Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the synthesis of all glycerolipids. It is the committed and rate-limiting step and is redundant in Saccharomyces cerevisiae, mammals, and plants. GPAT controls the formation of lipid intermediates that serve not only as precursors of more-complex lipids but also as intracellular signaling molecules. Saccharomyces cerevisiae possesses two GPATs, encoded by the GAT1 and GAT2 genes. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate-limiting GPAT step. We are particularly interested in identifying molecular determinants mediating lipid metabolic pathway partitioning; therefore, as a starting point, we have performed a detailed study of Gat1p and Gat2p cellular localization. We have compared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Our results indicate Gat1p and Gat2p overlap mostly in their localization and are in fact microsomal GPATs, localized to both perinuclear and cortical endoplasmic reticula in actively proliferating cells. A more detailed analysis suggests a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Furthermore, overexpression of these enzymes in the absence of endogenous GPATs induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth. In addition, our studies also uncovered a dynamic posttranslational regulation of Gat1p and Gat2p and a compensation mechanism through phosphorylation that responds to a cellular GPAT imbalance.The first step in the synthesis of almost all membrane phospholipids and neutral glycerolipids is catalyzed by glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15). This enzyme transfers a fatty acid from fatty acyl coenzyme A to the sn-1 position of glycerol-3-phosphate to produce lysophosphatidic acid (LysoPA). LysoPA is further acylated at the sn-2 position by a separate acyltransferase to produce phosphatidic acid (PA). PA can be either (i) dephosphorylated to produce diacylglycerol (DAG) or (ii) converted to CDP-DAG. These lipids not only are precursors of all glycerolipids but also are dynamic components of signal transduction systems that control cell physiology. Regulated interconversion of signaling lipids like LysoPA, PA, and DAG transmits information in part by their biophysical properties (5) and through lipid-lipid and lipid-protein interactions (18,23,29). The mechanisms of the regulation of PA biosynthesis, of the rate-limiting GPAT step, and of lipid metabolic pathway partitioning are not known (8,12).GPATs are present in bacteria, fungi, plants, and animals. We and others have previously identified a unique gene pair in Saccharomyces cerevisiae, YKR067W (GAT1/GPT2) and YBL011W (GAT2/SCT1), and demonstrated that they code for the major GPATs in this organism (32, 34). Bioinformatic approaches, using a region conserved betw...

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