Abstract
Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ∼5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.
Highlights
Somes? Studies from our laboratory [5, 6] and by others [7,8,9,10] are consistent with this conclusion, but studies using purified proteasomes and substrates of well-defined structure have been limited and have not determined the kinetic parameters associated with proteasome action
We could focus effectively and on the following question: Is substrate domain stability a significant determinant of proteasome action? We examined the effects of folding stability on turnover using two folded domains, the I27 module of the titin protein and mammalian dihydrofolate reductase (DHFR)
Design and Structure of Substrates Used in These Studies— Our previous studies of the native ubiquitin-independent proteasome substrate ornithine decarboxylase (ODC) provided the basis for the design of the substrates we generated and utilized
Summary
Proteasome molarity was calculated using a molecular weight 2,400 kDa. In Vitro Degradation Assay—Degradation reactions containing radiolabeled protein substrates were carried out at 30 °C by 50 nM 26 S proteasome in a buffer (25 mM HEPES pH 7.5, 100 mM KCl, 20 mM MgCl2, and 10% glycerol) containing 2 mM DTT, 5 mM ATP, and an ATP regenerating system (3 mM phospho(enol)pyruvic acid, 2 mM NADH, and 0.25 units of pyruvate kinase/lactate dehydrogenase mix (Sigma)). Degradation was initiated by substrate addition and time-dependent degradation was assessed by periodically removing 10 l volumes from the reaction and precipitation in 140 l of 20% TCA for 30 min on ice. After 30 min centrifugation at 18,000 ϫ g, the supernatant, containing the peptide products of proteolysis, was added to a scintillation vial for to quantification of radioactivity. Proteasome-specific ATP hydrolysis rates were calculated as (proteasomes ϩ substrate rate) Ϫ (substrate-only rate)
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