A vacuolar cysteine endopeptidase (SH-EP) that digests seed storage globulin: Characterization, regulation of gene expression, and posttranslational processing

A cysteine endopeptidase named SH-EP was isolated as one of the major hydrolytic enzymes involved in the mobilization of storage globulin in the cotyledon of germinated seeds of Vigna mungo. The enzyme, by itself or in combination with a serine endopeptidase, digests the globulin subunits to smaller peptides in vitro. Protein immunoblot analysis with antiserum raised against SH-EP showed that this enzyme is synthesized in the cotyledon after the onset of imbibition and increases until day 4 and decreases thereafter. Application of plant hormones and growth regulators, as well, as amino acids as proteolytic endproducts, appears to affect the development of SH-EP to a limited extent in the cotyledon of the germinated seeds. SH-EP of 33 kDa is synthesized on membrane-bound polysomes as a large, inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of a signal peptide. The amino-terminal propeptide region of the intermediate is cleaved further to form the 33-kDa mature enzyme by a multistep processing. The 43-kDa intermediate is also subjected to the cleavage of the carboxy-terminal decapeptide containing a Lys-Asp-Glu-Leu tail, which is known as a retention signal for the endoplasmic reticulum lumen. An asparaginyl endopeptidase, designated VmPE-1, was isolated as one of the processing enzymes responsible for the cleavage of the amino-terminal propeptide region of the precursor to SH-EP.