Membrane Extraction of HMG CoA Reductase as Determined by Susceptibility of Lumenal Epitope to In Vitro Protease Digestion.

Autor:	Morris LL; Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9046, USA., DeBose-Boyd RA; Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390-9046, USA. Russell.DeBose-Boyd@utsouthwestern.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2017; Vol. 1583, pp. 193-199.
DOI:	10.1007/978-1-4939-6875-6_14
Abstrakt:	Although many aspects of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway have been elucidated, methods to detect and examine intermediate steps in the process are lacking. Here, we describe the use of a protease protection assay to study the metabolically regulated ERAD substrate HMG CoA reductase. Studies utilizing this assay reveal that ubiquitinated reductase becomes extracted across the ER membrane prior to its cytosolic release and proteasomal degradation through reactions mediated by distinct AAA-ATPases. A similar approach could be applied to other substrates to determine whether membrane extraction is an intermediate step in their ERAD.
Databáze:	MEDLINE
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