Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

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 betaVPE 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: alphaVPE, betaVPE, and/or gammaVPE. More than 90% of VPE activity is abolished in the alphavpe-3 seeds, and no VPE activity is detected in the alphavpe-1/betavpe-3/gammavpe-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 betavpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alphavpe-1 and gammavpe-1) and double (alphavpe-1/gammavpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alphaVPE and gammaVPE, are not necessary for precursor processing in the presence of betaVPE, but partly compensates for the deficiency in betaVPE in betavpe-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 betaVPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement betaVPE activity in this processing.