Abstract
SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.
Highlights
The endoplasmic reticulum (ER)1 is the port of entry of proteins into the endomembrane system
Based on analysis of the heterologous expression of SH-EP and a KDEL deletion mutant of SH-EP in insect Sf9 cells and subcellular fractionation of cotyledon cells, it was proposed that the KDEL-tail of SH-EP functions to store the enzyme as a transient zymogen in the ER, and that the conversion of the 43-kDa SH-EP into the 42-kDa form in/at the ER is accompanied by the removal of the C-terminal propeptide containing the KDEL-tail [20]
This protease activity was inhibited by E-64 but not by diisopropyl fluorophosphate, pepstatin A, or EDTA (Fig. 1, B and C), suggesting that the enzyme involved in this proteolysis is a cysteine protease whose active site cysteine can be irreversibly bound to the epoxy group of E-64
Summary
The endoplasmic reticulum (ER)1 is the port of entry of proteins into the endomembrane system. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form.
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