Folding-dependent in vitro protein splicing of the Saccharomyces cerevisiae VMA1 protozyme.
VMA1 translational product undergoes excision of a 50-kDa intervening segment (VDE: VMA1-derived endonuclease) and religation of the flanking regions to create a 69-kDa catalytic subunit of vacuolar membrane H+-ATPase. VDEs conjugated with polypeptides at both N- and C-terminal ends were expressed in Escherichia 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 demonstrate in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction efficiently occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed.
Kawasaki
M
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Makino
S
S
Matsuzawa
H
H
Satow
Y
Y
Ohya
Y
Y
Anraku
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004792:Enzyme Precursors; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae