Topology of the microsomal glycerol‐3‐phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae
Topology of the microsomal glycerol‐3‐phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae
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酿酒酵母微粒体甘油-3-磷酸酰基转移酶 Gpt2p/Gat1p 的拓扑结构
DOI：
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发表时间：
2012
影响因子：
3.6
通讯作者：
A. Conzelmann
中科院分区：
文献类型：
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作者：
M. Pagác;Hector M. Vazquez;Arlette Bochud;C. Roubaty;Cécile Knöpfli;Christine Vionnet;A. Conzelmann
所有甘油磷脂都是由磷脂酸制成的，根据传统观点，磷脂酸是在ER的胞质表面产生的。在酵母中，磷脂酸通过两个酰基辅酶A依赖性酰化反应从头合成。第一种由两种同源的甘油-3-磷酸酰基转移酶Gpt 2 p/Gat 1 p和Sct 1 p/Gat 2 p中的一种催化，第二种由两种1-酰基-sn-甘油-3-磷酸酰基转移酶Slc 1 p和Ale 1 p/Slc 4p中的一种催化。为了研究Gpt 2 p的生物起源和拓扑结构，我们观察了在各种跨膜螺旋之后插入的双拓扑结构报告子的位置。此外，使用微粒体，我们探讨了天然的和取代的半胱氨酸残基的膜不渗透的烷化剂的可及性和测试的蛋白酶的敏感性插入到Gpt 2 p的各种表位标签。最后，我们测定了酰基转移酶活性对靶向赖氨酸残基的膜不透性试剂的敏感性。通过所有这些标准，我们发现Gpt 2 p及其功能相关的赖氨酸的最保守的基序是面向ER腔。因此，磷脂酸在酵母中生物合成的第一步似乎发生在ER腔中，底物可能必须穿过ER膜。
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.
脂质拓扑发生。
影响因子：
6.5
作者：
Bell,RM;Ballas,LM;Coleman,RA
通讯作者：
Coleman,RA