Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides.
A vacuolar processing enzyme (VPE) responsible for maturation of various vacuolar proteins is synthesized as an inactive precursor. To clarify how to convert the VPE precursor into the active enzyme, we expressed point mutated VPE precursors of castor bean in the pep4 strain of Saccharomyces cerevisiae. A VPE with a substitution of the active site Cys with Gly showed no ability to convert itself into the mature form, although a wild VPE had the ability. The mutated VPE was converted by the action of the VPE that had been purified from castor bean. Substitution of the conserved Asp-Asp at the putative cleavage site of the C-terminal propeptide with Gly-Gly abolished both the conversion into the mature form and the activation of the mutated VPE. In vitro assay with synthetic peptides demonstrated that a VPE exhibited activity towards Asp residues and that a VPE cleaved an Asp-Gln bond to remove the N-terminal propeptide. Taken together, the results indicate that the VPE is self-catalytically maturated to be converted into the active enzyme by removal of the C-terminal propeptide and subsequent removal of the N-terminal one.
Hiraiwa
N
N
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Nishimura
M
M
Hara-Nishimura
I
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics; D006031:Glycosylation; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases