Probing novel elements for protein splicing in the yeast Vma1 protozyme: a study of replacement mutagenesis and intragenic suppression.
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 Vma1p protozyme where the 50-kD site-specific endonuclease (VDE) is excised from the 120-kD precursor containing the N- and G-terminal regions of the catalytic subunit of the vacuolar H(+)-ATPase. In this work, 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 splicing junction genetically interacts with three hydrophobic residues preceding the N-terminal splicing junction. This is the first evidence showing that the N-terminal portion of the V-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.
Nogami
S
S
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Satow
Y
Y
Ohya
Y
Y
Anraku
Y
Y
D000595:Amino Acid Sequence; D004706:Endodeoxyribonucleases / Q000235:genetics*; D007532:Isoleucine / Q000502:physiology; D016296:Mutagenesis; D019154:Protein Splicing / Q000235:genetics*; D006180:Proton-Translocating ATPases; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013489:Suppression, Genetic; D014443:Tyrosine / Q000502:physiology; D014633:Valine / Q000502:physiology
D029701:Saccharomyces cerevisiae Proteins; D007532:Isoleucine; D014443:Tyrosine; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D014633:Valine