AAA+ ATPases are involved in a wide array of critical cellular events in all kingdoms of life and frequently rely on adaptor proteins to modulate substrate specificity. CIpX is an AAA+ ATPase that unfolds protein substrates and translocates them into the proteolytic chamber of CIpP for degradation. The delivery protein SspB dramatically enhances the CIpXP degradation of proteins with a specific C-terminal peptide sequence called the ssrA-tag. SspB is a homodimer that binds two ssrA-tagged substrates and presents them to CIpXP for degradation. Our current view is that SspB is loosely tethered to CIpX by flexible C-terminal linkers that end with a CIpX binding (XB) sequence, allowing it to sequentially deliver both substrate molecules for degradation. However, it is not known whether the SspB dimer comes apart during the delivery process, whether both substrates are in fact delivered, whether both XB regions in the dimer are critical for interactions with CIpX, or whether SspB can function as a monomer. I propose to answer these questions by using mutant proteins defective in specific properties together with biochemical and biophysical assays to test and refine our model for how this fascinating biological system works. The studies proposed herein will define the role of dimerization in SspB function. In addition to CIpX, many other members of the AAA+ ATPases interact with oligomeric adapter proteins including SNARE/NSF. Understanding the role of SspB dimerization in its function with CIpXP could broadly impact our understanding of the interactions of other oligomeric adapters with AAA+ ATPases.
