312:	Posttranslational removal of the carboxyterminal KDEL of the cysteine proteinase SH-EP occurs prior to maturation of the enzyme.

Authors:	Okamoto, Takashi(A)Satoh, Kouji(A)Minamikawa, Takao(A)Edward , Gerald(B)Vakharia, Vikram (B)Herman, Eliot(C)
Affiliations:	(A): Department of Biological Sciences, Tokyo Metropolitan University (B): Center for Agricultural Biotechnology, University of Maryland (C): Climate Stress Laboratory, USDA/ARS
Presenter:	Satoh, Kouji , okamoto-takashi@c.metro-u.ac.jp

SH-EP is a cysteine proteinase from germinated mung bean (Vigna mungo) that possesses a carboxyterminal ER retention sequence, KDEL. In order to examine the function of the ER retention sequence we expressed a full-length cDNA of SH-EP and a minus-KDEL control in insect Sf-9 cells using the baculovirus system. Our observations on the synthesis, processing and trafficking of SH-EP in Sf-9 cells suggest that the KDEL ER-retention sequence is posttranslationally removed either while the protein is still in the ER or immediately after its exit from the ER, resulting in the accumulation of proSH-EP minus its KDEL signal. It is this intermediate form that appears to progress through the endomembrane system and is subsequently processed to form mature active SH-EP. The removal of an ER retention may regulate protein delivery to a functional site and present an alternative role for ER retention sequences in addition to their well established role in maintaining the protein composition of the ER lumen. A putative proteinase involved in the cleavage of the carboxyterminal propeptide containing the KDEL tail was purified from microsomes prepared from cotyledons of germinated V. mungo seeds. The enzyme, termed ERPE (ER Processing Enzyme), had a molecular mass of 32 kDa, and the enzymatic activity was inhibited by E-64. Sucrose gradient analysis indicated that ERPE was co-fractionated with BiP.