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 flankin g regions to create a 69-kDa catalytic subunit of vacuolar membrane H-ATPase. VDEs conjugated with polypeptides at both N- and C-terminal e nds were expressed in Escherichia coli and examined for their ability to catalyze self-splicing. Processed VDE was found in soluble pools, w hile unspliced precursors accumulated in insoluble pools, forming incl usion bodies. We demonstrate in vitro protein splicing by refolding of the denatured precursor molecules. The processing reaction efficientl y occurs with the purified precursor peptide. VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed. (C) 1996 Academic Press, Inc.