{"doi":"10.1074/jbc.m900813200","title":"Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation","abstract":null,"journal":"Journal of Biological Chemistry","year":2009,"id":588442,"datarank":3.0638796474628487,"base_score":4.1588830833596715,"endowment":4.1588830833596715,"self_citation_contribution":0.6238324625039509,"citation_network_contribution":2.440047184958898,"self_endowment_contribution":0.6238324625039509,"citer_contribution":2.440047184958898,"corpus_percentile":null,"corpus_rank":null,"citation_count":63,"citer_count":62,"citers_with_citation_signal":57,"citers_with_endowment":57,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1505432,"name":"Shuh-ichi Nishikawa","orcid":null,"position":1,"is_corresponding":false},{"id":1505433,"name":"Toshiya Endo","orcid":"0000-0001-8548-1584","position":2,"is_corresponding":false},{"id":1505431,"name":"Machiko Sakoh-Nakatogawa","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation","abstract":"The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway. The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; MHD, mannosidase homology domain; EDEM, ER degradation enhancing α-mannosidase-like protein; DTT, dithiothreitol; CPY, caboxypeptidase Y; CPY*, a mutated version of caboxypeptidase Y; PMSF, phenylmethylsulfonyl fluoride; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; PDI, protein disulfide isomerase; WT, wild type. 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; MHD, mannosidase homology domain; EDEM, ER degradation enhancing α-mannosidase-like protein; DTT, dithiothreitol; CPY, caboxypeptidase Y; CPY*, a mutated version of caboxypeptidase Y; PMSF, phenylmethylsulfonyl fluoride; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; PDI, protein disulfide isomerase; WT, wild type. is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1Buck T.M. Wright C.M. Brodsk J.L. Semin. Cell Dev. Biol. 2007; 18: 751-761Crossref PubMed Scopus (65) Google Scholar, 2Anelli T. Sitia R. EMBO J. 2008; 27: 315-327Crossref PubMed Scopus (454) Google Scholar). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3Creighton T.E. Zapun A. Darby N.J. Trends. Biotech. 1995; 13: 18-23Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 4Molinari M. Helenius A. Nature. 1999; 402: 90-93Crossref PubMed Scopus (271) Google Scholar), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5Sevier C.S. Kaiser C.A. Antioxid. Redox. Signal. 2006; 8: 797-811Crossref PubMed Scopus (91) Google Scholar, 6Appenzeller-Herzog C. Ellgaard L. Biochim. Biophys. Acta. 2008; 1783: 535-548Crossref PubMed Scopus (302) Google Scholar). In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (232) Google Scholar). The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man9GlcNAc2 oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (299) Google Scholar). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar, 11Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar, 12Hirao K. Natsuka Y. Tamura T. Wada I. Morito D. Natsuka S. Romero P. Sleno B. Tremblay L.O. Herscovics A. Nagata K. Hosokawa N. J. Biol. Chem. 2006; 281: 9650-9658Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 13Olivari S. Cali T. Salo K.E. Paganetti P. Ruddock L.W. Molinari M. Biochem. Biophys. Res. Commun. 2006; 349: 1278-1284Crossref PubMed Scopus (142) Google Scholar, 14Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 15Clerc S. Hirsch C. Oggier D.M. Deprex P. Jakob C. Sommer T. Aebi M. J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (199) Google Scholar), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Bhamidipati A. Denic V. Quan E.M. Weissman J.S. Mol. Cell. 2005; 16: 741-751Abstract Full Text Full Text PDF Scopus (189) Google Scholar, 17Kim W. Spear E.D. Ng D.T. Mol. Cell. 2005; 16: 753-764Abstract Full Text Full Text PDF Scopus (149) Google Scholar, 18Szathmary R. Bielmann R. Nita-Lazar M. Burda P. Jakob C.A. Mol. Cell. 2005; 16: 765-775Abstract Full Text Full Text PDF Scopus (164) Google Scholar). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1). After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19Nishikawa S. Fewell S.W. Kato Y. Brodsky J.L. Endo T. J. Cell Biol. 2001; 153: 1061-1070Crossref PubMed Scopus (255) Google Scholar). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20Gilbert H.F. J. Biol. Chem. 1997; 272: 29399-29402Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 238: 38-42Google Scholar, 22Gillece P. Luz J.M. Lennarz W. de la Cruz F.J. Römisch K. J. Cell Biol. 1999; 147: 1443-1456Crossref PubMed Scopus (151) Google Scholar, 23Nørgaard R. Westphal V. Tachibana C. Alsøe L. Horst B. Winther J.R. J. Cell Biol. 2001; 152: PubMed Scopus Google Scholar). The yeast PDI, two of which have a as by a C-terminal the ER its PDI a disulfide with its substrate an intermolecular disulfide bond between the cysteine residues of the of PDI and the substrate of PDI as an interactions between the C-terminal domain of Mnl1p and PDI intermolecular disulfide PDI is for formation of the essential disulfide bond in the of Mnl1p, which in turn stabilizes and the for interactions between Mnl1p and PDI in the ERAD be and used in this are J.S. Mol. Cell Biol. 8: PubMed Scopus Google Scholar), J.S. Mol. Cell Biol. 8: PubMed Scopus Google Scholar), S. Endo T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and J. Cell Biol. PubMed Scopus Google Scholar). Lennarz Cell. Full Text PDF PubMed Scopus Google is a from K. Römisch Yeast cells were in yeast and or yeast with The was as A the was by K. M. 1995; PubMed Scopus Google as a with and The by of the and of the was into and were of the was by and the was Mnl1p was generated as The was by yeast as a with and The was with and and into the of P. PubMed Google to A was between the and the of the by to A for the sequence was by with and T. K. C. N. Endo T. Mol. Biol. Cell. PubMed Scopus Google as a The was with and into the of to The of was into the of I. A. Y. Y. 8: PubMed Scopus Google to A series of the Mnl1p the mutants for the conserved residues in the C-terminal domain of Mnl1p, and the Mnl1p were by as a and are from W. J. Lennarz and a series of mutants of PDI were by as a or was into and was by the on of and were as by and Brodsky Brodsky J.L. J. Cell Biol. PubMed Scopus Google Scholar). membrane were as Yeast cells from were in of and and by for The cells were converted to by in of 20 and for The were in of PMSF, and and by for with for two with an on In Fig. was from this Cell were with the as used in and for to The was for and the was used as the membrane or were in 20 and for with and on for and for to The was with 20 and and with or protein for more than The were with 20 and and with for In Fig. and were in for and the proteins were by with on for by for The were with and the proteins were in 20 and and for were by for The was with 20 and and with for The were with 20 and with for was to the to the mutants of Mnl1p. of Mnl1p with The signal sequence and the are in and from cells and a series of mutants and from with a were as in Fig. The and the by the intermolecular disulfide or and of the are indicated as and proteins in the in the of from or cells from a were by and with between the C-terminal domain of Mnl1p and PDI intermolecular disulfide sequence of Mnl1p with its fungal The are: C. and and residues are with and residues with were with from cells wild and a series of mutants from a and from with a were as in Fig. The and the by the intermolecular disulfide and of the are indicated as and that not form a are by membrane were from cells wild and a series of mutants from a and from with a in the of and were to as in Fig. were with or in 20 in the or of on for The was by of The were with for on and the proteins were by for The were with and were by for were in and and for The were with PMSF, and A. was to and the were for of were in 20 and or on for The were for to and cells were to and was to the or the of an of was and was in the of for on Cell were as by and M. and U. S. A. Scholar). of with and cells were in and by with proteins were by for by with Protein were in in the of or After on for for the were for and by a form of PDI, the were in for and the was with for proteins were with and in in the of or After on for for the were for and by Mnl1p an ER Protein with a was to in the yeast ER (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We first whether the the of Mnl1p functions as a cleavable signal sequence or a to the protein to the ER of from cells Mnl1p with the a which to with that Mnl1p N-linked carbohydrate chains which has was used as a Mnl1p an ER luminal protein but of the membrane with with Mnl1p and BiP were in the whereas an ER membrane protein, was in the and of the with of these proteins and that Mnl1p is an ER luminal protein, but not to the ER and BiP in the yeast a of the of the ER signal J. Cell Biol. PubMed Scopus Google Scholar), molecular from with different of than the N-terminal of Mnl1p functions as a cleavable signal sequence that the protein to the ER Mnl1p with Mnl1p facilitates ERAD of in the ER (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar), for its possible proteins with Mnl1p in the with were with and to with the PDI was found to be retained on the in was from a A and were to with the was in turn in the and in or BiP was not by the Yos9p and or in the not We whether the interactions between Mnl1p and PDI intermolecular disulfide of Mnl1p the proteins were by from the and with in the of which can to prevent disulfide was with the and by or by with and Mnl1p two molecular of by in to the of to the Mnl1p by a and but not and The were also by and that the to by intermolecular disulfide between Mnl1p and PDI. of the the of Mnl1p from a suggests that the Mnl1p with PDI and that of PDI with Mnl1p disulfide not and Disulfide