Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport.
Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport.
复制标题
线粒体磷酸转运蛋白。
DOI：
10.1021/bi002206i
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Wohlrab,H
中科院分区：
文献类型：
--
作者：
Phelps,A;Briggs,C;Haefele,A;Mincone,L;Ligeti,E;Wohlrab,H
线粒体磷酸转运蛋白的每个功能同源二聚体亚基有6个(A、−、F)跨膜螺旋，所有突变都涉及同源二聚体的亚基。在早期的研究中，位于TM螺旋的基质末端(Asp39/Helix A，Glu137/Helix C，Asp236/Helix E)或膜中心(His32/Helix A，Glu136/Helix C)的几个残基的保守替换产生了失活的单突变PTPs。其中一些残基被建议作为磷酸配体或作为质子共运输路径的一部分。我们现在证明，突变Ser158Thr不是TM螺旋的一部分，但位于TM螺旋C和D之间的矩阵环(Ile141−Ser171)中心附近，使PtP失活，因此也具有功能相关性。在膜的另一侧，TM螺旋D膜间隙末端的单一突变Glu192Asp产生了一种具有33%野生型活性的PTP。我们通过将这个突变添加到六个运输失活突变中构建了双突变。只有携带Asp39Asn、Glu137Gln或Ser158Thr的人才能检测到转运。我们的结论是，TM螺旋D可以与TM螺旋A和C以及基质环Ile141−Ser171相互作用，而Asp39、Glu137和Ser158对磷酸盐的运输不是必需的。由于我们的结果与线粒体运输蛋白(MTP)家族所有12个功能成员中存在的残基一致，因此它们导致了一个一般规则，即在7个不同的位置指定MTP残基类型。所有双突变PTP(除Ser158Thr外)的构象都与单突变PTP显著不同，这表明它们的脂质体掺入效率很低，而且需要较少的洗涤剂(Triton X-100)才能留在溶液中。这些显着的构象差异也暗示了TM螺旋D和E之间的相互作用。结果从TM螺旋运动和PTP单体/二聚体比率的变化方面进行了讨论。
The mitochondrial phosphate transport protein (PTP) has six (A−F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141−Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141−Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.