Multiple functional proteins are produced by cleaving Asn-Gln bonds of a single precursor by vacuolar processing enzyme.
Precursor-accumulating vesicles mediate transport of the precursors of seed proteins to protein storage vacuoles in maturing pumpkin seeds. We isolated the precursor-accumulating vesicles and characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97,310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CXXXC motifs, a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Both immunocytochemistry and immunoblots with anti-PV100 antibodies showed that <10-kDa proteins and the 50-kDa vicilin-like protein were accumulated in the vacuoles. To identify the mature proteins derived from PV100, soluble proteins of the vacuoles were separated, and their molecular structures were determined. Mass spectrometry and peptide sequencing showed that two Cys-rich peptides, three Arg/Glu-rich peptides, and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 and that all of these proteins had a pyroglutamate at their NH2 termini. To clarify the cleavage mechanism, in vitro processing of PV100 was performed with purified vacuolar processing enzyme (VPE). Taken together, these results suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins. It is likely that the Asn-Gln stretches not only provide cleavage sites for VPE but also produce aminopeptidase-resistant proteins. We also found that the Cys-rich peptide functions as a trypsin inhibitor. Our findings suggested that PV100 is converted into different functional proteins, such as a proteinase inhibitor and a storage protein, in the vacuoles of seed cells.
Yamada
K
K
Department of Cell Biology, National Institute for Basic Biology, Okazaki 444, Japan.
Shimada
T
T
Kondo
M
M
Nishimura
M
M
Hara-Nishimura
I
I
D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D019662:Cucurbitaceae / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004792:Enzyme Precursors; D010940:Plant Proteins; D001216:Asparagine; D003546:Cysteine Endopeptidases; C089278:vacuolar processing enzyme; D005998:Glycine