Isolation and characterization of filipin-resistant LM cell variants not auxotrophic for sterol.

Abstract

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 content. 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.