Inhibition of ubiquitin-mediated proteolysis by the Arabidopsis 26 S protease subunit S5a.
A variety of protease inhibitors have been used to study ubiquitin-dependent proteolysis by the 26 S protease. However, these inhibitors lack 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. MBP1 and S5a bind multiubiquitin chains with high affinity and presumably facilitate the recognition of ubiquitin conjugates by the 26 S protease. We show here that free MBP1 can be a potent inhibitor of ubiquitin-dependent proteolysis in several cell-free systems. When added to reticulocyte lysates or to Xenopus egg extracts, the plant protein effectively blocked the degradation of multiubiquitinated lysozyme and cyclin B, respectively. MBP1 did not enhance the removal of ubiquitin from lysozyme or affect the ability of the 26 S complex to hydrolyze fluorogenic peptides. These data suggest that the plant protein specifically interferes with the recognition of ubiquitin conjugates by the 26 S protease. Thus MBP1, human S5a, and their homologs should prove to be valuable reagents for investigating cellular events mediated by ubiquitin-dependent proteolysis.
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132, USA.
Q
Q
Deveraux
S
S
van Nocker
D
D
Mahaffey
R
R
Vierstra
M
M
Rechsteiner
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins; D002352:Carrier Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D016213:Cyclins / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D009113:Muramidase / Q000032:analysis / Q000378:metabolism; D009865:Oocytes / Q000502:physiology; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*; D014981:Xenopus
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D016213:Cyclins; C097202:MBP1 protein, Arabidopsis; D046911:Macromolecular Substances; D010940:Plant Proteins; D011994:Recombinant Proteins; D014452:Ubiquitins; D009113:Muramidase; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease