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Molecular biological studies on the mechanism of self protein splicing in the yeast VMA1 protozyme

酵母VMA1原酶自身蛋白剪接机制的分子生物学研究

基本信息

批准号：
07458156
负责人：
ANRAKU Yasuhiro
金额：
$ 4.1万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions. This unique autocatalytic reaction was first discovered in the yeast VMA1 protozyme where the 50 kDa site-specific endonuclease (VDE) is excised from the 120kDa precursor containing the N- and C-tereminal regions of the catalytic subumit of the vacuolar membrane ATPase. In this work we established a method for measuring in vitro protein splicing as follows : VDEs conjugated with various recombinant polypeptides at both N- and C-terminal ends were expressed in E.coli and examined for their ability to catalyze self splicing. Processed VDE was found in soluble pools, while unspliced precursors accumulated in insoluble pools, forming inclusion bodies. We demonstrated in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction eff … More iciently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed. Next, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the VMA1 protozyme, and found that these site-specific random mutations interfere with normal protein splicing to different extents. Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal aplicing junction genetically interacts with distal triplet residues preceding the N-terminal junction. This is the first evidence showing that the N-terminal portion of the vacuolar membrane ATPase subunit is involved in protein splicing. Our genetic evidence is consistent with a structural model that correctly aligns two parallel beta-strands ascribed to the triplets. This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions. Less

蛋白质剪接是一种引人注目的化学反应，其中通过切除内部蛋白质片段并连接侧翼区域，从单个前体多肽后序地产生两种蛋白质。这种独特的自催化反应首先在酵母VMA 1原酶中发现，其中50 kDa的位点特异性内切核酸酶（VDE）从含有液泡膜ATP酶催化亚基的N-和C-末端区域的120 kDa前体切除。在这项工作中，我们建立了一种方法，用于测量在体外蛋白质剪接如下：VDE共轭与各种重组多肽在N-和C-末端在大肠杆菌中表达，并检查其催化自我剪接的能力。加工的VDE被发现在可溶性池，而未剪接的前体积累在不溶性池，形成包涵体。我们证明了在体外蛋白质剪接的变性前体分子的重折叠。加工反应效果 ...更多信息与纯化的前体肽一起发生。仅由6个近端氨基酸和4个远端氨基酸包围的VDE被自催化加工。接下来，我们随机化保守的缬氨酸三联体残基的C-末端剪接交界处的VMA 1原酶的上游三个氨基酸，并发现这些位点特异性的随机突变干扰正常的蛋白质剪接不同程度。基因内抑制基因分析表明，这一特定的疏水三联体之前的C-末端applicing路口遗传相互作用与远端三联体残基之前的N-末端路口。这是首次证明液泡膜ATP酶亚基的N-末端部分参与蛋白质剪接。我们的遗传学证据与一个结构模型一致，该模型正确地排列了归因于三胞胎的两条平行β链。该模型描绘了两个保守区域之间的空间相互作用，这两个保守区域都位于剪接点的上游。少