DEGRADATION OF HMG-COA REDUCTASE IN-VITRO - CLEAVAGE IN THE MEMBRANE DOMAIN BY A MEMBRANE-BOUND CYSTEINE PROTEASE

We have recently shown that the endoplasmic reticulum (ER) membrane pr otein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is c leaved in isolated membrane fractions enriched for endoplasmic reticul um, Importantly, the cleavage rate is accelerated when the membranes a re prepared from cells that have been pretreated with mevalonate or st erols, physiological regulators of the degradation process in vivo (Mc Gee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1 996) J. Biol. Chem. 271, 25630-25638). In the current study, we furthe r characterize this in vitro cleavage of HMG-CoA reductase, E64, a spe cific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase clea vage in vitro. In contrast, lactacystin, ale inhibitor of the proteaso me, inhibits HMG-CoA reductase degradation in vivo but does not inhibi t the in vitro cleavage. Purified ER fractions contain lactacystin-sen sitive and E64-insensitive proteasome activity as measured by succinyl -Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the p roteasome from purified ER fractions by solubilization with heptylthio glucoside and observed that the detergent extracted, proteasome-deplet ed membrane fractions retain regulated cleavage of HMG-CoA reductase, This indicates that ER-associated proteasome is not involved in degrad ation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prep ared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The si zes 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 re ductase can be cleaved in the membrane-span 8 region by a cysteine pro tease(s) tightly associated with ER membranes.