Ubiquitin-ovomucoid fusion proteins as model substrates for monitoring degradation and deubiquitination by proteasomes.
Protein degradation by 26S proteasomes requires the coordinated action of multiple binding and catalytic activities to process ubiquitinated protein substrates. For the purpose of studying conjugate degradation independently of substrate targeting and unfolding steps, we have developed substrates based on an N-terminal fusion of ubiquitin to an irreversibly unfolded protein, the 83 amino acid HA epitope-tagged first domain of chicken ovomucoid. Fluorescent labeling of the six cysteines in the ovomucoid moiety (OM) with Lucifer Yellow iodoacetamide yields UbOM(LY); the ubiquitin in the fusion protein can be extended by the addition of a K48-linked polyubiquitin chain to form Ub(n)OM(LY). UbOM(LY) derivatives provide versatile substrates to monitor both protein degradation and deubiquitination by 26S proteasomes in vitro. Comparisons of polyubiquitin conjugates of unfolded OM(LY) with folded dihydrofolate reductase (DHFR) in degradation assays can help resolve and identify the rate-limiting steps in proteasome degradation.
Yao
Tingting
T
Stowers Institute For Medical Research, Kansas City, MO 64110, USA.
Cohen
Robert E
RE
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004926:Escherichia coli; D007700:Kinetics; D010059:Ovomucin / Q000235:genetics / Q000378:metabolism*; D025821:Polyubiquitin / Q000096:biosynthesis; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; D010059:Ovomucin; D046988:Proteasome Endopeptidase Complex