AN ASPARTIC PROTEINASE PRESENT IN SEEDS CLEAVES ARABIDOPSIS-2-S ALBUMIN PRECURSORS IN-VITRO

The Arabidopsis thaliana 2 S albumins are examples of vacuolar protein s which undergo intensive posttranslational processing. An in vitro pr ocessing assay to screen for processing enzymes present in seeds was d eveloped using an in vitro synthesized 2 S albumin precursor as the su bstrate. A protease was characterized which cleaved the substrate into two fragments with molecular weights (as determined from their migrat ion distance on SDS-polyacrylamide gel) corresponding to those of the small and large subunits of Arabidopsis 2 S albumin. The pH optimum of this protease activity, its inhibition by pepstatin A, and partial se quence data led to the conclusion that the protease under study was an aspartic proteinase. Synthetic peptides representing two 2 S albumin propeptides allowed the determination of the in vitro cleavage sites, and suggested that the protease activity is capable, in vitro, of clea ving the amino-terminal propeptide as well as the internal propeptide linking the two subunits. Alterations of the amino acids in and around the cleavage sites, made to study the specificity of the protease act ivity, suggest that both structural and sequence determinants are impo rtant in cleavage site recognition.