Clipping along.

In this issue of JMB, Rajendar and Lucius report a study of themechanism of polypeptide translocation catalyzed by the Escherichia coli ClpA enzyme—the motor component of the ATP-dependent protease, ClpAP. ATP-dependent proteases play important roles in protein turnover and share a common architecture across organisms. ClpAP is composed of the hexameric AAA+ motor protein ClpA and the tetradecameric serine protease ClpP. The motor component, ClpA, couples the energy from ATP binding and hydrolysis to enzyme-catalyzed protein unfolding and polypeptide translocation. ClpA moves proteins targeted for degradation into the central cavity of the proteolytic component, ClpP, a barrel-shaped protease. ClpA, in the absence of ClpP, also functions to form active RepA monomers from inactive dimers. To date, mechanistic information about the enzymatic activities of ClpA has been inferred from studies of the activity of the full ClpAP complex. This is because the ClpP protease activity or entry of a polypeptide substrate into the ClpP cavity was used to detect translocation indirectly. In the absence of ClpP, ClpA will simply move along the polypeptide “track,” leaving it essentially unchanged, except that it may become unfolded. Furthermore, most of these studies used steady-state kinetic methods that are not generally sensitive to the fast translocation rates since the steady-state rates are limited by slower processes, such as substrate binding and product dissociation. Thus, a transient kinetic method is needed to examine the faster rates of ClpA motor enzyme translocation along a polypeptide in the absence of the proteolytic component, ClpP. Rajendar and Lucius have solved this problem by developing a real-time stopped-flow method to examine ClpA translocation that uses polypeptides that are end-labeled with a fluorophore that undergoes an intensity change when ClpA arrives at the end of the polypeptide. By using this method under conditions that ensure a single round of translocation, they have examined the kinetics and mechanism of polypeptide translocation by ClpA in the absence of ClpP. Methods similar to this have been used to study translocation along nucleic acids of motor proteins, such as helicases. Rajendar and Lucius1 show that the ClpAmotor translocates along a polypeptide chainwith directional bias from the Cterminus to the N-terminus. Although fluorescence-