title: Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana.
abstract: The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.
authors.affiliation: Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Tomoo
authors.initials: T
authors.lastname: Shimada
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamada
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kataoka
authors.forename: Satoru
authors.initials: S
authors.lastname: Nakaune
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Koumoto
authors.forename: Miwa
authors.initials: M
authors.lastname: Kuroyanagi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tabata
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Kato
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
authors.forename: Motoaki
authors.initials: M
authors.lastname: Seki
authors.forename: Masatomo
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Maki
authors.initials: M
authors.lastname: Kondo
authors.forename: Mikio
authors.initials: M
authors.lastname: Nishimura
authors.forename: Ikuko
authors.initials: I
authors.lastname: Hara-Nishimura
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D012639:Seeds / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D029681:Arabidopsis Proteins; D017931:DNA Primers