In vitro analysis of Hrd1p-mediated retrotranslocation of its multispanning membrane substrate 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase.

ABSTRACT: Endoplasmic reticulum (ER)-associated degradation (ERAD) is responsible for the ubiquitin-mediated destruction of both misfolded and normal ER-resident proteins. ERAD substrates must be moved from the ER to the cytoplasm for ubiquitination and proteasomal destruction by a process called retrotranslocation. Many aspects of retrotranslocation are poorly understood, including its generality, the cellular components required, the energetics, and the mechanism of transfer through the ER membrane. To address these questions, we have developed an in vitro assay, using the 8-transmembrane span ER-resident Hmg2p isozyme of HMG-CoA reductase fused to GFP, which undergoes regulated ERAD mediated by the Hrd1p ubiquitin ligase. We have now directly demonstrated in vitro retrotranslocation of full-lengt

SUBMITTER: Garza RM 

PROVIDER: S-EPMC2685653 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature