Squalene synthase-deficient mutant of Chinese hamster ovary cells.
Squalene synthase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) converts farnesyl pyrophosphate to squalene, the first metabolic step committed solely to the biosynthesis of sterols. Using a fluorescence-activated cell sorting technique designed to screen for cells defective in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, we isolated a squalene synthase-deficient mutant of Chinese hamster ovary cells. The mutant cell line, designated SSD, exhibits less than 7% of the squalene synthase activity of the parental cell line, CHO-HMGal. Both the SSD and the parental cells stably express HMGal, a model protein for studying the regulated degradation of HMG-CoA reductase, which consists of the membrane domain of HMG-CoA reductase fused to bacterial beta-galactosidase (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). In this study, the regulatory effects of mevalonate and compactin on the activity levels of HMGal are substantially reduced in SSD cells as compared to the parental cell line. In lipid-poor medium, SSD cell growth is arrested. The rate of [3H]acetate incorporation into cholesterol for the mutant SSD cells is less than 2% of the rate for the parental cells. However, the incorporation of [3H] squalene into sterols is essentially wild type for SSD cells. When the mutant SSD cells are fed [3H]acetate, radioactivity accumulates in farnesol, much of which is secreted into the medium. By growing SSD cells in lipid-poor medium, a revertant cell type, designated SSR, was isolated. In every assay performed the revertant SSR cells exhibited a phenotype that was essentially wild type, demonstrating that the SSD mutant phenotype was the result of a single mutation.
Bradfute
D L
DL
Department of Biological Sciences, Stanford University, California 94305-5020.
Silva
C J
CJ
Simoni
R D
RD
D000085:Acetates / Q000378:metabolism; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000201:enzymology*; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D006224:Cricetinae; D005204:Farnesol / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000172:deficiency*; D005434:Flow Cytometry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008798:Mevalonic Acid / Q000494:pharmacology; D016296:Mutagenesis
D000085:Acetates; C012258:mevastatin; D005204:Farnesol; D002784:Cholesterol; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D013186:Farnesyl-Diphosphate Farnesyltransferase; D008798:Mevalonic Acid