. M. (1996). Sls1p, an endoplasmic reticulum component, is involved in the protein translocation process in the yeast Yarrowia lipolytica.J. Biol. Chem. 271,11668 -11675.[Abstract/Free Full Text]

Boisramé, A., Kabani, M., Beckerich, J.-M., Hartmann, E. and Gaillardin, C. M. (1998). Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica. J. Biol. Chem. 273,30903 -30908.[Abstract/Free Full Text]

Bon, E., Casarégola, S., Blandin, G., Llorente, B., Neuvéglise, C., Munsterkotter, M., Guldener, U., Mewes, H.-W., Dujon, B. and Gaillardin, C. (2002). Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns. Nucleic Acid Res. (in press).

Broughton, J., Swennen, D., Wilkinson, B. M., Joyet, P., Gaillardin, C. and Stirling, C. J. (1997). Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery. J. Cell Sci. 110,2715 -2727.[Abstract/Free Full Text]

Chen, Y., le Cahérec, F. and Chuck, S. L. (1998). Calnexin and other factors that alter translocation affect the rapid binding of ubiquitin to ApoB in the Sec61 complex. J. Biol. Chem. 273,11887 -11894.[Abstract/Free Full Text]

Degen, E. and Williams, D. B. (1991). Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibility molecules. J. Cell. Biol. 112,1099 -1115.[Abstract]

Deshaies, R. J. and Schekman, R. (1987). A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum. J. Cell Biol. 105,633 -645.[Abstract]

Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. and Schekman, R. (1988). A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature 332,800 -805.[CrossRef][Medline]

Elagöz, A., Callejo, M., Armstrong, J. and Rokeach, L. A. (1999). Although calnexin is essential in S. pombe, its highly conserved central domain is dispensable for viability. J. Cell Sci. 112,4449 -4460.[Abstract/Free Full Text]

Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R. and Kepes, F. (1993). The yeast SSS1 gene is essential for secretory protein translocation, and encodes a highly conserved protein of the endoplasmic reticulum. EMBO J. 12,4083 -4093.[Abstract]

Fabre, E., Nicaud, J.-M. Lopez, M. C. and Gaillardin, C. (1991). Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease. J. Biol. Chem. 266,3782 -3790.[Abstract/Free Full Text]

Fields, S. and Songs, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340,245 -246.[CrossRef][Medline]

Finke, K., Plath, K., Panzner, S., Prehn, S., Rapoport, T. A., Hartmann, E. and Sommer, T. (1996). A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae. EMBO J. 15,1482 -1494.[Abstract]

Görlich, D., Prehn, S., Hartmann, E., Kalies, K.-U. and Rapoport, T. A. (1992). A mammalian homolog of Sec61p and SECYp is associated with ribosomes and nascent polypeptides during translocation. Cell 71,489 -503.[Medline]

Görlich, D. and Rapoport, T. A. (1993). Protein translocation into proteoliposomes reconstitued from purified components of the endoplasmic reticulum membrane. Cell 75,615 -630.[Medline]

Hanein, D., Matlack, K. E., Jungnickel, B., Plath, K., Kalies, K.-U., Miller, K. R., Rapoport, T. A. and Akey, C. W. (1996). Oligomeric rings of the Sec61p complex induced by ligands required for protein translocation. Cell 87,721 -732.[Medline]

Hartmann, E., Sommer, T., Prehn, S., Görlich, D., Jentsch, S. and Rapoport, T. A. (1994). Evolutionary conservation of components of the protein translocation complex. Nature 367,654 -657.[CrossRef][Medline]

James, P., Halladay, J. and Craig, E. A. (1996). Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144,1425 -1436.[Abstract/Free Full Text]

Jannatipour, M. and Rokeach, L. A. (1995). The Schizosaccharomyces pombe homologue of the chaperone calnexin is essential for viability. J. Biol. Chem. 270,4845 -4853.[Abstract/Free Full Text]

Kabani, M., Boisramé, A., Beckerich, J.-M. and Gaillardin, C. (2000). A highly representative two-hybrid genomic library for the yeast Yarrowia lipolytica.Gene 241,309 -315.[CrossRef][Medline]

Kabani, M., Beckerich, J.-M. and Gaillardin, C. (2000). Sls1p stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum. Mol. Cell. Biol. 20,6923 -6934.[Abstract/Free Full Text]

Kalies, K.-U., Görlich, D. and Rapoport, T. A. (1994). Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex. J. Cell Biol. 126,925 -934.[Abstract]

Kalies, K.-U., Rapoport, T. A. and Hartmann, E. (1998). The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation. J. Cell Biol. 141,887 -894.[Abstract/Free Full Text]

Laird, V. and High, S. (1997). Discrete cross-linking products identified during membrane protein biosynthesis. J. Biol. Chem. 272,1983 -1989.[Abstract/Free Full Text]

Madzak, C., Tréton, B. and Blanchin-Roland, S. (2000). Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J. Mol. Microbiol. Biotechnol. 2,207 -215.[Medline]

Maftahi, M., Gaillardin, C. and Nicaud, J.-M. (1996). Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae.Yeast 12,859 -868.[CrossRef][Medline]

Matlack, K. E., Misselwitz, B., Plath, K. and Rapoport, T. A. (1999). BiP acts as a molecular ratchet during posttranslational transport of prepro-alpha factor across the ER membrane. Cell 97,553 -564.[Medline]

Mothes, W., Prehn, S. and Rapoport, T. A. (1994). Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane. EMBO J. 13,3973 -3982.[Abstract]

Nicchitta, C. V. and Blobel, G. (1993). Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell 73,989 -998.[Medline]

Panzner, S., Dreier, L., Hartmann, E., Kostka, S. and Rapoport, T. A. (1995). Posttranslational protein transport in yeast rconstitued with a purified complex of Sec proteins and Kar2p. Cell 81,561 -570.[Medline]

Richard, M., de Groot, P., Courtin, O., Poulain, D., Klis, F. and Gaillardin, C. (2002). GPI7 affects cell-wall protein anchorage in Saccharomyces cerevisiae and Candida albicans. Microbiology 148,2125 -2133.[Abstract/Free Full Text]

Rothblatt, J. A. and Meyer, D. I. (1986). Secretion in yeast: translocation and glycosylation of prepro-{alpha}-factor in vitro occur via an ATP-dependent posttranslational mechanism. EMBO J. 5,1031 -1036.

Simon, S. M. and Blobel, G. (1991). A protein-conducting channel in the endoplasmic reticulum. Cell 65,371 -380.[Medline]

Swennen, D., Joyet, P. and Gaillardin, C. (1997). Cloning the Yarrowia lipolytica homologue of the Saccharomyces cerevisiae SEC62 gene. Curr. Genet. 31,128 -132.[Medline]

Travers, K. J., Patil, C. K., Wodicka, L., Lockhart, D. J., Weissman, J. S. and Walter, P. (2000). Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101,249 -258.[Medline]

Walter, P. and Blobel, G. (1980). Purification of membrane-associated protein complex required for protein translocation across the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 77,7112 -7116.[Abstract]





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