title: Gp78 cooperates with RMA1 in endoplasmic reticulum-associated degradation of CFTRDeltaF508.
abstract: Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRDeltaF508, by specifically promoting ubiquitylation of CFTRDeltaF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRDeltaF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRDeltaF508 and catalyzes further polyubiquitylation of CFTRDeltaF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRDeltaF508.
authors.affiliation: Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Morito
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Hirao
authors.forename: Yukako
authors.initials: Y
authors.lastname: Oda
authors.forename: Nobuko
authors.initials: N
authors.lastname: Hosokawa
authors.forename: Fuminori
authors.initials: F
authors.lastname: Tokunaga
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Cyr
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Iwai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nagata
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C505032:CFTR protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C555000:AMFR protein, human; C555002:Amfr protein, mouse; C529960:RNF5 protein, mouse; D060285:Receptors, Autocrine Motility Factor; C498235:Syvn1 protein, mouse; D044767:Ubiquitin-Protein Ligases