Overproduction of a M r 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells

We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP+ oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [35 S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band ofM r 92,000 and a minor band ofM r 63,000. We conclude that theM r 92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i ) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii ) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii ) Isolation and solubilization of [35 S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of theM r 92,000 protein and the appearance of two proteins ofM r 52,000 and 38,000. (iv ) Analysis of cells labeled for 30 min with [35 S]methionine, well under the half-life of HMG-CoA reductase, revealed only theM r 92,000 protein to be present in total cell extract. (v ) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase ofM r 62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982)Proc. Natl. Acad. Sci. USA 79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO4 gel electrophoresis. Analysis of C100 cells labeled with [35 S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is theM r 92,000, rather than theM r 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.