Abstract
The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases. Here we report the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae. Disruption of the open reading frame YBL011w, corresponding to a gene previously identified as a choline transporter suppressor (SCT1), resulted in a substantial decrease of total cellular G-3-P acyltransferase activity. A yeast strain disrupted at the open reading frame YKR067w, which encodes a protein closely related to Sct1p, also exhibited a dramatic reduction in G-3-P acyltransferase activity. Molecular characterizations of the genes revealed that a missense mutation in YKR067w accounted for a defect in the activities of the G-3-P acyltransferase in the yeast mutant strain TTA1. Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity. These results indicate that YKR067w and YBL011w, designated herein as GAT1 and GAT2(SCT1), respectively, are yeast G-3-P acyltransferase genes. Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferases. The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P acyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors. In contrast, Gat2p(Sct1p) displayed preference toward 16-carbon fatty acids. The most notable of the altered phospholipid compositions of the gat1Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidylinositol ratio. This did not appear to affect the mutants as no growth defect was found. However, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.
Highlights
The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases
It is widely accepted that the initial step of glycerolipid biosynthesis in yeast is mediated by a G-3-P/ DHAP dual substrate acyltransferase (10, 12), and that multiple isoforms of G-3-P acyltransferase are present in yeast (11, 12)
Based on our general interest in eukaryotic fatty acyltransferases, we pursued the identification of G-3-P acyltransferase genes after examining some of the available information on yeast mutants related to phospholipid synthesis
Summary
G-3-P, glycerol 3-phosphate; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PA, phosphatidic acid; LPA, lysophosphatidic acid; PCR, polymerase chain reaction; DHAP, dihydroxyacetone phosphate; DTT, dithiothreitol; TLC, thin layer chromatography. The ise ctr[1] double mutant cannot grow on high inositol medium even in the presence of a choline supplement (14) Such a growth defect is apparently caused by a reduced synthesis of phosphatidylcholine (PC). One could envisage Sct1p as being a positive factor involved in the biosynthetic pathway leading to PC synthesis This would appear to be a plausible explanation if a small amount of choline is available, as this seems to be the case in ctr[1]. As evident in the studies of other acyltransferases, identification of these two initial enzymes of the glycerolipid pathway is crucial for understanding the regulation of phospholipid biosynthesis in yeast and will, it is hoped, facilitate similar endeavors in other eukaryotes, including plants
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