The initial step of the glycerolipid pathway - Identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae

The initial step of phospholipid biosynthesis in yeast is carried out throu gh the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosp hate by stereospecific sn-1 acyltransferases. Here we report the identifica tion of two key fatty acyltransferases of the glycerolipid biosynthesis pat hway in Saccharomyces cerevisiae. Disruption of the open reading frame YBLO 11w, 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 exhib ited a dramatic reduction in G-3-P acyltransferase activity. Molecular char acterizations of the genes revealed that a missense mutation in YKR067w acc ounted for a defect in the activities of the G-3-P acyltransferase in the y east mutant strain TTA1. Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity. These results indicate that YKR 067w and YBLO11w, designated herein as GAT1 and GAT2(SCT1), respectively, a re yeast G-3-P acyltransferase genes. Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyaceto ne phosphate dual substrate-specific sn-1 acyltransferases. The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P a cyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors. In contrast, Gat2p(Sct1p) displayed preference toward 16-ca rbon fatty acids. The most notable of the altered phospholipid compositions of the gat1 Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidyl-inositol ratio. This did not appear to affect the mutants as no growth defect was found. H owever, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.