Yeast Dgk1p diacylglycerol kinase is regulated by the transcription factor Reb1p

Abstract

In the yeast Saccharomyces cerevisiae, Dgk1p diacylglycerol (DAG) kinase catalyzes the CTP‐dependent phosphorylation of DAG to form phosphatidate. The enzyme activity is required for phospholipid synthesis during growth resumption from stationary phase when de novo fatty acid synthesis is inhibited. Growth resumption is accompanied by the induction of DAG kinase activity, which implicates a potential transcriptional regulation of DGK1. The promoter of DGK1 contains a consensus sequence TTACCCG (−167 to −173) for the binding of the transcription factor Reb1p. Using purified recombinant His6‐Reb1p, we performed electrophoretic mobility shift assays to examine the interaction of Reb1p with the potential Reb1p binding site. The interaction of His6‐Reb1p with a radiolabeled oligonucleotide probe was dependent on the concentration of His6‐Reb1p, and non‐labeled probe competed with this interaction. Mutations in the Reb1p binding site reduced this interaction. The DGK1 gene with a mutation in the Reb1p binding site (DGK1reb1) was expressed in dgk1Δ mutant cells and caused a low constitutive level of DGK1 mRNA, Dgk1p, and DAG kinase activity. In addition, the dgk1Δ cells expressing DGK1reb1 showed a defect in growth resumption from stationary phase and the mobilization of triacylglycerol for membrane phospholipid synthesis was compromised. Supported by NIH grant GM‐28140.