Interaction of Kar2p and Sls1p Is Required for Efficient Co-translational Translocation of Secreted Proteins in the YeastYarrowia lipolytica (1998)

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation. The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation. endoplasmic reticulum signal recognition particle ribosome-associated membrane protein alkaline extracellular protease polyacrylamide gel electrophoresis. To initiate their pathway, secretory proteins are first targeted to the endoplasmic reticulum (ER)1 membrane in eukaryotic organisms. In mammalian cells, a cytoplasmic particle (theSignal Recognition Particle or SRP) first recognizes these proteins when the signal peptide emerges from the ribosome and then ensures their delivery to the ER membrane through its interaction with the SRP receptor (1Walter P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7112-7116Crossref PubMed Scopus (302) Google Scholar). SRP binding to the nascent chain-ribosome complex causes a translational pause that is released upon docking of SRP (2Walter P. Blobel G. Cell. 1983; 34: 525-533Abstract Full Text PDF PubMed Scopus (164) Google Scholar). As translation resumes, the complex is transferred to the translocation site where crossing through the ER membrane takes place. Sec61α, one of the three polypeptides of the translocation pore, is a polytopic membrane protein; the two others, Sec61β and γ, span the ER membrane once. Three to four units of this hetero-oligomer are needed to form the aqueous pore (3Hannein D. Matlack K.E.S. Jungnickel B. Plath K. Kalies K-U. Miller K.R. Rapoport T.M. Akey C.W. Cell. 1996; 87: 721-732Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In vitro reconstitution experiments, using purified mammalian components, show that the SRP receptor and the Sec61 complex are sufficient to achieve translocation of some preproteins, while the TRAM protein is required for translocation of other preproteins (4Görlich D. Rapoport T.A. Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (521) Google Scholar).In vivo, many other soluble or membrane proteins could be involved in this process to adjust the translocation rate to cell growth.In the model yeast, Saccharomyces cerevisiae, components involved in SRP-dependent targeting are not essential, and several secretory proteins were shown to cross the ER membrane post-translationally (5Brown J.D. Hann B.C. Medzihradszzy K.F. Niwa M. Burlingame A.L. Walter P. EMBO J. 1994; 13: 4390-4400Crossref PubMed Scopus (98) Google Scholar, 6Rothblatt J.A. Meyer D.I. EMBO J. 1986; 5: 1031-1036Crossref PubMed Scopus (80) Google Scholar). This translocation mode relies both on cytosolic chaperones whose binding delays preproteins folding (7Deshaies R.J. Koch B.D. Werner-Washburne M. Craig E.A. Schekman R. Nature. 1988; 332: 800-805Crossref PubMed Scopus (994) Google Scholar) and on membrane proteins that ensure specific insertion of secretory proteins at the translocation site. A heptameric complex containing the trimeric Sec61 complex and four other polypeptides, Sec62p, Sec63p, Sec71p, and Sec72p, allows in vitro post-translational translocation of several preproteins in the presence of the lumenal chaperone Kar2p and ATP (8Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar). All these polypeptides were previously identified in genetic screens and through biochemical approaches (9Rothblatt J.A. Deshaies R.J. Sanders S.L. Daum G. Schekman R. J. Cell Biol. 1989; 109: 2641-2652Crossref PubMed Scopus (229) Google Scholar,10Feldheim D. Yoshimura K. Admon A. Schekman R. Mol. Biol. Cell. 1993; 4: 931-939Crossref PubMed Scopus (68) Google Scholar). A contribution of proteins of the Hsp70 family has been described for two preprotein transport machineries. The Kar2p and mitochondrial matrix mHsp70 proteins, respectively, promote translocation across the endoplasmic reticulum membrane and across the mitochondrial inner membrane in yeast. While the transmembrane protein Sec63p was identified as the Kar2p-binding partner in the ER membrane (11Lyman S.K. Schekman R. J. Cell Biol. 1995; 131: 1163-1171Crossref PubMed Scopus (98) Google Scholar), two components of the Tim complex, Tim44 and Tim17, were found to function as membrane anchor for mHsp70 (12Schneider H.-C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Brunner M. Neupert W. Nature. 1994; 371: 768-774Crossref PubMed Scopus (331) Google Scholar, 13Bömer U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar).In the yeast Yarrowia lipolytica, inactivation of the two genes encoding the 7 S RNA component of the SRP is lethal (14He F. Yaver D. Beckerich J-M. Ogrydziak D. Gaillardin C. Curr. Genet. 1990; 17: 289-292Crossref PubMed Scopus (25) Google Scholar), whereas deletion of the SRP54 and SEC65 genes result in very low growth (15Lee H. Ogrydziak D.M. Yeast. 1997; 13: 499-513Crossref PubMed Scopus (13) Google Scholar). SRP is also essential in the yeastSchizosaccharomyces pombe (16Brennwald P. Liao X. Holm K. Porter G. Wise J.A. Mol. Cell. Biol. 1988; 8: 1580-1590Crossref PubMed Scopus (59) Google Scholar). Study of Y. lipolytica conditional lethal 7 S RNA mutants provided the firstin vivo evidence of SRP involvement in the targeting step of the co-translational translocation process (17He F. Beckerich J.-M. Gaillardin C.M. J. Biol. Chem. 1992; 267: 1932-1937Abstract Full Text PDF PubMed Google Scholar). Pursuing this genetic approach, secondary mutations that led to synthetic lethality in combination with the 7 S RNA mutation were selected. This screen allowed cloning of the SLS1 gene whose product, located in the lumen of the endoplasmic reticulum, was shown to participate in preprotein translocation (18Boisramé A. Beckerich J-M. Gaillardin C.M. J. Biol. Chem. 1996; 271: 11668-11675Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To gain insights in Sls1p function, putative associations with known ER resident proteins were tested. In the present paper, we show that Sls1p directly interacts with the chaperone protein Kar2p. We also show by a fractionation procedure that the majority of translocation pores in Y. lipolytica are linked to ribosomes and that Sls1p and Kar2p partially co-fractionate with ribosome-bound Sec61p. To investigate the structure/function relationship, we constructed different Sls1p mutant proteins and compared their ability to interact with Kar2p with their capacity to promote secretory protein translocation. One of these mutant proteins, Sls1−5p, that does not further associate with Kar2p confers a decrease in the synthesis of a secreted protein, suggesting that such an interaction is required for efficient co-translational translocation.DISCUSSIONWe defined solubilization conditions for Y. lipolyticamicrosomes that allowed us in a subsequent fractionation experiment to show that the major translocation pore component, Sec61p, distributes between two one not linked to ribosomes and the other to ribosomes as in S. S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar). The two in the of these two Indeed, from 70 to 80% of Sec61p is present in the in Y. lipolytica, whereas this to in S. Sec62p, to the membrane protein complex required for post-translational translocation (8Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar), is in the first and is from the that this translocation complex involved in co-translational translocation In these amounts of the chaperone protein Kar2p co-fractionate with Sec61p in the suggesting that the two proteins are in the Kar2p present in this is co-immunoprecipitated by Sec61p of Kar2p in co-translational translocation was described in the yeast S. J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and the chaperone protein as a mammalian translocation pore B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). We also the of a lumenal protein identified in Y. lipolytica, and that of this protein are also to the Sec61p population. the two-hybrid we then that Sls1p directly interacts with Kar2p and thus a partner of this chaperone then allowed us to that the peptide sequence from to is required for Sls1p function in co-translational translocation and for Sls1p interaction with the chaperone protein Kar2p. The decrease of the co-translational translocation rate in a is not linked to a growth as in the suggesting that the protein a function, is of the in the of the alkaline extracellular protease and the of Kar2p in the of Sls1p (18Boisramé A. Beckerich J-M. Gaillardin C.M. J. Biol. Chem. 1996; 271: 11668-11675Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were not further in a the mutant and the protein allows us to a model for Sls1p function in the translocation Sls1p could first to the at the lumenal of the translocation The preprotein could then be directly transferred to the chaperone protein, Kar2p that is to the translocation site through its with in that the crossing of the Sls1p could be to the translocation complex through its interaction with Kar2p that thus first to the preprotein and then to Sls1p. Sls1p and Kar2p could as as has been for Tim44 and mHsp70 U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar). an could a of secretory for the majority of on the ribosomes be sufficient to ensure their across the ER membrane Blobel G. