Homodimeric mitochondrial phosphate transport protein. Transient subunit/subunit contact site between the transport relevant transmembrane helices A.
Homodimeric mitochondrial phosphate transport protein. Transient subunit/subunit contact site between the transport relevant transmembrane helices A.
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同二聚体线粒体磷酸转运蛋白。
DOI：
10.1021/bi036148n
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Wohlrab,Hartmut
中科院分区：
文献类型：
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作者：
Phelps,Anne;Wohlrab,Hartmut
酵母（Saccharomyces cerevisiae）线粒体磷酸转运蛋白（PTP）亚基的三个Cys被Ser取代。在酵母中表达了7个突变体（单、双和完全替换Cys），并从线粒体中纯化了同源二聚体突变体PTPs并进行了重组。这些重组突变体催化的pH梯度依赖性净磷酸盐（Pi）运输摄取速率（初始条件：1 mM [Pi]e， pHe 6.80; 0 mM [Pi]i, pHi 8.07）与野生型蛋白相似，范围为15 ~ 80 μmol Pi/min mg PTP蛋白。有氧培养基仅抑制ptp与保守的（酵母和牛）Cys28催化的Pi摄取速率。这种蛋白脂质体的抑制作用为84 ~ 95%，可被二硫苏糖醇完全逆转。野生型和所有Cys28突变蛋白的转运被mersalyl抑制了90%以上。仅含Cys300或Cys134的突变蛋白催化的转运敏感性较低，而不含Cys突变蛋白催化的转运被mersalyl抑制了40%。当从纯化的单个Cys突变蛋白中去除二硫苏糖醇时，只有具有Cys28的突变蛋白在非还原SDS聚丙烯酰胺凝胶中显示为同型二聚体。因此，与功能相关的跨膜螺旋A与功能同二聚体PTP中另一个亚基的跨膜螺旋A部分密切接触，cys28与两个内膜表面的距离大约相等。研究结果首次确定了同二聚体线粒体运输蛋白的两个亚基之间的跨膜螺旋接触位点，而且还确定了一个锁定位置阻断运输的接触位点。结果与两种可用的二级转运蛋白结构（乳糖渗透酶，甘油-3-磷酸转运蛋白）以及抑制剂ADP/ATP载体复合物单体的低分辨率投影结构和高分辨率结构有关。
The three Cys of the yeast (Saccharomyces cerevisiae) mitochondrial phosphate transport protein (PTP) subunit were replaced with Ser. The seven mutants (single, double, and complete Cys replacements) were expressed in yeast, and the homodimeric mutant PTPs were purified from the mitochondria and reconstituted. The pH gradient-dependent net phosphate (Pi) transport uptake rates (initial conditions: 1 mM [Pi]e, pHe 6.80; 0 mM [Pi]i, pHi 8.07) catalyzed by these reconstituted mutants are similar to those of the wild-type protein and range from 15 to 80 μmol Pi/min mg PTP protein. Aerobic media inhibit only the Pi uptake rates catalyzed by PTPs with the conserved (yeast and bovine) Cys28. This inhibition in the proteoliposomes is 84−95% and can be completely reversed by dithiothreitol. Transport by the wild type as well as by all mutant proteins with Cys28 is more than 90% inhibited by mersalyl. Transport catalyzed by mutant proteins with only Cys300 or only Cys134 is less sensitive, and that catalyzed by the no Cys mutant shows 40% inhibition by mersalyl. When dithiothreitol is removed from purified single Cys mutant proteins, only the mutant protein with Cys28 appears as a homodimer in a nonreducing SDS polyacrylamide gel. Thus, the function relevant transmembrane helix A, with Cys 28 about equidistant from the two inner membrane surfaces, is in close contact with parts of transmembrane helix A of the other subunit in the functional homodimeric PTP. The results identify for the first time not only a transmembrane helix contact site between the two subunits of a homodimeric mitochondrial transport protein but also a contact site that if locked into position blocks transport. The results are related to two available secondary transporter structures (lactose permease, glycerol-3-phosphate transporter) as well as to a low resolution projection structure and a high resolution structure of monomers of inhibitor ADP/ATP carrier complexes.