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.
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Acknowledgments
This work was supported by NIH grants HL20948 and GM090216 to R.D.B.
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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: Gelissen, I., Brown, A. (eds) Cholesterol Homeostasis. Methods in Molecular Biology, vol 1583. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6875-6_14
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DOI: https://doi.org/10.1007/978-1-4939-6875-6_14
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