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

Abstract

Summary 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 end-products, 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.