Substrate recognition by the ClpA chaperone component of ClpAP protease

ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone an d regulatory component of Clp-AP protease, We explored the mechanism of pro tein recognition by ClpA using a high affinity substrate, RepA, which is ac tivated for DNA binding by ClpA and degraded by ClpAP, By characterizing Re pA derivatives with N- or C-terminal deletions, we found that the N-termina l portion of RepA is required for recognition. More precisely, RepA derivat ives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly. Thus, ClpA recognizes an N-term inal signal in RepA beginning in the vicinity of amino acids 10-15, Moreove r, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interac tions between ClpA and RepA We constructed fusions of RepA and green fluore scent protein, a protein not recognized by ClpA, and found that the N-termi nal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-termin al amino acids of RepA are degraded more efficiently in vitro and are notic eably stabilized in vivo in clpA Delta and clpP Delta strains compared with wild type.