Novel Interactions of Yeast Glycerol‐3‐Phosphate Acyltransferases

Abstract

Glycerol‐3‐phosphate acyltransferases (GPATs) catalyze the first committed and rate limiting step in the de novo synthesis of glycerophospholipids and neutral triacylglycerols. GPATs transfer an acyl group from acyl‐coenzyme A to the sn‐1 position of glycerol‐3‐phosphate to produce 1‐acyl glycerol‐3‐phosphate (lysophosphatidic acid). Lysophosphatidic acid can then be further acylated by a second acyltransferase to produce phosphatidic acid, a signalling lipid and key intermediate in lipid biosynthesis. In the model organism yeast Saccharomyces cerevisiae two GPATs, Gpt2 and Sct1, have been identified. Both are integral membrane proteins located to the endoplasmic reticulum (ER), differentially enriched in ER domains. Interestingly, we have shown that Gpt2 (but not Sct1) is responsible for channeling oleate (C18:1) into triacylglycerol that is eventually stored in lipid droplets. Furthermore, our studies on oleate metabolism unveiled novel ER structures that surround lipid droplets and are differentially enriched in Gpt2 while devoid of Sct1. In addition, we also uncovered a dynamic post‐translational regulation of Gpt2 and Sct1 through phosphorylation that responds to growth phase, carbon source and cellular GPAT imbalance. The aim of this work was to understand how GPAT interactions with other proteins contribute to their spatial distribution in the ER and to lipid synthesis partitioning. In order to identify potential protein interactors, Gpt2 and Sct1 were overproduced in cells deleted for endogenous GPATs and then isolated from lysates and microsomes using co‐immuno‐affinity and Ni‐NTA affinity chromatography purification techniques. Potential interactors co‐purifying with Gpt2 or Sct1 were identified through liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Our results suggest that Gpt2 and Sct1 differentially associate with ER sites where inter‐organelle transport of lipids occurs. Of importance are the contact sites between ER‐mitochondria, ER‐lipid droplets and the nuclear‐vacuolar junction.Support or Funding InformationThis work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ.