Isolation and characterization of temperature-sensitive cdsl mutants of Saccharomyces cerevisiae

The essential CDS1 gene of S. ccrcvisiae encoders CDP-diacylglycerol (CDP-DAG) synthase. Variation in CDS1 expression results in changes in activities of other phospholipid biosynthetic enzymes, and consequently alters the apparent initial rate of synthesis and steady-state level of individual phospholipids. To study the molecular mechanism by which ('DP-DAG synthase levels affect phospholipid metabolism, two temperature-sensitive mutants of 1 lie CDS1 gene. rdsl-5 and cdst-31, were constructed by a PCR based mutagenesis approach. A null cdsl mutant bearing the cdsl-5 mutation on a multi-copy plasmid has undetectable CDP-DAG synthase activity in vitro even at permissive temperatures; however these cells do not excrete inositol, indicating the m vivo activity is above the threshold necessary to prevent inositol excretion. The cdsl-31 transformant of the null mutant has reduced activity which is still above the wild-type level. At the restrictive temperature, cdsl-5 mutant cells cease growth within one generation and display significant morphological changes. Introduction of the cdsl-5 mutant on a multi-copy plasmid into wild type yeast conferred the temperature-sensitive phenotype indicating this mutant gene is dominant negative. The rapid arrest of cells carrying cdsl-5 indicates that CDP-DAG may be critical for functions in addition to being an intermediate in bulk phospholipid synthesis.