Isolation and characterization of filipin-resistant

A series of LM cell variants resistant to filipin, but not auxotrophic for sterol, was isolated by plating mutagenized, filipin-treated cells on soft agar medium containing no sterol. Cloned variants were assayed for growth in the presence and absence of sterol or unsaturated fatty acid. Filipin-resistant clones whose growth rate was unaffected by the addition of sterol to the medium were further analyzed. Variants were cultured in minimal medium, and plasma membranes prepared from these cultures were analyzed for sterol content, phospholipid head group composition, and unsaturated fatty acyl content. All variants examined showed a decrease in membrane sterol in conjunction with an increase in unsaturated fatty acyl chains in the membrane phospholipids. In addition, several variants exhibited alterations in phospholipid head group composition, including changes in the phosphatidylcholine/phosphatidylethanolamine ratio, or a decrease in sphingomyelin cont e n t . l These observations imply that several different metabolic lesions can give rise to decreased plasma membrane sterol content (and hence to filipin resistance). The range of phospholipid alterations observed in these sterol prototrophs emphasizes the complex interrelationship between membrane sterol and phospholipid structure.-Rintoul, D. A., N. Neungton, and D. F. Silbert. Isolation and characterization of filipin-resistant L M cell variants not auxotrophic for sterol. J. Lipid Res. 1982. 23: 405-409. Supplementary key words phospholipid metabolism plasma membranes unsaturated fatty acid metabolism It has long been recognized that eukaryotic cellular membranes are composed of many different species of lipid (l), and that particular types of membranes from the same cell often vary widely in lipid composition (1, 2). This heterogeneity extends both to lipid classes, e.g., sterols and various types of phospholipids (2); and even to the level of molecular species, e.g., disaturated vs. diunsaturated species of glycerophospholipids (3-5). The function of this structural variability is still relatively mysterious, although some progress has been made in recent years. One approach to the analysis of this heterogeneity has been to isolate variants that are defective in synthesis of certain membrane lipids, and ascertain the effects of this loss on the other membrane lipids. A particularly fruitful approach in this regard has been the analysis of L M cell variants which are auxotrophic for sterol, first reported by Saito, Chou, and Silbert ( 6 ) . The selection technique used to obtain these variants relied on the cytotoxic effect of low concentrations of filipin, a polyene antibiotic which binds to membrane sterol and presumably damages the membrane (7). Various clones of L M cells resistant to this treatment have been characterized; most of them are true sterol auxotrophs and do not proliferate in the absence of exogenous sterol. These mutants have been very useful in analysis of the role of sterol in plasma membrane structure and function (5, 8, 9). However, this selection scheme also gave rise to a number of variant clones which did not require exogenous sterol, but which were filipin-resistant nonetheless. One of these variants, known as S1, has been previously characterized (5, 6 ) ; it seems to be a leaky auxotroph that can synthesize sufficient sterol to grow, albeit slowly, in the absence of exogenous sterol. In the present paper, we analyze several additional filipin-resistant, sterol prototrophic variants in an attempt to gain some further insight into the role of sterol in eukaryotic plasma membrane structure. MATERIALS AND METHODS