Abstract
ClpA, a member of the Clp/Hsp100 family of ATPases, is both an ATP-dependent molecular chaperone and the regulatory component of ClpAP protease. We demonstrate that chaperone and protease activities occur concurrently in ClpAP complexes during a single round of RepA binding to ClpAP and ATP-dependent release. This result was substantiated with a ClpA mutant, ClpA(K220V), carrying an amino acid substitution in the N-terminal ATP binding site. ClpA(K220V) is unable to activate RepA, but the presence of ClpP or chemically inactivated ClpP restores its ability to activate RepA. The presence of ClpP simultaneously facilitates degradation of RepA. ClpP must remain bound to ClpA(K220V) for these effects, indicating that both chaperone and proteolytic activities of the mutant complex occur concurrently. ClpA(K220V) itself is able to form stable complexes with RepA in the presence of a poorly hydrolyzed ATP analog, adenosine 5'-O-(thiotriphosphate), and to release RepA upon exchange of adenosine 5'-O-(thiotriphosphate) with ATP. However, the released RepA is inactive in DNA binding, indicating that the N-terminal ATP binding site is essential for the chaperone activity of ClpA. Taken together, these results suggest that substrates bound to the complex of the proteolytic and ATPase components can be partitioned between release/reactivation and translocation/degradation.
Highlights
Sition of bacteriophage Mu DNA [7,8,9], disaggregates O aggregates in vitro [10], and activates the plasmid RK2 DNA replication initiator protein, TrfA, by dissociating dimers into monomers [11]
The N-terminal site is involved in the assembly of ClpA hexamers, and the C-terminal site is involved in ATP hydrolysis and degradation of proteins
ClpA(K220V) Lacks Chaperone Activity but Degrades RepA in Conjunction with ClpP—Our first evidence to support the hypothesis that the chaperone and proteolytic activities of ClpAP are concurrent came from the study of a ClpA mutant
Summary
Materials—ATP, ATP␥S,1 and Triton X-100 were obtained from Roche Molecular Biochemicals. [␥-32P]ATP was from ICN Pharmaceuticals, Inc. RepA Activation Assay—Reaction mixtures contained (in 20 l) Buffer A (20 mM Tris1⁄7HCl, pH 7.5, 100 mM KCl, 40 mM NaCl, 10 mM MgCl2, 5 mM dithiothreitol, 0.1 mM EDTA), 1 mM ATP, 100 g/ml bovine serum albumin, 1.4 pmol of ClpA, and 0.03 pmol of RepA, unless indicated otherwise. RepA Degradation Assay—Reaction mixtures were assembled in 50 l of Buffer B (20 mM Tris1⁄7HCl, pH 7.5, 10 mM magnesium acetate, 100 mM KCl, 5 mM dithiothreitol, 0.005% Triton X-100 (v/v)) containing 4 mM ATP, 0.8 pmol of ClpA or ClpA mutant proteins, 4.7 pmol of [3H]RepA, and 0.3 pmol of ClpP. Gel Filtration Chromatography of RepA1⁄7ClpA Complexes—To isolate RepA1⁄7ClpA complexes, reaction mixtures (70 l) containing 500 pmol of [3H]RepA, 100 pmol of ClpA or ClpA(K220V), and 1 mM ATP␥S in Buffer A containing 5% (v/v) glycerol were incubated for 15 min at 23 °C. To isolate RepA released from complexes, RepA1⁄7ClpA(wt) and RepA1⁄7ClpA(K220V) complexes were formed as described above with 60 pmol of [3H]RepA and
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
