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

蛋白质剪接是一种引人注目的化学反应，在这个过程中，通过切除内部蛋白质片段和连接侧翼区域，从单一前体多肽翻译后产生两种蛋白质。这种独特的自催化反应首先在酵母VMA1原酶中发现，其中50 kDa位点特异性内切酶(VDE)从120 kDa的前体中切下，该前体含有液泡膜ATPase催化亚单位的N-和C-末端区域。在这项工作中，我们建立了一种检测体外蛋白质剪接的方法：在大肠杆菌中表达与N-端和C-端各种重组多肽结合的VDEs，并检测它们催化自身剪接的能力。加工的VDE存在于可溶性池中，而未剪接的前体积累在不溶性池中，形成包涵体。我们演示了通过变性前体分子的重折叠在体外进行蛋白质剪接。…的加工反应更有效地出现在纯化的前体多肽中。VDE只由6个近端和4个远端氨基酸组成，是自动催化处理的。接下来，我们对VMA1原酶C末端剪接接头上游的三个氨基酸保守的valine三联体残基进行了随机化，发现这些位点特异性的随机突变不同程度地干扰了正常的蛋白质剪接。基因内抑制分析表明，在C-末端连接之前的这个特殊的疏水三联体与N-末端连接之前的远端三联体残基在遗传上相互作用。这是首次有证据表明液泡膜ATPase亚基的N端部分参与了蛋白质剪接。我们的遗传证据与一个结构模型是一致的，该模型正确地将两条平行的β-链归因于三胞胎。该模型描述了两个保守区域之间的空间相互作用，这两个区域都位于剪接连接的上游。较少

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