Abstract
The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.
Highlights
ClpXP is an ATP-dependent protease that catalyzes unfolding and degradation of misfolded proteins as well as tagged native and non-native proteins [1, 2]
We have found that isolated human mitochondrial ClpP is stable as a heptamer and remains a monodisperse species (s20,w 7.0 S; Mapp 169, 200) at concentrations >3 mg/ml
Peptidase activity does not require ATP hydrolysis, suggesting that ClpA and ClpX exert an allosteric effect on the conformation of ClpP, either improving peptide access to the active sites or enhancing the catalytic efficiency of the active sites
Summary
Protein Expression and Purification—Native hClpP, hClpP-⌬C, in which the C-terminal domain was deleted, and hClpX proteins were. Proteins (hClpX and hClpP) were added to the same buffer used for gel filtration in a final volume of 50 l, 10 l was taken for reference, and 40 l was loaded into the cartridge, which was centrifuged at 1000 ϫ g to allow ϳ30 l to pass through. To obtain a buffer baseline, the protein was removed from the assembled cell at the end of a run, and the right channel was flushed repeatedly before reloading with dialysate; a scan was recorded after the cell came to equilibrium at 3500 rpm and 12 °C. The final washed pellet was suspended in 50 mM Tris/HCl, pH 7.5, containing 10 mM MgCl2, 0.1 M KCl, and 10% (v/v) glycerol, with protease inhibitor mixture (Sigma). Debris was removed by centrifugation at 12,000 rpm for 1 h
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