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

Abstract

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 of M(r) 92,000 and a minor band of M(r) 63,000. We conclude that the M(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 the M(r) 92,000 protein and the appearance of two proteins of M(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 the M(r) 92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of M(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 NaDodSO(4) 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 the M(r) 92,000, rather than the M(r) 63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.