3-Hydroxy-3-methylglutaryl coenzyme A reductase is sterol-dependently cleaved by cathepsin L-type cysteine protease in the isolated endoplasmic reticulum

We have recently shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an endoplasmic reticulum (ER) membrane protein, is degraded in ER membranes prepared from sterol pretreated cells and that such degradatio n is catalyzed by a cysteine protease within the reductase membrane domain. The use of various protease inhibitors suggested that degradation of HMG-C oA reductase in vitro is catalyzed by a cathepsin L-type cysteine protease, Purified ER contains E-64-sensitive cathepsin L activity whose inhibitor s ensitivity was well matched to that of HMG-CoA reductase degradation in vit ro. CLIK-148 (cathepsin L inhibitor) inhibited degradation of HMG-CoA reduc tase in vitro. Purified cathepsin L also efficiently cleaved HMG-CoA reduct ase in isolated ER preparations. To determine whether a cathepsin L-type cy steine protease is involved in sterol-regulated degradation of HMG-CoA redu ctase in vivo we examined the effect of E-64d, a membrane-permeable cystein e protease inhibitor, in living cells, While lactacystin, a proteasome-spec ific inhibitor, inhibited sterol-dependent degradation of HMG-CoA reductase , E-64d failed to do so. In contrast, degradation of HMG-CoA reductase in s onicated cells was inhibited by E-64d, CLIK-148, and leupeptin but not by l actacystin, Our results indicate that HMG-CoA reductase is degraded by the proteasome under normal conditions in living cells and that it is cleaved b y cathepsin L leaked from lysosomes during preparation of the ER, thus clar ifying the apparently paradoxical in vivo and in vitro results. Cathepsin L -dependent proteolysis was observed to occur preferentially in sterol-pretr eated cells, suggesting that sterol treatment results in conformational cha nges in HMG-CoA reductase that make it more susceptible to such cleavage. ( C) 2001 Academic Press.