AAA+ molecular motors involved in protein quality control are at the heart of many biological functions. Here I will discuss our efforts on two model AAA+ driven motors, prokaryotic ClpA and ClpB. ClpA is a hexameric ring motor that uses the energy from ATP binding/hydrolysis to processively translocate a polypeptide substrate through its axial channel for one of two purposes: protein remodeling or ATP dependent proteolysis. When catalyzing proteolysis, ClpA associates with the tetradecameric serine protease, ClpP, to form the ATP dependent protease ClpAP, which is architecturally identical to the 26 S proteasome in humans. ClpB, on the other hand, has the unique ability to disrupt protein aggregates in vivo and does not associate with any known protease. Due to the structural similarity between ClpA and ClpB it has long been proposed that ClpB processively translocates a polypeptide through the axial channel of its hexameric ring structure; as do ClpA and ClpX. However, for ClpB and related enzymes, this has been difficult to show because ClpB does not covalently modify the substrate on which it translocates. We have developed a transient state polypeptide translocation strategy that is sensitive to processive translocation catalyzed by ClpA in the absence of proteolytic degradation catalyzed by ClpP. Using this approach, we have shown that ClpA employs a different molecular mechanism when translocating polypeptide in the absence of the protease vs. when it is associated with the protease. Applying this approach to ClpB, we have shown that ClpB takes, at most, two translocation steps on the polypeptide substrate before rapid dissociation. These results reveal ClpB to be a non‐processive translocase that is not likely to fully translocate a polypeptide through its axial channel during protein disaggregation. These findings and the application of these approaches have broad impact on the examination of many AAA+ molecular motors that do not covalently modify the substrate on which they operate.Support or Funding InformationNSF MCB‐1412624This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.