Phosphatidate biosynthesis in the yeast Saccharomyces cerevisiae.

Abstract

The properties of phosphatidate-synthesizing enzymes dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) in Saccharomyces cerevisiae total membrane fraction were studied. Though similar with respect to thermal stability and optimum temperature, their pH optima and sensitivity to N-ethylmaleimide differed. S. cerevisiae total membrane fraction also exhibited NADPH-acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) activity. Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Aerobically grown yeast showed twice the activity of anaerobically grown cells for all three enzymes. Reductase activity in both wild type and tpa1 mutant cells grown on a non-fermentable carbon source was about 2-fold greater than in glucose-grown cells. Activity increased as wild type glucose-grown cells entered the stationary phase but was constitutively high on non-fermentable carbon sources throughout the growth phase. The subcellular localization of these enzymes was studied by both differential and density-gradient centrifugations. Upon differential centrifugation GPAT, DHAPAT and reductase activity was found mainly in the light mitochondrial (LM) fraction. On Nycodenz density gradient centrifugation of the LM fraction, these three phosphatidate-synthesizing enzymes were localized to the smooth endoplasmic reticulum and not in peroxisomes. Reductase solubilized from dried commercial baker's yeast with CHAPS detergent was partially purified by a regimen of gel filtration, anion exchange and affinity chromatographies. A single major 60 kDa band was obtained upon SDS-PAGE. The partially purified enzyme prefers its physiological palmitoyl DHAP substrate to hexadecyl DHAP. Its apparent K$\sb{\rm m}$ for NADPH is similar to that of the mammalian enzyme. The reductase is inhibited by NADP$\sp+$ and strongly inhibited by palmitoyl CoA. Since S. cerevisiae lacks ether glycerolipids and alkyl DHAP synthase yet exhibits both DHAPAT and reductase activities, the acyl DHAP pathway likely contributes to non-ether glycerolipid biosynthesis in this organism.