ClpA mediates directional translocation of substrate proteins into the ClpP protease

The intracellular degradation of many proteins is mediated in an ATP-depend ent manner by large assemblies comprising a chaperone ring complex associat ed coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the ch aperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP prote ase. Because the axial passageway into the proteolytic chamber is narrow, i t seems likely that unfolded substrate proteins are threaded from the chape rone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins hearing the COOH-terminal ssrA recognition ele ment, each labeled near the NH2 or COOH terminus with fluorescent probes. T ime-dependent changes in both fluorescence anisotropy and fluorescence reso nance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as accepters were measured to monitor translocation of th e substrates from ClpA into ClpP. We observed for both substrates that ener gy transfer occurs 2-4 s sooner with the COOH-terminally labeled molecules than with the NH2-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein enteri ng ClpP first.