Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.
Mol Microbiol. 2012 Dec;86(5):1156-66. doi: 10.1111/mmi.12047. Epub 2012 Oct 12.
All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl-CoA-dependent acylation reactions. The first is catalysed by one of the two homologous glycerol-3-phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1-acyl-sn-glycerol-3-phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.
所有甘油磷脂都是由磷脂酸合成的，根据传统观点，磷脂酸是在 ER 的细胞质表面生成的。在酵母中，磷脂酸是通过两种酰基辅酶 A 依赖性酰化反应从头合成的。第一个反应由两种同工型甘油-3-磷酸酰基转移酶 Gpt2p/Gat1p 和 Sct1p/Gat2p 之一催化，第二个反应由两种 1-酰基-sn-甘油-3-磷酸酰基转移酶 Slc1p 和 Ale1p/Slc4p 之一催化。为了研究 Gpt2p 的生物发生和拓扑结构，我们观察了插入各种跨膜螺旋后的双拓扑报告基因的位置。此外，我们使用微粒体探测了天然和取代半胱氨酸残基对膜不可渗透的烷化剂的可及性，并测试了插入 Gpt2p 中的各种表位标签的蛋白酶敏感性。最后，我们测定了酰基转移酶活性对针对赖氨酸残基的膜不可渗透试剂的敏感性。通过所有这些标准，我们发现 Gpt2p 及其功能相关赖氨酸的最保守基序朝向 ER 腔。因此，酵母中磷脂酸生物合成的第一步似乎发生在 ER 腔中，并且底物可能必须穿过 ER 膜。
