Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin - Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase

Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, ha s a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhi bited a high cytolytic sensitivity to lysenin, but treatment with sphingomy elinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lysenin-resistant variants from mutagenized CH O cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wildtype cells. One mutant str ain was severely defective in sphingomyelin synthesis but not glycosphingol ipid synthesis, and another strain (designated LY-B) was incapable of de no vo synthesis of any sphingolipid species and had no activity of serine palm itoyltransferase (SPT; EC 2.3.1.50) catalyzing the first step of sphingolip id biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, an d transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT a ctivity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protei n to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 pro tein antibody co-immunoprecipitated both SPT activity and the wild-type LCB 1 protein with the LCB2 protein. Thus, cell surface sphingomyelin ia; essen tial for lysenin-induced cytolysis, and lysenin is a useful tool for isolat ion of sphingomyelin-deficient mutants, Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.