INHIBITION OF UBIQUITIN-MEDIATED PROTEOLYSIS BY THE ARABIDOPSIS 26-S PROTEASE SUBUNIT S5A

A variety of protease inhibitors have been used to study ubiquitin-dep endent proteolysis by the 26 S protease. However, these inhibitors lac k complete specificity and thus affect ubiquitin-independent pathways as well, We recently identified an Arabidopsis protein, MBP1, that is homologous to subunit 5a (S5a) of the human 26 S protease complex, MBP 1 and S5a bind multiubiquitin chains with high affinity and presumably facilitate the recognition of ubiquitin conjugates by the 26 S protea se, We show here that free MBP1 can be a potent inhibitor of ubiquitin -dependent proteolysis in several cell-free systems, When added to ret iculocyte lysates or to Xenopus egg extracts, the plant protein effect ively blocked the degradation of multiubiquitinated lysozyme and cycli n B, respectively. MBP1 did not enhance the removal of ubiquitin from lysozyme or affect the ability of the 26 S complex to hydrolyze fluoro genic peptides, These data suggest that the plant protein specifically interferes with the recognition of ubiquitin conjugates by the 26 S p rotease, Thus MBP1, human S5a, and their homologs should prove to be v aluable reagents for investigating cellular events mediated by ubiquit in-dependent proteolysis.