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). In the presence of Sls1−5p, the of the preprotein from one partner to the other be interaction is This study thus evidence of Sls1p involvement in the co-translational translocation process that relies on its with does not the domain present in Sec63p, the partner of Kar2p in the yeast ER membrane (11Lyman S.K. Schekman R. J. Cell Biol. 1995; 131: 1163-1171Crossref PubMed Scopus (98) Google Scholar). the that Tim44 a very domain of with the and that at with U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar), that of the Hsp70 protein family in translocation are not to the protein Moreover, the complex does not at a we can that Kar2p to at a partner in the translocation This partner could be a of the S. Sec63p as the two proteins were shown to be involved in co-translational translocation J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) or membrane protein with to Kar2p function in the translocation pore at an of the co-translational translocation process B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of Sls1p with secretory protein and on ATP of the complex function in translocation be in an in vitro translocation system. To initiate their pathway, secretory proteins are first targeted to the endoplasmic reticulum (ER)1 membrane in eukaryotic organisms. In mammalian cells, a cytoplasmic particle (theSignal Recognition Particle or SRP) first recognizes these proteins when the signal peptide emerges from the ribosome and then ensures their delivery to the ER membrane through its interaction with the SRP receptor (1Walter P. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7112-7116Crossref PubMed Scopus (302) Google Scholar). SRP binding to the nascent chain-ribosome complex causes a translational pause that is released upon docking of SRP (2Walter P. Blobel G. Cell. 1983; 34: 525-533Abstract Full Text PDF PubMed Scopus (164) Google Scholar). As translation resumes, the complex is transferred to the translocation site where crossing through the ER membrane takes place. Sec61α, one of the three polypeptides of the translocation pore, is a polytopic membrane protein; the two others, Sec61β and γ, span the ER membrane once. Three to four units of this hetero-oligomer are needed to form the aqueous pore (3Hannein D. Matlack K.E.S. Jungnickel B. Plath K. Kalies K-U. Miller K.R. Rapoport T.M. Akey C.W. Cell. 1996; 87: 721-732Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). In vitro reconstitution experiments, using purified mammalian components, show that the SRP receptor and the Sec61 complex are sufficient to achieve translocation of some preproteins, while the TRAM protein is required for translocation of other preproteins (4Görlich D. Rapoport T.A. Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (521) Google Scholar).In vivo, many other soluble or membrane proteins could be involved in this process to adjust the translocation rate to cell In the model yeast, Saccharomyces cerevisiae, components involved in SRP-dependent targeting are not essential, and several secretory proteins were shown to cross the ER membrane post-translationally (5Brown J.D. Hann B.C. Medzihradszzy K.F. Niwa M. Burlingame A.L. Walter P. EMBO J. 1994; 13: 4390-4400Crossref PubMed Scopus (98) Google Scholar, 6Rothblatt J.A. Meyer D.I. EMBO J. 1986; 5: 1031-1036Crossref PubMed Scopus (80) Google Scholar). This translocation mode relies both on cytosolic chaperones whose binding delays preproteins folding (7Deshaies R.J. Koch B.D. Werner-Washburne M. Craig E.A. Schekman R. Nature. 1988; 332: 800-805Crossref PubMed Scopus (994) Google Scholar) and on membrane proteins that ensure specific insertion of secretory proteins at the translocation site. A heptameric complex containing the trimeric Sec61 complex and four other polypeptides, Sec62p, Sec63p, Sec71p, and Sec72p, allows in vitro post-translational translocation of several preproteins in the presence of the lumenal chaperone Kar2p and ATP (8Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar). All these polypeptides were previously identified in genetic screens and through biochemical approaches (9Rothblatt J.A. Deshaies R.J. Sanders S.L. Daum G. Schekman R. J. Cell Biol. 1989; 109: 2641-2652Crossref PubMed Scopus (229) Google Scholar,10Feldheim D. Yoshimura K. Admon A. Schekman R. Mol. Biol. Cell. 1993; 4: 931-939Crossref PubMed Scopus (68) Google Scholar). A contribution of proteins of the Hsp70 family has been described for two preprotein transport machineries. The Kar2p and mitochondrial matrix mHsp70 proteins, respectively, promote translocation across the endoplasmic reticulum membrane and across the mitochondrial inner membrane in yeast. While the transmembrane protein Sec63p was identified as the Kar2p-binding partner in the ER membrane (11Lyman S.K. Schekman R. J. Cell Biol. 1995; 131: 1163-1171Crossref PubMed Scopus (98) Google Scholar), two components of the Tim complex, Tim44 and Tim17, were found to function as membrane anchor for mHsp70 (12Schneider H.-C. Berthold J. Bauer M.F. Dietmeier K. Guiard B. Brunner M. Neupert W. Nature. 1994; 371: 768-774Crossref PubMed Scopus (331) Google Scholar, 13Bömer U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar). In the yeast Yarrowia lipolytica, inactivation of the two genes encoding the 7 S RNA component of the SRP is lethal (14He F. Yaver D. Beckerich J-M. Ogrydziak D. Gaillardin C. Curr. Genet. 