Research Article 4947 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: [email protected])

Accepted 24 September 2002 Journal of Cell Science 115, 4947-4956 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00187

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

Introduction displays strong homology with the Escherichia coli SecY In eukaryotic cells, the protein secretory pathway initiates with protein (Görlich et al., 1992). The integral Sec61α protein transport across the endoplasmic reticulum (ER) membrane. contains several transmembrane domains that were found in Two major pathways have been described. In the first one, the proximity to the nascent chains during their transfer and were preprotein is not released in the cytoplasm but is directly shown to contribute to the hydrophilic environment reported in transferred into the ER lumen in a process called co- translocation pores (Mothes et al., 1994). Sec61β and Sec61γ translational translocation. The ribosome-nascent chain were co-purified in complex with the Sec61α polypeptide in complex is recognized and targeted to the ER membrane by mammals (Görlich and Rapoport, 1993). In S. cerevisiae, the the signal recognition particle (Walter and Blobel, 1980) and γ subunit, Sss1p, was isolated as a suppressor of the sec61-2 docks at the translocation site. Translation then provides the temperature-sensitive mutation (Esnault et al., 1993). This energy for precursor progression through the ER membrane single transmembrane domain protein is related to the SecE (Nicchitta and Blobel, 1993). In the second pathway, which has subunit of E. coli translocase (Hartmann et al., 1994). Although been well studied in the yeast Saccharomyces cerevisiae, both polypeptides are encoded by essential genes in the yeast, proteins are translocated post-translationally in an unfolded the third one, Sbh1p (Sec61β homolog) (Panzner et al., 1995), state (Rothblatt and Meyer, 1986). In this mode, cytosolic heat does not display an essential function (Finke et al., 1996). A shock proteins (HSP) belonging to the HSP70 family interact second trimeric complex was identified in S. cerevisiae with the nascent protein to prevent its folding (Deshaies et al., comprising Ssh1p (a Sec61p homolog) and Sbh2p (another 1988), and the translocation machinery specifically recognises Sec61β homolog) proteins together with Sss1p. In vitro studies the secretory protein at the ER membrane and actively pulls indicate that this second Sec61 complex was specialized in the the precursor into the lumen of the ER (Panzner et al., 1995; co-translational translocation pathway (Finke et al., 1996). Matlack et al., 1999). Yeast Sss1p and mammalian Sec61γ proteins are highly The translocation sites that allow transport of hydrophilic conserved and were shown to be functionally interchangeable proteins across a hydrophobic membrane are aqueous channels (Esnault et al., 1993). By contrast, Sbh1p and Sec61β show (Simon and Blobel, 1991), formed by the oligomerization of a poor homology and are not related to the third component of trimeric complex, the Sec61 complex (Hanein et al., 1996). E. coli translocation apparatus, SecG. Until now, no precise The first subunit of this complex, Sec61α, was initially function has been attributed to the β subunit of the Sec61 identified in S. cerevisiae as Sec61p (Deshaies and Schekman, complex in either yeast or higher eukaryotic cells. 1987); it was later discovered in mammalian cells too and We addressed the function of Sec61 in the yeast Yarrowia 4948 Journal of Cell Science 115 (24) lipolytica. The SEC61 gene had been previously cloned by couples. Sec61β-62 and sec61β-R822 contain a 16-bases 5′ extension reverse genetics (Broughton et al., 1997) and was shown to corresponding to a restriction site for AscI; sec61β-R242 and sec61β- complement a null mutation in the S. cerevisiae SEC61 gene. 401 contain, respectively, a EcoRI and BamHI restriction site (Table Antibodies recognizing this translocon component gave us 1). A second amplification was performed using 50 ng of each purified β β access to the ribosome-associated membrane protein (RAMP) fragment as template and primers sec61 -R242 and sec61 -401. The fraction in Y. lipolytica and provided biochemical evidence for 600 base pair amplified fragment was digested with EcoRI and BamHI and cloned in pINA300′, restricted with the same enzymes. The the predominance of the co-translational mode of translocation recombinant plasmid, pINA1325, was linearized using the AscI in this yeast (Boisramé et al., 1998). In order to gain insights enzyme before transformation of the Y. lipolytica wild-type strain. into the role of the Sec61β subunit, we decided to clone and to pINA1330, containing the SBH1-coding sequence fused in frame characterize it. No secretory defect was associated with a SBH1 with the binding domain of Gal4p in pAS2∆∆, was constructed for gene interruption in Yarrowia, and we obtained for the first screening of the Y. lipolytica two-hybrid library (James et al., 1996). time evidence for an association of Sbh1p with the ER- The SBH1 open reading frame was excised from pINA1328 by a NcoI membrane chaperone protein calnexin, thus linking this and BamHI digestion and ligated into pAS2∆∆ cut with the same translocon component with folding and/or quality control of enzymes. The recombinant vector was transformed into the S. α secretory proteins. 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 Materials and Methods oligonucleotide called sbh1HA (see Table 1) was designed. Two Strains, plasmids and media fragments were amplified separately on the SBH1 cDNA using, The S. cerevisiae YKF16 strain matα, ∆sbh1::HIS3, ∆sbh2::ADE2, respectively, the sbh1-1/sbh1-R209 and sbh1HA/sbh1-2 primer his3-11, –15, leu2-3, –112, trp1-1, ura3-1, ade2-1, can1-100 was used couples. After restriction with SalI, they were ligated and an aliquot for cloning of the Y. lipolytica SBH1 cDNA (Finke et al., 1996). of the ligation mixture was used as the template for a new Interruption of the Y. lipolytica SBH1 gene was done in the 136463 amplification with the sbh1-1 and sbh1-2 primers. The final product strain MatB, scr1::ADE1, SCR2, his-1, leu2, ura3. The S. cerevisiae was then digested with NcoI and SphI and cloned in pINA300′ opened strain PJ694α MATα, trp1-901, leu2-3,112, ura3-52, his3-200, gal4∆, with the same enzymes. The recombinant plasmid, pINA1329, was gal80∆, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ (James linearized at the StuI site (upstream from the tag epitope) before et al., 1996) was chosen for screening of the Y. lipolytica two-hybrid integration at the SBH1 locus of the Y. lipolytica wild-type strain. The library (Kabani et al., 2000). recombination event leads to a tandem of SBH1 sequences: the first PINA1326 corresponds to the pFL61 SBH1-complementing vector contains the HA epitope under the SBH1 gene transcriptional and (Swennen et al., 1997) cloned in the S. cerevisiae ∆sbh1, ∆sbh2 strain. translational regulatory elements, and the second is devoid of pINA1328 is a derivative of the Y. lipolytica integrative URA3 plasmid promoter and translation initiation codon. Expression of a tagged pINA300′ that contains the 325 base pair SBH1 cDNA amplified using Sbh1p protein was confirmed with anti-HA antibodies for three the two primers: sbh1-1 and sbh1-2 (Table 1) and cloned at the NcoI transformants. and SphI sites. pINA1332 and pINA1331 correspond, respectively, to a pAS2∆∆ To create the inactivated copy of SBH1, an upstream fragment two-hybrid plasmid containing the ScSBH2 coding sequence fused in corresponding to nucleotides 62 to 242 of the SBH1 gene and a frame with the Gal4p-binding domain and a pINA1269 vector downstream fragment from nucleotides 401 to 822 were amplified expressing the ScSBH2 open reading frame under the control of the separately on wild-type genomic DNA using, respectively, the Y. lipolytica strong hp4d promoter (Madzak et al., 2000). The SBH2 sec61β-62/sec61β-R242 and sec61β-401/sec61β-R822 primer open reading frame was amplified with two primers: scsbh2-1 and

Table 1. List of the different oligonucleotides used in this study Name Sequence Plasmid sbh1-1 CCG GCC ATG GTT CCC GGA GGC CCC GCT GCC pINA1328 pINA1329 sbh1-2 ACATGCATGCATTATAGATATGTTTTAGAGTCC pINA1328 pINA1329 sec61β-62 CCC CGG CGC GCC CCC CGA TGG AGT GAG TAT GAC ACC GC pINA1325 sec61β-R242 CCG GAA TTC AAC ATC CAC CCC AAA CAT pINA1325 sec61β-401 CGC GGA TCC CGC CCC ACA TCC TTC ACA pINA1325 sec61β-R822 GGG GGG CGC GCC GGG GCG GTA CTC TGT TCG GCT TAT pINA1325 sbh1HA G GCT GTC GAC TAC CCA TAC GAT GTT CCA GAT TAC GCT CCC GTA TTG GTC ATG GTG C pINA1329 sbh1-R209 CGGGGTCGACCTTGAGACCC pINA1329 scsbh2-1 CGG GAT CCT AAG AAT GGC AGC TTC AGT TCC pINA1331 pINA1332 scsbh2-2 GGG GTA CCT CGA GAC TGA TAT TAT ATA ATG TGT GTA AA pINA1331 pINA1332 Cnx1-1 CGCAGATCTCCCTTCTACATTGCCGACCCC Cnx1-2 GCGGAATTCGGATAGCGGAGACGGG

Italics indicate a restriction site. Bold characters correspond to the template. The HA epitope is underlined. Sbh1p characterization 4949 scsbh2-2 (Table 1) that contain a BamHI for the first one and a KpnI After a slow centrifugation at 2000 g, supernatant was further and XhoI restriction sites for the second. After digestion of the centrifuged at 18,000 g for 30 minutes. The pellet was then solubilized fragment either by BamHI and XhoI or BamHI and KpnI, the digested in 1 ml of PBS plus anti-protease plus Triton X100 2% at room products were, respectively, ligated with the pAS2∆∆ vector cut with temperature for 20 minutes, and a solubilized supernatant, BamHI and SalI and pINA1269 opened with BamHI and KpnI. The corresponding to proteins from membranous compartments, was recombinant plasmid, pINA1331, was linearized in the LEU2 gene by obtained after a new 30 minutes centrifugation at 18,000 g. For an ApaI restriction before transformation of the ∆sbh1 strain. immunoprecipitation, 200 µl of this sample was diluted five times in Y. lipolytica strains were grown in YPD complete medium (1% PBS either with anti-HA, anti-c-myc or anti-Sec62 antibodies, and yeast extract, 1% bacto-peptone, 1% glucose) or YNB minimal complexes were recovered with protein-A sepharose beads. Sepharose medium (0.17% yeast nitrogen base without ammonium sulfate and beads were washed three times with 500 µl of PBS and precipitates without amino acids, 1% glucose, 0.1% proline); supplements were were eluted in 50 µl of sample buffer (100 mM Tris-HCl pH 6.8, 2% added to a final concentration of 0.01%. Induction of the alkaline 2 β-mercaptoethanol, 20% glycerol, 4% SDS, 0.02% Bromophenol extracellular protease was performed using GPP medium (2% blue) for 20 minutes at 65°C. Samples were then applied on a 8% glycerol, 0.17% yeast nitrogen base without ammonium sulfate and polyacrylamide denaturing gel, and proteins were transferred onto a without amino acids, 0.3% proteose peptone, 50 mM phosphate nitrocellulose membrane after migration. Anti-calnexin antibodies buffer, pH 6.8). were used as primary antibodies, peroxidase-conjugated anti-IgG antibodies as secondary ones, and detection was realized using the ECL method (Amersham). 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 Results increasing amounts of SDS. Plates were incubated at 28°C for 48 Cloning of the SBH1 cDNA from Y. lipolytica hours. The temperature-sensitive S. cerevisiae strain ∆sbh1 ∆sbh2 was transformed with pools B and C of the Y. lipolytica cDNA Y. lipolytica two-hybrid library screening expression library (Swennen et al., 1997). Ura+ transformants The PJ69-4α strain was transformed with the recombinant plasmid were first selected on minimal medium at permissive pINA1330. About 2×109 cells grown in rich medium were mixed with temperature and then replica-plated on rich medium and 1.5-3.5×108 cells of each library (Kabani et al., 2000), which incubated at 38°C. Among 100,000 clones, eight were able to corresponds to a ratio of 10:1 (bait:prey). Cells were sedimented by grow at this temperature. To test if the temperature resistance centrifugation and resuspended in 4 ml of YPD for each pool, which was due to the resident plasmid, Ura– segregants were obtained were plated on rich medium and incubated overnight at 28°C for on rich medium at 20°C and then checked for reappearance of mating. Cells were harvested in 30 ml of YPD for each pool and plated the thermosensitive phenotype. Six transformants displayed a on minimal medium plus methionine and uracile for selection of temperature-sensitive growth when the pFL61 recombinant Leu+, Trp+, His+ and Ade+ diploids. plasmid was lost. Plasmids from these S. cerevisiae clones were extracted and used to retransform the initial ∆sbh1 ∆sbh2 Antibodies mutant. All plasmids rescued the growth defect of this strain. Polyclonal anti-HA antibodies from Santa Cruz Biotechnology and Restriction analysis of the different plasmids using the NotI anti-c-myc antibodies from Upstate Biotechnology were used. For enzyme revealed two types of insert of either 350 or 450 base calnexin, Kar2p and Sec62p, a fusion protein with the glutathione S- pairs. cDNA inserts were sequenced using primers that transferase was expressed in E. coli, purified on a gluthatione column hybridize in the PGK promoter and terminator of pFL61. Four and used to immunize rabbits as previously described (Boisramé et different ORF sequences were obtained and compared to al., 1996). The CNX1 open reading frame from nucleotide 840 to databases. The first one, found into two clones, had no nucleotide 1471 (see Fig. 4A) was amplified using the primers Cnx1- homologue in the databases; the second one, present into two 1 and Cnx1-2 (Table 1) and restricted with BglII and EcoRI for clones, corresponded to a vacuolar ATPase subunit; the third cloning at the BamHI and EcoRI sites of pGEX-2T. Anti-Sec62p one was in inverse orientation relative to the PGK promoter antibodies were raised against a fragment corresponding to amino acids 236 to 381. and was not studied further; and the last one was the Y. lipolytica SBH1 cDNA.

Cell extract preparation and analysis Membrane-enriched extracts were prepared as follow: cells from 200 The Y. lipolytica SBH1 gene and the Sbh1p protein ml of an overnight culture in YPD were lysed in 2 ml of phosphate In order to subclone the SBH1 gene in a Y. lipolytica integrative saline buffer (PBS) in the presence of anti-protease and glass beads. vector, an amplification was performed on genomic DNA using

10 20 30 40 50 | | | | | 1 Sbh1p Y. lipolytica MSTSAQVPGGPAAQMKRRNNAQRQEAKASQRPTSTRSVGAGGSSSTMLKL 50 2 Sbh2p S. cerevisiae -MAASVPPGGQRILQKRRQAQSIKEKQAKQTPTSTRQAGYGGSSSSILKL 49 Fig. 1. Alignment of the Y. lipolytica Sbh1p 3 Sbh1p S. cerevisiae -MSSPTPPGGQRTLQKRKQGSSQKVAASAPKKNTN------SNNSILKI 42 protein and the two Sbh2p and Sbh1p proteins from S. cerevisiae. Identical amino acids in the three proteins are in black. 60 70 80 90 | | | | Identical amino acids in YlSbh1p and 1 Sbh1p Y. lipolytica YTDESQGLKVDPVVVMVLSLGFIFSVVALHILAKVSTKLLG 91 ScSbh2p or YlSbh1p and ScSbh1p are in red 2 Sbh2p S. cerevisiae YTDEANGFRVDSLVVLFLSVGFIFSVIALHLLTKFTHII-- 88 and blue. 3 Sbh1p S. cerevisiae YSDEATGLRVDPLVVLFLAVGFIFSVVALHVISKVAGKLF- 82 4950 Journal of Cell Science 115 (24) 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. Fig. 2. (A) Comparison at 28°C on rich medium of the growth and ∆ Interruption of the SBH1 coding sequence colonies morphology of the Y. lipolytica wild-type and sbh1 strains. (B) Comparison of the SDS resistance of the Y. lipolytica wild-type Since the promoter and downstream sequences of the Y. and ∆sbh1 strains. 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). Sbh1p plays a role in the quality control process that allow Integration at the SBH1 locus of Yarrowia was confirmed by retention of misfolded proteins in the ER either to ensure their Southern blot analysis for three Ura+ transformants among ten. normal folding or to target them to a degradation pathway. The Y. lipolytica ∆sbh1 strain growth phenotype was then First, the two Y. lipolytica wild-type and ∆sbh1 strains were tested at three temperatures and compared to the wild-type incubated in the presence of 10 µg/ml of Tunicamycine for parental strain to detect a temperature-sensitive phenotype. No three hours. Growth curves were similar for the two strains difference in the growth rates between the two strains was and comparable to the untreated cultures. Intracellular observed at 18, 28 or 32°C. The only visible phenotype was proteins were extracted from cell pellets and levels of the ER the colonial aspect on solid medium: indeed, after a one-week chaperone protein, Kar2p, and its cofactor Sls1p (Kabani incubation, ∆sbh1 colonies appeared smooth in contrast to the et al., 2001; Travers et al., 2000) were estimated by 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. Fig. 3. Levels of Kar2p in Y. lipolytica wild-type and ∆sbh1 strains treated with tunicamycine. Exponential cultures were treated for 3 µ ∆ hours with 10 g/ml tunicamycine before intracellular protein Sbh1 strain is impaired in the unfolded protein extraction. Same amounts of total protein were separated by SDS- response PAGE, transferred onto nitrocellulose and blotted using anti-kar2p Since Sbh1p and Cnx1p interact (see below), we supposed that antibodies. Sbh1p characterization 4951 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), Fig. 4. Intracellular level of precursor forms of the alkaline extracellular protease in Y. lipolytica wild-type and ∆sbh1 strains. the two others (lanes 2 and 3) accumulated intracellular Total intracellular protein was extracted from wild-type cells (lane precursors as already described. Only the mature form was 1), wild-type cells transformed with the mutated copy of the XPR2 detected in the supernatant of all the tested strains (data not gene (lanes 2 and 3), ∆sbh1 cells (lane 4) and ∆sbh1 cells expressing shown). This observation is in accordance with an absence of the mutant protease (lanes 5 and 6). Proteins were analyzed by SDS- accumulation of unfolded or misfolded precursors in the ER of PAGE and blotted with anti-AEP antibodies. the ∆sbh1 strain. immunoblotting. As shown in Fig. 3, although the amount of Screening of the two-hybrid library for partners of Sbh1p Kar2p was induced two to three times in the wild-type strain, In order to elucidate the Sbh1p function, we chose to look for its level was unchanged in the sbh1 null mutant. A similar partners interacting with this Sec61 complex subunit using result was obtained for Sls1p. Such an observation could the two-hybrid system (Fields and Songs, 1989). The PJ69- indicate that the ∆sbh1 strain does not accumulate 4α strain, transformed with plasmid pINA1330 encoding the unglycosylated proteins. fusion protein Gal4BD-Sbh1p, was mated with aliquots of the In order to test this hypothesis, a mutated copy of the gene three pools of the library constructed in pGAD-C1 to C3 encoding the reporter protein, alkaline extracellular protein plasmids in PJ69-4A (James et al., 1996). The number of (AEP), was integrated at the XPR2 locus of the two strains diploids obtained was comparable for the three pools (about using pINA317. This copy encodes a protease with a mutation 15×106 each), and was sufficient to ensure a good representation of the 4×106 PJ694A clones present in each pool. Diploid cells were directly plated on minimal A medium devoid of leucine, tryptophane, histidine and adenine and incubated at 30°C. After seven days, 80, 9 Number Position of the Pool of Number of and 35 Ade+, His+ clones were isolated, respectively, for fusion point the library sequences 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 1 - 8 3 1 retained. The total number of candidates was thus 836. 2 14 1 1 Yeast colonies were purified on minimal medium before 3 230 1 9 amplification of the inserted genomic fragments by 4 302 3 1 PCR using two primers flanking the cloning site 5 339 1 9 and sequencing. About two hundred sequences, representative of each subgroup, were analyzed using the GCG package (University of Wisconsin, Madison, WI). Cnx1p 1 582 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 B candidate protein was repeatedly obtained in this screening. Name of Position The open reading frame and its protein product in Y. the deletion lipolytica are presented in Fig. 6A. YlCnx1p displays a potential sequence signal between amino acids 1 and 18, ∆SacI 397 a large lumenal domain that is highly conserved (see Fig. 6B), a potential transmembrane domain lying between ∆SalI 460 amino acids 495 and 524 and a short cytoplasmic domain ∆XhoI 130-473 containing many acidic residues. Fig. 5. (A) Schematic representation of the different genomic fragments encoding the Y. lipolytica calnexin protein selected in the two-hybrid Analysis of the Sbh1p-Cnx1p interaction 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 In a second step, a deletion analysis was performed to map the Gal4p activating domain is indicated. (B) Schematic representation of the interacting domain in each of the partners. A truncated the different deletions of Cnx1p tested for their interaction with Sbh1p in Gal4BD-Sbh1p protein that eliminates the transmembrane the two-hybrid assay. helix and the 13 terminal amino-acid lumenal residues was 4952 Journal of Cell Science 115 (24) constructed using the SalI restriction site. Expression A of the fusion protein in S. cerevisiae was controlled 1 ATTCAGAAACAGAACAAAATGCGACTTTCCAAATTGGCCGTTTCTTCGGTGCTGGCCGCC for by western blot analysis and was similar to the 1 M R L S K L A V S S V L A A full-length hybrid protein (data not shown). Unlike 61 GTTGCTTGTGCTCAGGACGCCGACGCTGCTGCTGATGCTGCTCCTTCCTCTCAGGTTGAG ∆ 15 V A C A Q D A D A A A D A A P S S Q V E the entire protein, Gal4BD-Sbh1p C was unable to 121 CACCCCGAATTTACTCCTTACACTGGAGCTGTGACGGGCTTCTTTGAGCAGTTCCTGGAT interact with full-length Gal4AD-Cnx1p in the two- 35 H P E F T P Y T G A V T G F F E Q F L D hybrid assay (compare sectors 1 and 3 in Fig. 7), 181 GGCCACAAGTGGCAAAAGTCGTCGGCTATGAAGGACGACGAGTTTTCTTACGTCGGCGAA 55 G H K W Q K S S A M K D D E F S Y V G E suggesting that the C-terminal tail of Sbh1p is 241 TGGGCCGTGGAGGAACCCTATGTGTTCCCCGGCTTCAAGGGAGACAAGGGTCTTGTCGTC required for interaction. 75 W A V E E P Y V F P G F K G D K G L V V For calnexin, we knew from the screening of the 301 AAGTCTCCTGCTGCTCACCACGCCATCACCACCGCCTTTGACACTCCCATCAACAACAAG 95 K S P A A H H A I T T A F D T P I N N K two-hybrid library that the interaction domain was 361 GGCAAGACTCTGGTGGTGCAGTACGAGGTCAAGCTGCAGAAGGGACTCGAGTGCGGAGGT located downstream from amino acid 339. Three 115 G K T L V V Q Y E V K L Q K G L E C G G deletions were made by digestion: the first one, called 421 GCTTACGTCAAGCTGCTGTCTGCTGAAGTTAACGCCGATGATAAGGGCGTTGAGGAGTTC 135 A Y V K L L S A E V N A D D K G V E E F ∆SacI, eliminates amino acids 397 to 576 in the 481 TCTTCCGAAACTCCTTACCAGATCATGTTTGGCCCTGACAAATGTGGATCCACCAACAAG protein; the second one, ∆SalI, starts at amino acid 155 S S E T P Y Q I M F G P D K C G S T N K 460 and continues to the end; and the third one, 541 GTCCACTTCATTGTCAAGCGACCTCTTCCCGATGGTACCTACGAGGAGAAGCACCTTGTT 175 V H F I V K R P L P D G T Y E E K H L V ∆XhoI, fuses in frame amino acid 130 to amino acid 601 TCTCCCGCCCACGCCCGTCTCAACAAGCTCACCAACCTCTACACCCTGGTGATCCGACCC 473 (Fig. 5B). None of these Gal4AD-Cnx1p-deleted 195 S P A H A R L N K L T N L Y T L V I R P 661 AAGAACGAGTTTGAGATCCGAATTAACGGAAACGTTGTCAAGACCGGCAACCTGCTAGAG proteins was able to reconstitute an active Gal4p 215 K N E F E I R I N G N V V K T G N L L E activator when co-expressed with Gal4BD-Sbh1p 721 GAGGGTCTGTTCAAGCCCTCTTTCAACCCTCCTGCTGAGATTGATGATCCCGAGGACACA (data not shown). This indicates that amino acids 460 235 E G L F K P S F N P P A E I D D P E D T 781 AAGCCCGCCGACTGGGTTGAGGAGCCTTACATGCCCGATCCTGAGCAGGCCGAGAAGCCC to 473, at least are required for the interaction with 255 K P A D W V E E P Y M P D P E Q A E K P Sbh1p, whereas the transmembrane domain and the 841 GCCGACTGGGACGAGAAGGCTCCCTTCTACATTGCCGACCCCGAGGCTGTCATGCCCGCC C-terminal tail of Cnx1p present in the last deletion 275 A D W D E K A P F Y I A D P E A V M P A 901 GACTGGCAGGAGGATACCCCGGATTACATTGTGGATCCTGAGGCCTTCAAGCCCGAGGAC are not sufficient. Considering these results, Sbh1p 295 D W Q E D T P D Y I V D P E A F K P E D and Cnx1p are thought to interact through their 961 TGGGACGACGAGGAGGATGGAGAGTGGGTTGCCCCCGAGATCCCTAACCCCGTTTGCGAG lumenal domains. 315 W D D E E D G E W V A P E I P N P V C E 1021 GAGATTGGTTGCGGTCCCTGGGTCGCCCCCAAGATCCAGAACCCCGACTACAAGGGTGTG 335 E I G C G P W V A P K I Q N P D Y K G V 1081 TGGTCTCAGCCCATGATCGAGAACCCCGACTACAAGGGTACCTGGGCCCCCAAGAAGATT Interaction between Sbh1p and Cnx1p in 355 W S Q P M I E N P D Y K G T W A P K K I Y. lipolytica 1141 CCCAACCCCAACTTCAAGGCTGACGAGCACGCCTCTGATCTTGAGCCCATTGGTGGTCTT 375 P N P N F K A D E H A S D L E P I G G L To validate the Sbh1p-Cnx1p interaction observed 1201 GGCTTCGAGCTCTGGACCATGCAGGAGGACATTCTGTTCGACAACATCTATGTCGGACAC in an heterologous context, we performed a co- 395 G F E L W T M Q E D I L F D N I Y V G H 1261 TCTGTCGATGAGGCCGAGGCCATTGGAAACGCCACCTTTGTGCCCAAGCTGGCTCTCGAG immunoprecipitation experiment on Y. lipolytica 415 S V D E A E A I G N A T F V P K L A L E protein extracts. A polyclonal serum was obtained 1321 GCCGAGGAGGAGAAGCTCTCTGGTCCCCAGAAGGAGACTGCTCCTTGGGATACTGATGAG against the Cnx1p protein that recognized a 80 kDa 435 A E E E K L S G P Q K E T A P W D T D E 1381 GGTGTTCTTTCCTCTGTCGACATGTTCCTTGCTGACCCCGTTTCTTTCGTTCTCGAGCGG product in a protein extract from a wild-type strain 455 G V L S S V D M F L A D P V S F V L E R extract (Fig. 8A, lane 1). Although calnexin is a 582 1441 GTTCTTGGTTTCTTTGAGGTCTTCTCTCAGGATCCCGTCTCCGCTATCCGAGAGGACCCC 475 V L G F F E V F S Q D P V S A I R E D P amino-acid long polypeptide, such an aberrant 1501 GTCGGTGCAGCAGCTTCTTTCGGTCTCCTTCTCATCACGTCTGCTACCGCCTTTGGTCTC migration was already described (Degen and 495 V G A A A S F G L L L I T S A T A F G L Williams, 1991). A solubilized supernatant of a 1561 CTCAATGTGATCATCTTCCTCCTGTTCGGCAAGAAGAAGCAGTCGGCTGCTCCCGCCAAG 515 L N V I I F L L F G K K K Q S A A P A K membrane-enriched fraction was prepared from a Y. 1621 AAGACCAAGAAGACCGGTGACGGTCCTTCTAAGCTCACCAAGGCCGACGTTGTTGAGGCC lipolytica strain expressing the HA-tagged Sbh1p 535 K T K K T G D G P S K L T K A D V V E A protein (see Materials and Methods). The sample 1681 GAGGCTGAGGCTGAGCAGGCTGCTGAGACCGTCGTTGCCTCTGGTGTCGATGATGGTCTG 555 E A E A E Q A A E T V V A S G V D D G L was diluted in phosphate buffer with salt and 1741 CCGAGCTCAAGAAGCGAAAGGGCTTAGTTTAATTTATGTATAATAGAGCTGATTGAAAAG incubated with anti-HA antibodies in the presence 575 P S S R S E R A * of protein-A sepharose for 2 hours at 4°C. The 1801 TCCCCGCACGGTTCTGTGCACTCACATCAACATTGGTACGTACTACTGTGTTGGGTCTTG immunoprecipitate was further analysed by SDS- 1861 TGTTCGTAATATTCTTGATTGATTGATAACATAGGGTTTTTT PAGE, blotted with anti-Cnx1p antibodies and Fig. 6. (A) Nucleotidic sequence of the CNX1 gene from Y. lipolytica and compared with crude extracts. As shown in Fig. 8A amino acid translation. The start and stop codons are in bold and the SacI lane 2, calnexin was detected in the anti-HA (positions 1207 and 1743), SalI (position 1396) and XhoI sites (positions 406 precipitate when the tagged version of Sbh1p and 1432) are both in italic and underlined. The signal sequence (amino acids 1 was present, but no Cnx1p was observed if to 18) and the transmembrane helix (amino acids 495 to 524) are in bold. The immunoprecipitation was performed on a wild-type different fusion points found in the two-hybrid library are both in bold and extract (Fig. 8A, lane 3). underlined. (B) Amino acid alignment of YlCnx1p and calnexins from Aspergillus nidulans (ANCNX), Schizosaccharomyces pombe (SPCNX), Homo In order to show that the Sbh1p-calnexin sapiens (HSCNX) and Saccharomyces cerevisiae (SCCNX). 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 proteins were independently immunoprecipitated using anti- prepared from a SEC61-c-myc-tagged strain, and the two c-myc and anti-Sec62p antibodies. Western blot analysis of Sbh1p characterization 4953

B as shown for YlSbh1p, the S. * 20 * 40 * 60 cerevisiae SBH2 coding sequence YLCNX : MRLSKLAVSSVLAAVACAQDADAAADAAP------SSQVEHPEFTPYTGAVTG------: 47 ∆∆ ANCNX : MRFNAALTSALVSSASIMGYAHAEETEKKPET------TSLAEKPTFTP-TSIEAP------: 49 was cloned into the pAS2 vector. SPCNX : MKYGKVSFLALLCSLYVRGSLADPE------SEQEPLVFNP-TEVKAP------: 41 The S. cerevisiae strain containing HSCNX : MEGKWLLCMLLVLGTAIVEAHDGHDDDVIDIEDDLDDVIEEVEDSKPDTTAPPSSPKVTYKAPVPTGEV : 69 the pGADCNX1 two-hybrid vector SCCNX : MKFSAYLWWLFLNLALVKGTSLLSN------VTLAEDSFWEHFQAYTNT------: 43 was transformed with the * 80 * 100 * 120 * 1 recombinant plasmid, pINA1332. YLCNX : -FFEQFLDG--HKWQKSSAMKDD-----EFSYVGEWAVEEPYVFPGFKGDKGLVVKSPAAHHAITTAFD : 108 ANCNX : -FLEQFTDDWDSRWTPSHAKKEDSKSEEDWAYVGEWSVEEPTVLKGMEGDKGLVVKNVAAHHAISAKFP : 117 Co-expression of the Gal4BD- SPCNX : -LVEQFQGAWSERWIPSHAKRFVN-GIEEMSYVGEWTVEESSGPGALKGEAGLVMKDEAAHHAISYEFD : 108 ScSbh2p and Gal4AD-Cnx1p fusion HSCNX : YFADSFDRGTLSGWILSKAKKDDT-DDEIAKYDGKWEVEEMKE-SKLPGDKGLVLMSRAKHHAISAKLN : 136 proteins allowed growth on minimal SCCNX : ------KHLNQEWITSEAVNNEG----SKIYGAQWRLS-QGRLQGSAWDKGIAVRTGNAAAMIGHLLE : 100 medium devoid of leucine, 40 * 160 * 180 * 200 tryptophane, histidine and adenine, YLCNX : TPINN-KGKTLVVQYEVKLQKGLECGGAYVKLLSAEVNADDKGVEEFSSETPYQIMFGPDKCG-STNKV : 175 ANCNX : KKIDN-KDKTLVVQYEVKPQNSLVCGGAYLKLLQDNK---QLHLDEFSNASPYVIMFGPDKCG-ATNKV : 181 indicating that the two proteins SPCNX : EPINE-PEKDLVVQYEVNPEEGLNCGGAYLKLLAEPT------HGEMSNSIDYRIMFGPDKCG-VNDRV : 169 interact to reconstitute a functional HSCNX : KPFLF-DTKPLIVQYEVNFQNGIECGGAYVKLLSKTP---ELNLDQLHDKTPYTIMFGPDKCG-EDYKL : 200 Gal4p activator (data not shown). SCCNX : TPINVSETDTLVVQYEIKLDNSLTCGGAFIKLMSGFMN-VEALKHYAPDTEGVELVFGPDYCAPEINGV : 168 The control strain expressing the * 220 * 240 * 260 * Gal4BD-ScSbh2p hybrid protein YLCNX : HFIVKRPLP-DGTYEEKHLVSP---AHARLN-KLTNLYTLVIRP-KNEFEIRINGNVVKTGNLLEEGL- : 237 ANCNX : HFIFRHKNPKTGEYEEKHLKAP---PAARTS-KVTSVYTLVVNP-DQTFQILIDGESVKEGSLLED--- : 242 with the Gal4p-activating domain SPCNX : HFIFKHKNPLTGEYSEKHLDSR---PASLLKPGITNLYTLIVKP-DQTFEVRINGDVVRQGSLFYD--- : 231 alone did not grow on the same HSCNX : HFIFRHKNPKTGIYEEKHAKRPDADLKTYFTDKKTHLYTLILNP-DNSFEILVDQSVVNSGNLLND--- : 265 SCCNX : QFAINKVDKITHESKLRYLQEMP--LSKLTDTSQSHLYTLIIDESAQSFQILIDGKTVMVREHIEDKKK : 235 medium. Considering this positive result, we 280 * 300 * 320 * 340 expressed the S. cerevisiae SBH2 YLCNX : --FKPSFNPPAEIDDPEDTKPADWVEEPYMPDPEQAEKPADWDEKAPFYIADPEAVMPADWQEDTPDYI : 304 ANCNX : --FNPPVNPEKEIDDPKDKKPADWVDEAKIPDPE-ATKPEDWDEEAPFEIVDEEATIPEDWLEDEPTSI : 308 coding sequence in Y. lipolytica under SPCNX : --FIPPVLPPVEIYDPEDIKPADWVDEPEIPDPN-AVKPDDWDEDAPRMIPDPDAVKPEDWLEDEPLYI : 297 the control of the hp4d promoter HSCNX : --MTPPVNPSREIEDPEDRKPEDWDERPKIPDPE-AVKPDDWDEDAPAKIPDEEATKPEGWLDDEPEYV : 331 SCCNX : VNFEPPITPPLMIPDVSVAKPHDWDDRIRIPDPE-AVKLSDRDERDPLMIPHPDGTEPPEWNSSIPEYI : 303 (Madzak et al., 2000). We confirmed by immunoblotting that the * 360 * 380 * 400 * heterologous protein was detectable YLCNX : VDPEAFKPEDWDDEEDGEWVAPEIPNPVCEEIG-CGPWVAPKIQNPDYKGVWSQPMIENPDYKGTWAPK : 372 ANCNX : PDPEAEKPEDWDDEEDGDWVPPTVPNPKCQDASGCGPWSPPMKKNPDYKGKWSAPLIDNPAYKGPWAPR : 377 in a total protein extract from one of SPCNX : PDPEAQKPEDWDDEEDGDWIPSEIINPKCIEGAGCGEWKPPMIRNPNYRGPWSPPMIPNPEFIGEWYPR : 366 the Leu+ transformants. This HSCNX : PDPDAEKPEDWDEDMDGEWEAPQIANPRCESAPGCGVWQRPVIDNPNYKGKWKPPMIDNPSYQGIWKPR : 400 SCCNX : LDPNAQKPSWWKELEHGEWIPPMIKNPLCTAERGCGQQIPGLINNAKYKGPGELNEIINPNYMGEWHPP : 372 transformed strain recovered the ability to form rough colonies on rich 420 * 440 * 460 * 480 solid medium. We first showed that YLCNX : KIPNPNFKADEHASDLE-PIGGLGFELWTMQEDILFDNIYVGHSVDEAEAIGNATFVPKLALEAE---- : 436 ANCNX : KIANPAYFEDKTPSNFE-PMGAIGFEIWTMQNDILFDNIYVGHSAEDAEKLRQETFDVKHPIELA---- : 441 the Sbh2p protein co-fractionated SPCNX : KIPNPDYFDDDHPSHFG-PLYGVGFELWTMQPNIRFSNIYVGHSIEDAERLGNETFLPKLKAERELLSK : 434 with calnexin in a membrane- HSCNX : KIPNPDFFEDLEPFRMT-PFSAIGLELWSMTSDIFFDNFIICADRRIVDDWANDGWGLKKAADGA---A : 465 SCCNX : EIENPLYYEEQHPLRIENVISGVILEFWSGSPNMLISNIYVGKNVTEAQIIGNKTWLMRDRAFRG---- : 437 enriched extract of the Y. lipolytica recombinant strain (data not shown). * 500 * 520 * 540 * YLCNX : EEKLSGP--QKETAP---WDTDEGVLSSVDMFLADPVSFVLERVLGFFEVFSQDPVSAIREDPVGAAAS : 500 An immunoprecipitation experiment ANCNX : EEEANKP--KPEEK------AAEPSVS----FKEDPVGHIKEKVDNFVRLSKQDPINAVKQVPD-VAGG : 497 was then performed using a SPCNX : QESMEKQSMHVDEES---NQILEKFLDVYDIIKAKLPPNVAEKVDYYVETIIETPEIGIAIVAV--LGS : 498 solubilized supernatant of a HSCNX : EPGVVGQMIEAAEERPWLWVVYILTVALPVFLVILFCCSGKKQTSGMEYKKTDAPQPDVKEEEEEKEEE : 534 SCCNX : ------SDGPTERKFMNSRLGN : 453 membrane-enriched fraction and anti- Sbh2p antibodies. As shown in Fig. 560 * 580 * 600 * 620 YLCNX : FGLLLITSATAFGLLNVIIFLLFGKKKQSAAPAKKTKKTGDGPSKLTKADVVEAEAEAEQAAETVVASG : 569 8C, YlCnx1p was co-precipitated ANCNX : LAAVLVT------MILVIVGAVGASTPAPAPAKKGKE-AAGATKEKTGAASSSSADTGKGGATKRTT- : 557 with ScSbh2p (Fig. 8C, lane 2). The SPCNX : LTAVILT------CYFYFFASSSPASLSTGTTEAEKEQQEKFKQETETEK-IDVSYAPETESPT- : 555 reverse experiment was performed, HSCNX : KDKGDEE------EEGEEKLEEKQKSDAEEDGGTVSQEEEDRKPKAEEDEILNRSPRNRK- : 588 SCCNX : LQTTFHN------ERESPNPFDRIIDRILEQPLKFVLTAAVVLLTTSVLCCVVFT--- : 502 and ScSbh2p was revealed in an anti- Cnx1p precipitate (data not shown). YLCNX : VDDGLPSSRSERA : 582 ANCNX : ------RSSAE-- : 562 These results suggest that the S. SPCNX : ------AKNED- : 560 cerevisiae protein behaves similarly to HSCNX : ------PRRE-- : 592 Sbh1p in Y. lipolytica. immunoprecipitates using anti-calnexin antibodies allowed Discussion detection of Cnx1p in anti-c-myc precipitate (Fig. 8B, lane 2) Until recently the precise function of Sec61β was unclear. but not in anti-Sec62p precipitate (Fig. 8B, lane 3). This result Previous results suggested that this subunit was the least confirms that a pool of calnexin is located closer to the essential one for the function of the trimeric Sec61 complex minimal translocation apparatus formed by the Sec61 involved in protein translocation. Indeed, in the yeast S. complex. cerevisiae, mutants lacking either the α (Sec61p) or γ subunit (Sss1p) are not viable (Deshaies and Schekman, 1987; Esnault et al., 1993), whereas deletion of the two genes (SBH1 and ScSbh2p is a functional homologue of YlSbh1p SBH2) encoding the β subunit conferred a growth defect only To determine if ScSbh2p was able to interact with YlCnx1p, at high temperature (Finke et al., 1996). The growth phenotype 4954 Journal of Cell Science 115 (24) ribosome-binding site in reconstitution experiments (Kalies et al., 1994), Sec61β-depleted proteoliposomes showed the same affinity for ribosome binding as proteoliposomes containing