Mechanical Protein Unfolding and Translocation by AAA+ Proteases

In all cells, protein degradation regulates key processes and maintains protein quality control. The AAA+ protease ClpXP is one of the best-studied examples of the ATP-dependent machinery responsible for intracellular proteolysis. A ring-shaped hexamer of the ClpX ATPase first recognizes and denatures folded protein substrate, then translocates the unfolded polypeptide into the barrel-shaped ClpP peptidase for degradation. In recent studies, dual-laser optical trapping was used to dissect the molecular events resulting in protein degradation by single molecules of ClpXP (Aubin-Tam et al. 2011, Maillard et al. 2011). This technique allows for the detection of small events (nm scale) with high temporal resolution (sub-ms scale). Here, we adapt our single-molecule assay to study a related AAA+ protease ClpAP and to address whether the similar architecture shared by all AAA+ proteases indicates a common mechanism of protein denaturation and degradation. Unlike ClpX, ClpA contains two ATPase modules per hexamer subunit and represents a major subclass of double-ring protein remodeling enzymes. We find similarities but also differences in mechanochemical activity between single- and double-ring ATPases, which contribute to their distinct activities in protein unfolding and degradation. We anticipate that the methods used to study ClpXP and ClpAP at the single-molecule level can be readily applied to examine the activity of other AAA+ proteases and protein remodeling enzymes.Aubin-Tam ME, Olivares AO, Sauer RT, Baker TA, Lang MJ (2011). Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145: 257-67.Maillard RA, Chistol G, Sen M, Righini M, Tan J, Kaiser CM, Hodges C, Martin A, Bustamante C (2011). ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145: 459-69.