Membrane extraction of HMG CoA reductase as determined by susceptibility of lumenal epitope to in vitro protease digestion

Lindsey L. Morris, Russell A. DeBose-Boyd

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages193-199
Number of pages7
Volume1583
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1583
ISSN (Print)10643745

Keywords

  • ER-associated degradation
  • Lumenal
  • Membrane extraction
  • Protease
  • Trypsin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Morris, L. L., & DeBose-Boyd, R. A. (2017). Membrane extraction of HMG CoA reductase as determined by susceptibility of lumenal epitope to in vitro protease digestion. In Methods in Molecular Biology (Vol. 1583, pp. 193-199). (Methods in Molecular Biology; Vol. 1583). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6875-6_14