Regulation of Yeast Glycerol‐3‐Phosphate Acyltransferase Gpt2p/Gat1p Through Phosphorylation

Abstract

Glycerol‐3‐phosphate acyltransferases (GPAT) catalyze the acylation of glycerol‐3‐phosphate, the first and rate limiting reaction in de novo synthesis of phosphatidic acid (PA). GPAT redundancy led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism S. cerevisiae two GPATs have been identified, Gpt2 and Sct1. Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid normally induces lipid particle accumulation, cells lacking GPT2 failed to accumulate these neutral lipids storage compartments, exhibited a strong growth defect, and cell viability was reduced. The fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under this growth condition attracted our interest. We have identified 3 highly conserved phosphorylation sites in Gpt2 by mass spectrometry, localized to a Ser rich region in the carboxy‐end. Ser to Ala triple mutations as well as deletion of the Ser rich region, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, both Gpt2 mutant forms retain enzymatic activity sufficient to support life of cells lacking Sct1. Microscopic inspection of phosphomutants revealed changes in lipid droplet formation. In line, cells overproducing these mutants exacerbated this phenotype, suggesting that lack of Gpt2 phosphorylation leads to abnormal accumulation of lipid droplets and carbon source consumption. The role of the Gpt2 serine‐rich carboxy‐end in mediating protein‐protein interactions is currently investigated