Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis

doi:10.1074/jbc.271.41.25630

Degradation of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Endoplasmic Reticulum Membranes Is Accelerated as a Result of Increased Susceptibility to Proteolysis*

From the ^‡ Department of Biological Sciences, Stanford University, Stanford, California 94305-5020 and the
^″ Research Center for Biological Function, The Kitasato Institute, Tokyo 108, Japan

^‴ Supported by National Institutes of Health Grant 5 R01 HL26502-15. To whom correspondence should be addressed:
Dept. of Biological Sciences, Stanford University, Stanford, CA 94305-5020
. Tel.: 415-725-7001; Fax: 415-725-5807; E-mail: rdsimoni{at}leland.stanford.edu

↵^∥ Present address: Niigata University School of Medicine, Niigata, Japan.

Abstract

The endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to regulated degradation when cells are presented with an excess of sterols or mevalonate. In this report, we demonstrate the degradation of HMG-CoA reductase in ER membranes prepared from cells which have been pretreated with mevalonate or sterols prior to membrane purification. Degradation of HMG-CoA reductase in membranes prepared from pretreated cells is more rapid than in membranes prepared from cells which have received no regulatory molecules. In vitro degradation is blocked by protease inhibitors previously shown to inhibit reductase degradation in vivo and is specific for intact HMG-CoA reductase. The lumenal contents of the ER membranes are dispensible for the regulated proteolysis and the proteases responsible for reductase degradation are stably associated with the ER membrane. Regulated proteolysis of HMG-CoA reductase is inhibited by lactacystin, a newly defined inhibitor of the multicatalytic protease, the proteasome, and in vitro degradation of reductase correlates with the presence of proteasome subunits in purified ER membranes. The ubiquitin system for protein degradation, which has recently been shown to be required for the degradation of several ER membrane proteins, is not required for the degradation of HMG-CoA reductase. Finally, we conclude that the regulated proteolysis of HMG-CoA reductase in response to regulatory molecules such as mevalonate or sterols is mediated by increased susceptibility of the reductase to ER proteases, rather than the induction of a new proteolytic activity.

Footnotes

↵^§ Supported by National Institutes of Health Fellowship 1 F32 GM17363-01.
↵^¶ Supported in part by National Cancer Institute Training Grant ST32 CA09302.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

HMG-CoA

3-hydroxy-3-methylglutaryl coenzyme A

MEM

minimum essential medium

FCS

fetal calf serum

LPS

lipid-poor serum

ALLN

N-acetyl-leucyl-leucyl-norleucinal

PMSF

phenylmethylsulfonyl fluoride

APMSF

(4-amidophenyl)methanesulfonyl fluoride

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

CHO

Chinese hamster ovary

E64

N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine

CFTR

cystic fibrosis transmembrane conductance regulator.
- Received June 4, 1996.
- Revision received July 22, 1996.