1990; 17: 289-292Crossref PubMed Scopus (25) Google Scholar), whereas deletion of the SRP54 and SEC65 genes result in very low growth (15Lee H. Ogrydziak D.M. Yeast. 1997; 13: 499-513Crossref PubMed Scopus (13) Google Scholar). SRP is also essential in the yeastSchizosaccharomyces pombe (16Brennwald P. Liao X. Holm K. Porter G. Wise J.A. Mol. Cell. Biol. 1988; 8: 1580-1590Crossref PubMed Scopus (59) Google Scholar). Study of Y. lipolytica conditional lethal 7 S RNA mutants provided the firstin vivo evidence of SRP involvement in the targeting step of the co-translational translocation process (17He F. Beckerich J.-M. Gaillardin C.M. J. Biol. Chem. 1992; 267: 1932-1937Abstract Full Text PDF PubMed Google Scholar). Pursuing this genetic approach, secondary mutations that led to synthetic lethality in combination with the 7 S RNA mutation were selected. This screen allowed cloning of the SLS1 gene whose product, located in the lumen of the endoplasmic reticulum, was shown to participate in preprotein translocation (18Boisramé A. Beckerich J-M. Gaillardin C.M. J. Biol. Chem. 1996; 271: 11668-11675Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To gain insights in Sls1p function, putative associations with known ER resident proteins were tested. In the present paper, we show that Sls1p directly interacts with the chaperone protein Kar2p. We also show by a fractionation procedure that the majority of translocation pores in Y. lipolytica are linked to ribosomes and that Sls1p and Kar2p partially co-fractionate with ribosome-bound Sec61p. To investigate the structure/function relationship, we constructed different Sls1p mutant proteins and compared their ability to interact with Kar2p with their capacity to promote secretory protein translocation. One of these mutant proteins, Sls1−5p, that does not further associate with Kar2p confers a decrease in the synthesis of a secreted protein, suggesting that such an interaction is required for efficient co-translational translocation. defined solubilization conditions for Y. lipolyticamicrosomes that allowed us in a subsequent fractionation experiment to show that the major translocation pore component, Sec61p, distributes between two one not linked to ribosomes and the other to ribosomes as in S. S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar). The two in the of these two Indeed, from 70 to 80% of Sec61p is present in the in Y. lipolytica, whereas this to in S. Sec62p, to the membrane protein complex required for post-translational translocation (8Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar), is in the first and is from the that this translocation complex involved in co-translational translocation In these amounts of the chaperone protein Kar2p co-fractionate with Sec61p in the suggesting that the two proteins are in the Kar2p present in this is co-immunoprecipitated by Sec61p of Kar2p in co-translational translocation was described in the yeast S. J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and the chaperone protein as a mammalian translocation pore B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). We also the of a lumenal protein identified in Y. lipolytica, and that of this protein are also to the Sec61p population. the two-hybrid we then that Sls1p directly interacts with Kar2p and thus a partner of this chaperone then allowed us to that the peptide sequence from to is required for Sls1p function in co-translational translocation and for Sls1p interaction with the chaperone protein Kar2p. The decrease of the co-translational translocation rate in a is not linked to a growth as in the suggesting that the protein a function, is of the in the of the alkaline extracellular protease and the of Kar2p in the of Sls1p (18Boisramé A. Beckerich J-M. Gaillardin C.M. J. Biol. Chem. 1996; 271: 11668-11675Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were not further in a the mutant and the protein allows us to a model for Sls1p function in the translocation Sls1p could first to the at the lumenal of the translocation The preprotein could then be directly transferred to the chaperone protein, Kar2p that is to the translocation site through its with in that the crossing of the Sls1p could be to the translocation complex through its interaction with Kar2p that thus first to the preprotein and then to Sls1p. Sls1p and Kar2p could as as has been for Tim44 and mHsp70 U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar). an could a of secretory for the majority of on the ribosomes be sufficient to ensure their across the ER membrane Blobel G. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). In the presence of Sls1−5p, the of the preprotein from one partner to the other be interaction is This study thus evidence of Sls1p involvement in the co-translational translocation process that relies on its with does not the domain present in Sec63p, the partner of Kar2p in the yeast ER membrane (11Lyman S.K. Schekman R. J. Cell Biol. 1995; 131: 1163-1171Crossref PubMed Scopus (98) Google Scholar). the that Tim44 a very domain of with the and that at with U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar), that of the Hsp70 protein family in translocation are not to the protein Moreover, the complex does not at a we can that Kar2p to at a partner in the translocation This partner could be a of the S. Sec63p as the two proteins were shown to be involved in co-translational translocation J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) or membrane protein with to Kar2p function in the translocation pore at an of the co-translational translocation process B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of Sls1p with secretory protein and on ATP of the complex function in translocation be in an in vitro translocation system. We defined solubilization conditions for Y. lipolyticamicrosomes that allowed us in a subsequent fractionation experiment to show that the major translocation pore component, Sec61p, distributes between two one not linked to ribosomes and the other to ribosomes as in S. S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar). The two in the of these two Indeed, from 70 to 80% of Sec61p is present in the in Y. lipolytica, whereas this to in S. Sec62p, to the membrane protein complex required for post-translational translocation (8Panzner S. Dreier L. Hartmann E. Kostka S. Rapoport T.A. Cell. 1995; 81: 561-570Abstract Full Text PDF PubMed Scopus (320) Google Scholar), is in the first and is from the that this translocation complex involved in co-translational translocation In these amounts of the chaperone protein Kar2p co-fractionate with Sec61p in the suggesting that the two proteins are in the Kar2p present in this is co-immunoprecipitated by Sec61p of Kar2p in co-translational translocation was described in the yeast S. J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar), and the chaperone protein as a mammalian translocation pore B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). We also the of a lumenal protein identified in Y. lipolytica, and that of this protein are also to the Sec61p population. the two-hybrid we then that Sls1p directly interacts with Kar2p and thus a partner of this chaperone A then allowed us to that the peptide sequence from to is required for Sls1p function in co-translational translocation and for Sls1p interaction with the chaperone protein Kar2p. The decrease of the co-translational translocation rate in a is not linked to a growth as in the suggesting that the protein a function, is of the in the of the alkaline extracellular protease and the of Kar2p in the of Sls1p (18Boisramé A. Beckerich J-M. Gaillardin C.M. J. Biol. Chem. 1996; 271: 11668-11675Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were not further in a the mutant and the protein allows us to a model for Sls1p function in the translocation Sls1p could first to the at the lumenal of the translocation The preprotein could then be directly transferred to the chaperone protein, Kar2p that is to the translocation site through its with in that the crossing of the Sls1p could be to the translocation complex through its interaction with Kar2p that thus first to the preprotein and then to Sls1p. Sls1p and Kar2p could as as has been for Tim44 and mHsp70 U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar). an could a of secretory for the majority of on the ribosomes be sufficient to ensure their across the ER membrane Blobel G. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). In the presence of Sls1−5p, the of the preprotein from one partner to the other be interaction is This study thus evidence of Sls1p involvement in the co-translational translocation process that relies on its with Kar2p. Sls1p does not the domain present in Sec63p, the partner of Kar2p in the yeast ER membrane (11Lyman S.K. Schekman R. J. Cell Biol. 1995; 131: 1163-1171Crossref PubMed Scopus (98) Google Scholar). the that Tim44 a very domain of with the and that at with U. Meijer M. Maarse A.C. Hönlinger A. Dekker P.J.T. Pfanner N. Rassow J. EMBO J. 1997; 16: 2205-2216Crossref PubMed Scopus (55) Google Scholar), that of the Hsp70 protein family in translocation are not to the protein Moreover, the complex does not at a we can that Kar2p to at a partner in the translocation This partner could be a of the S. Sec63p as the two proteins were shown to be involved in co-translational translocation J. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) or membrane protein with to Kar2p function in the translocation pore at an of the co-translational translocation process B.D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of Sls1p with secretory protein and on ATP of the complex function in translocation be in an in vitro translocation system. We M. for the and P. for the and