of Mnl1p by cysteine residues in its which are are in the and in the C-terminal domain of Mnl1p. the cysteine residues involved in the intermolecular disulfide bond with PDI, a series of mutants of Mnl1p to in which of the was by and their disulfide bond formation by by with and The and of the in and and of the and in the of the two and and/or of Mnl1p form intermolecular disulfide with PDI, and the and of to with disulfide and the of the that the of the Mnl1p for different mutants but not The and mutants than the wild Mnl1p, that the to the Mnl1p with and as and the to the one a disulfide bond between and as The and mutants of PDI have in the in one of the two domains Fig. and still have disulfide activity B. Tachibana C. Winther J.R. J. Cell Biol. 1997; PubMed Scopus Google Scholar, E.M. Ellgaard L. Weissman J.S. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). Mnl1p in cells the or PDI mutants only the for the Mnl1p that of and both of the two domains in PDI. the for the Mnl1p that and were only In interactions of Mnl1p with PDI and/or in the C-terminal domain of Mnl1p disulfide formation between and in the N-terminal by PDI. PDI with Mnl1p as as Disulfide formation of the disulfide bond between and Mnl1p to with PDI the intermolecular disulfide bond and/or We whether the intermolecular disulfide bond is for the prolonged interactions between Mnl1p and PDI. this membrane from cells in the of Mnl1p is in the that the not contain the by a disulfide bond the form of is still in the that the disulfide bond between and was more by than the intermolecular disulfide bond between and PDI. the were with with in the of and to with the PDI was still with the of interactions between Mnl1p and PDI of the intermolecular disulfide bond PDI was with and mutants as as the wild protein and of PDI for the which is less in formation of the disulfide bond between and PDI was not with the or mutants and that the disulfide bond is essential for prolonged association of PDI with Mnl1p in the of and residues in Mnl1p are involved in interactions with PDI the intermolecular disulfide and of Mnl1p are in the C-terminal which is not conserved in EDEM the C-terminal domain is only found in fungal of Mnl1p and does not show any homology to known protein residues in the of and are conserved among the fungal proteins Mnl1p and with of of these residues with to their interactions with PDI. the of in the C-terminal domain of Mnl1p on formation of the disulfide bond between and in the and intermolecular disulfide bond and/or with PDI. these not interactions of with PDI and the and mutants are in formation of the disulfide interactions with PDI and the of and the and mutants to form the disulfide bond between and as in of only form of the and Fig. the not in with PDI but was in introduction of the disulfide bond between and the interactions of the C-terminal domain mutants of Mnl1p with PDI PDI was not with or of and In the which could form the disulfide bond, the disulfide bond was more to by than in wild of mutants molecular and they were not with that they are of the Mnl1p. that the conserved residues in the C-terminal domain including and are for interactions with PDI, which is essential for formation between and in the of Mnl1p, is not whether these residues are involved in the interactions with PDI or folding of the C-terminal The intermolecular disulfide bond and/or of Mnl1p with PDI also facilitates the association of the C-terminal domain of Mnl1p with PDI to form the disulfide bond in the the disulfide bond between and is to prolonged association of PDI with Mnl1p as Disulfide between and for the Mnl1p in the of the disulfide in Mnl1p in the Mnl1p is for the ERAD of misfolded including CPY*, a of (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar). protein was with the was degraded in wild cells with a of whereas in the Mnl1p, was and degraded with a of as (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). However, of the or in the not the ERAD In of the mutants the ERAD of the the in which the disulfide bond was only was the ERAD of the was that the disulfide bond formation between and in the is essential for the ERAD activity of Mnl1p. is the role of the disulfide bond between and of Mnl1p in its ERAD We the of the in Mnl1p by membrane from cells wild or Mnl1p were with and to with of wild and Mnl1p generated of with with and only of the and the and mutants were to the disulfide bond between and is essential for the stably folded of the MHD, which important for the ERAD activity of Mnl1p. that the disulfide bond to prolonged association of PDI with Mnl1p the that associated PDI also the ERAD Mnl1p. In EDEM a which was proposed to misfolded proteins for their retrotranslocation to the for degradation R. J. K. Thomas D.Y. Nagata K. 2008; PubMed Scopus Google Scholar). However, is not whether PDI in yeast can take the role of in the ERAD whether PDI with can function as a as as an in the ER is in (5Sevier C.S. Kaiser C.A. Antioxid. Redox. Signal. 2006; 8: 797-811Crossref PubMed Scopus (91) Google Scholar, 6Appenzeller-Herzog C. Ellgaard L. Biochim. Biophys. Acta. 2008; 1783: 535-548Crossref PubMed Scopus (302) Google Scholar). We first to the of PDI in yeast cells by of yeast cells with and with the in the of We the Kaiser C.A. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google that PDI by AMS, that PDI was in the and However, of the of PDI with with two and mutants with that the of not be to modified We used of B. J. Cell Biol. PubMed Scopus Google to with After by with the and which with Mnl1p, the with and two modified and and modified and the for the were for the than the for could of for PDI and with two modified of the of Mnl1p. that a of PDI is in a which have in the AMS, in In the found that the disulfide bond between and in the of Mnl1p is essential for the ERAD and its formation PDI in the ER which is stably associated with Mnl1p. The association of PDI with Mnl1p conserved residues in the C-terminal domain of Mnl1p and is by the intermolecular disulfide or in the C-terminal domain of Mnl1p. Mnl1p the C-terminal domain to form a disulfide bond between and as (supplemental Fig. PDI, which has both and is in turn for its association with Mnl1p (supplemental Fig. The disulfide bond in the MHD, stabilizes the prolonged association of PDI with Mnl1p. of the S. Hirsch C. Oggier D.M. Deprex P. Jakob C. Sommer T. Aebi M. J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (199) Google that Mnl1p interacts with PDI its C-terminal which with Fig. of disulfide bond formation of Mnl1p by PDI first the C-terminal domain of Mnl1p and step to step PDI an intermolecular disulfide bond with or of Mnl1p step to step PDI a disulfide bond between and in the of Mnl1p step to step the disulfide bond between and in turn stabilizes association of PDI with and the intermolecular disulfide bond between PDI and or of Mnl1p is whereas association of PDI and Mnl1p step to step The for the of PDI to the C-terminal domain of Mnl1p for introduction of the disulfide bond in the is not The C-terminal domain of Mnl1p a for PDI to and with of PDI to the folding of the MHD, which is a for the disulfide bond formation between and the activity of PDI in the formation of the disulfide bond between and in the N-terminal the of the C-terminal domain (supplemental Fig. the of Mnl1p with a set of intermolecular disulfide can be is the role of the disulfide bond in the of Mnl1p in the Both and are conserved between Mnl1p and EDEM, whereas but not of Mnl1p is conserved in a yeast α1,2-mannosidase (supplemental Fig. S1). The disulfide is essential for the functional folded of the which is with its by The folded aided by the disulfide bond essential for Mnl1p functions as recognition and/or of the carbohydrate chains of the ERAD is to be EDEM proteins contain conserved and in the MHD, they the C-terminal domain found in Mnl1p and its fungal homologs (supplemental Fig. S1). EDEM proteins that are not conserved in Mnl1p. the of formation of the possible disulfide in EDEM proteins be different from that for Mnl1p revealed is the role of PDI stably associated with Mnl1p formation of the disulfide bond in the The intermolecular disulfide bond between Mnl1p and PDI for in the (supplemental Fig. that the does not represent a folding and/or disulfide bond of Mnl1p. the disulfide bond between and to the interactions between PDI and Mnl1p and the disulfide between PDI and Mnl1p (supplemental Fig. In PDI functions as a of protein and of J. R. T. T. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and a of the PDI is stably associated with interactions including a intermolecular disulfide bond for of P. Cell Biol. 2008; PubMed Scopus (152) Google Scholar). yeast PDI a function in a with Mnl1p. In this be to that retrotranslocation of proteins from the ER to the for degradation and of disulfide in mammals was found to disulfide of misfolded proteins, their ERAD R. J. K. Thomas D.Y. Nagata K. 2008; PubMed Scopus Google Scholar), and PDI in yeast was suggested to a role P. Luz J.M. Lennarz W. de la Cruz F.J. Römisch K. J. Cell Biol. 1999; 147: 1443-1456Crossref PubMed Scopus (151) Google Scholar). PDI stably associated with Mnl1p the ERAD by disulfide in misfolded substrate proteins that were or by Mnl1p. that PDI is not only in the but also in the form with the role of PDI as a for the ERAD substrate by Mnl1p. PDI associated with Mnl1p function as a to of the ERAD which is of the role of BiP in yeast (19Nishikawa S. Fewell S.W. Kato Y. Brodsky J.L. Endo T. J. Cell Biol. 2001; 153: 1061-1070Crossref PubMed Scopus (255) Google and with EDEM in cells Y. Hosokawa N. Wada I. Nagata K. PubMed Scopus Google Scholar). We and the members of the Endo for Römisch for J. 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