title: Key steps in ERAD of luminal ER proteins reconstituted with purified components.
abstract: Misfolded proteins of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we use purified components from Saccharomyces cerevisiae to analyze the mechanism of retrotranslocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process in which the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain and discriminates misfolded from folded polypeptides. Both Hrd1p and substrate are polyubiquitinated, resulting in the binding of Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the deubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of polyubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retrotranslocation in which Hrd1p forms a membrane conduit for misfolded proteins.
authors.affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Electronic address: alexander_stein@hms.harvard.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Stein
authors.affiliation: Department of Cell and Developmental Biology, CRG-Centre de Regulacio Genomica, Dr. Aiguader 88, 08003 Barcelona, Spain.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Ruggiano
authors.affiliation: Department of Cell and Developmental Biology, CRG-Centre de Regulacio Genomica, Dr. Aiguader 88, 08003 Barcelona, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Carvalho
authors.affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Electronic address: tom_rapoport@hms.harvard.edu.
authors.forename: Tom A
authors.initials: TA
authors.lastname: Rapoport
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D060746:Endoplasmic Reticulum-Associated Degradation; D017510:Protein Folding; D011510:Proteolipids / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination; D000074405:Valosin Containing Protein
chemical_list: D018797:Cell Cycle Proteins; D011510:Proteolipids; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; C104621:HRD1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C000592929:Otu1 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein