Animal cell mutants defective in sterol metabolism: a specific selection procedure and partial characterization of...

By using a chemically defined medium, a general and highly specific procedure was devised to select for mutant cells with less abundant or structurally altered sterol in their surface membranes. Within a certain concentration range, the polyene antibiotic filipin was shown to kill only cells with normal (as opposed to decreased) membrane sterol levels. Sterol-requiring derivatives of LM cells were isolated by chemical mutagenesis, filipin treatment, and cloning followed by replica plating in soft agar. Mutants (S1 and S2) are described which, when compared to normal cells, show decreased synthesis of demosterol in vivo from acetate and mevalonate relative to cell number or to fatty acid synthesis. When exogenous sterol is supplied, mutants S1 and S2 grow normally in suspension culture. However, when deprived of sterol supplement, mutant S1 grows slower than wild type cells and mutant S2 lyses within one to two generations. Gas/liquid chromatography revealed that the mutants contained a normal spectrum of fatty acids including unsaturated fatty acyl groups but, unlike wildtype cells, they have less abundant (mutant S1) or no (mutant S2) desmosterol in either the presence or absence of exogenous cholesterol. In vitro experiments with mevalonate as the substrate suggest that the defect in both mutants is in a demethylation reaction subsequent to lanosterol synthesis. The selection method developed here may permit the isolation of mutants with defective membrane incorporation of sterols and other polyisoprenoids as well as defective synthesis of these compounds.