The Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme
The Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme
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DOI：
10.1074/jbc.m112.421776
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发表时间：
2012-11-23
影响因子：
4.8
通讯作者：
Carman, George M.
中科院分区：
文献类型：
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作者：
Chae, Minjung;Han, Gil-Soo;Carman, George M.
磷脂酸盐磷酸酶(PAP)催化磷脂酸脱磷生成甘油二酯。在酿酒酵母中，PAP由PAH1、DPP1和LPP1编码。在pah1 Delta dpp1 Delta lpp1 Delta三重突变体中存在PAP活性，这表明编码该酶的另一个基因(S)。通过线粒体盐抽提、DE52、Affi-Gel Blue、苯基-琼脂糖凝胶、MonoQ和Superdex 200层析，从pah1 Delta dpp1 Delta lpp1 Delta三重突变体中纯化了PAP。对富含PAP的样品进行的液相色谱/串联质谱分析显示了多种可能的磷酸酶。通过对缺乏这两种蛋白的突变体的PAP活性分析，我们发现分子功能未知的App1基因具有类似于野生型细胞30%的PAP活性。在pah1 Delta dpp1 Delta lpp1 Delta突变体中过表达App1后，PAP活性增加了10倍。在大肠杆菌中异源表达的App1p显示的PAP活性证实了App1是该酶的结构基因。将app1 Delta突变引入到pah1 Delta dpp1 Delta lpp1 Delta三重突变体中，导致PAP活性完全丧失，表明酿酒酵母中不同的PAP酶是由app1、PAH1、DPP1和LPP1编码的。对缺乏PAP基因的细胞进行的脂类分析表明，Pah1p是唯一参与三酰甘油合成和磷脂合成调节的PAP。App1p显示了与内吞蛋白的相互作用，可能通过其PAP活性在囊泡运输中发挥作用。
Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1 Delta dpp1 Delta lpp1 Delta triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1 Delta dpp1 Delta lpp1 Delta triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers similar to 30% PAP activity of wild type cells. The overexpression of APP1 in the pah1 Delta dpp1 Delta lpp1 Delta mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1 Delta mutation into the pah1 Delta dpp1 Delta lpp1 Delta triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.