the intact Sec61 complex. More recent studies brought some insights into Sec61β function. First, a function was suggested for Sec61β 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, Sec61β, and other partners were observed using a membrane-permeable heterobifunctional reagent (Laird and High, 1997), suggesting that the β subunit favors the exit of transmembrane domains Fig. 7. Analysis of the interacting domain of Sbh1p with Cnx1p from the translocation site. Third, two studies revealed that using the two-hybrid system. All diploids are able to grow on Sec61β could be co-immunoprecipitated with secretory medium A, and only interaction between Sbh1p and Cnx1p allows proteins that failed to translocate across or to integrate into the growth on medium B (expression of two reporter genes is required). ER membrane and that were finally targeted to the proteasome 1 and 2, pAS2∆∆SBH1 + pGADCNX1(entire protein); 3, for degradation (Chen et al., 1998; Bebök et al., 1998). This pAS2∆∆SBH1∆SalI (deletion of the C-terminal domain) + suggested a possible function of Sec61β in selecting or pGADCNX1; 4, pAS2∆∆ + pGADCNX1. escorting the polypeptide to the cytosol. Using two complementary approaches, we have shown for displayed by the Y. lipolytica sbh1 null mutant is not very the first time that the β subunit of the Sec61 complex directly different from the wild-type strain (as shown in this study), associates with the ER chaperone, calnexin. The two proteins suggesting that in this yeast too the β subunit does not have an were described as membrane proteins with a single membrane- essential function although we can not totally exclude the spanning domain: although Sec61β exposes only a short C-tail existence of a second gene. Moreover, whereas Sec61α was to the lumen of the ER, calnexin displays a large N-terminal identified as the major component adjacent to the polypeptide lumenal domain (Kalies et al., 1998). The Sbh1p protein newly during its transfer across the ER membrane by a photocross- identified in Y. lipolytica shows a better homology with the linking approach (Mothes et al., 1994) and as the major Sbh2p component of the yeast S. cerevisiae and displays 42%

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. Sbh1p characterization 4955 identity to the human Sec61β protein. YlCnx1p is also well translational translocation of secreted proteins in the yeast Yarrowia conserved when compared to other calnexin sequences and lipolytica. J. Biol. Chem. 273, 30903-30908. displays 45% identity to human calnexin. Since the overall Bon, E., Casarégola, S., Blandin, G., Llorente, B., Neuvéglise, C., Munsterkotter, M., Guldener, U., Mewes, H.-W., Dujon, B. and primary structure of the polypeptides was conserved and a Gaillardin, C. (2002). Molecular evolution of eukaryotic genomes: transmembrane domain was predicted for each one, we may hemiascomycetous yeast spliceosomal introns. Nucleic Acid Res. (in press). assume topology conservation for these two proteins in Broughton, J., Swennen, D., Wilkinson, B. M., Joyet, P., Gaillardin, C. Yarrowia. and Stirling, C. J. (1997). Cloning of SEC61 homologues from Deletion analysis of the interacting domain between these Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation two partners strongly suggests that the association involves machinery. J. Cell Sci. 110, 2715-2727. regions localized in the ER lumen. These results could be put Chen, Y., le Cahérec, F. and Chuck, S. L. (1998). Calnexin and other factors together with the work done on the S. pombe Cnx1p protein that alter translocation affect the rapid binding of ubiquitin to ApoB in the that mapped the essential region of the protein to the terminal Sec61 complex. J. Biol. Chem. 273, 11887-11894. Degen, E. and Williams, D. B. (1991). Participation of a novel 88-kD protein 52 amino-acid residues of the lumenal domain (Jannatipour in the biogenesis of murine class I histocompatibility molecules. J. Cell. and Rokeach, 1995; Elagöz et al., 1999). This domain was also Biol. 112, 1099-1115. identified as sufficient for the formation of a complex including Deshaies, R. J. and Schekman, R. (1987). A yeast mutant defective at an the chaperone protein BiP, and the authors speculate that the early stage in import of secretory protein precursors into the endoplasmic essential function of Cnx1p could reside in its ability to reticulum. J. Cell Biol. 105, 633-645. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. and associate with proteins involved in protein folding (Elagöz et Schekman, R. (1988). A subfamily of stress proteins facilitates al., 1999). Our results reveal the existence of a new type of translocation of secretory and mitochondrial precursor polypeptides. Nature interaction for calnexin that involves the translocation pore. 332, 800-805. Calnexin was the unique Sbh1p-interacting protein Elagöz, A., Callejo, M., Armstrong, J. and Rokeach, L. A. (1999). 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