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

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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