Autoubiquitination of the Hrd1 Ligase Triggers Protein Retrotranslocation in ERAD.
Misfolded proteins of the ER are retrotranslocated to the cytosol, where they are polyubiquitinated, extracted from the membrane, and degraded by the proteasome. To investigate how the ER-associated Degradation (ERAD) machinery can accomplish retrotranslocation of a misfolded luminal protein domain across a lipid bilayer, we have reconstituted retrotranslocation with purified S. cerevisiae proteins, using proteoliposomes containing the multi-spanning ubiquitin ligase Hrd1. Retrotranslocation of the luminal domain of a membrane-spanning substrate is triggered by autoubiquitination of Hrd1. Substrate ubiquitination is a subsequent event, and the Cdc48 ATPase that completes substrate extraction from the membrane is not required for retrotranslocation. Ubiquitination of lysines in Hrd1's RING-finger domain is required for substrate retrotranslocation in vitro and for ERAD in vivo. Our results suggest that Hrd1 forms a ubiquitin-gated protein-conducting channel.
Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Electronic address: ryan_baldridge@hms.harvard.edu.
Ryan D
RD
Baldridge
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.
Tom A
TA
Rapoport
D000251:Adenosine Triphosphatases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D060746:Endoplasmic Reticulum-Associated Degradation; D017510:Protein Folding; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D011510:Proteolipids; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; C104621:HRD1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein