Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1* 210
Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1* 210
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DOI：
10.1074/jbc.m206993200
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发表时间：
2003-03
期刊：
影响因子：
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通讯作者：
T. Tada;M. Ohmori;H. Iida
中科院分区：
文献类型：
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作者：
T. Tada;M. Ohmori;H. Iida
酿酒酵母MID 1基因产物Mid 1由548个氨基酸残基组成，具有4个相对疏水的片段，命名为H1-H4，并且当在哺乳动物细胞中表达时作为Ca 2+可渗透的牵张激活通道发挥功能。在某些条件下，Mid 1与Cch 1（哺乳动物电压门控通道α1亚基的酵母同源物）合作。为了确定Mid 1功能所需的重要区域或氨基酸残基，我们采用体外定点诱变H3和H4的Mid 1和表达所得的突变基因在mid 1无效突变体中，以检查突变基因产物是否在体内有功能或没有。突变体Mid 1蛋白缺乏整个H3或H4段，H3 De或H4 De，没有补充的致死性和低Ca 2+积累活性的mid 1突变体，虽然他们的定位和内容似乎是正常的，这表明H3和H4所需的Mid 1功能本身。对H3和H4的单个氨基酸残基的单氨基酸交换实验表明，H3中20个残基中的10个和H4中23个残基中的14个对于Mid 1的正常功能是重要的。特别是，我们发现了四个严重的功能丧失突变，D341 E，F356 S，C373 D和C373 R，以及两个有趣的突变，导致高水平的Ca 2+积累，补充活性略低，G342 A和Y355 A。将讨论这些氨基酸残基的重要性。
The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1–H4, and functions as a Ca2+-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the α1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca2+ accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca2+accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.