Degradation of HMG-CoA Reductase in Vitro
CLEAVAGE IN THE MEMBRANE DOMAIN BY A MEMBRANE-BOUND CYSTEINE PROTEASE*
Abstract
We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996)J. Biol. Chem. 271, 25630–25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro.
In order to determine the site(s) of proteolysis of HMG-CoA reductasein vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein.
We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.
Footnotes
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↵* This work was supported by National Institutes of Health Grant HL26502.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.
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↵‡ Present address: Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan.
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↵§ To whom correspondence should be addressed: Dept. of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Tel.: 650-725-4817; Fax: 650-725-5807; E-mail:rdsimoni{at}leland.stanford.edu.
- Abbreviations:
- HMG-CoA
-
3-hydroxy-3-methylglutaryl coenzyme A
- ER
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endoplasmic reticulum
- MEM
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minimum essential medium
- FCS
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fetal calf serum
- LPS
-
lipid-poor serum
- ALLN
-
N-acetyl-leucyl-leucyl-norleucinal
- PMSF
-
phenylmethylsulfonyl fluoride
- PSI
-
carbobennzoxy-l-isoleucyl-γ-t-butyl-l-glutamyl-l-alanyl-l-leucinal
- MG115
-
carbobennzoxy-l-leucyl-l-leucyl-l-norvalinal
- E64
-
N-[N-(l-3-trans-carboxirane-2-carbonyl)-l-leucyl]-agmatine
- NEM
-
N-ethylmaleimide
- AMC
-
7amino-4-methylcoumarin
- sLLVY-AMC
-
succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin
- PAGE
-
polyacrylamide gel electrophoresis
- CHO
-
Chinese hamster ovary
- HTG
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heptylthioglucoside
- APMSF
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(4-amidinophenyl)-methanesulfonyl fluoride.
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- Received March 3, 1998.
- Revision received May 11, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
