Substrate recognition by the ClpA chaperone component of ClpAP protease.
ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease. We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP. By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition. More precisely, RepA derivatives 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-terminal signal in RepA beginning in the vicinity of amino acids 10-15. Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA. We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP. However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.
Hoskins
J R
JR
Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kim
S Y
SY
Wickner
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D049071:Endopeptidase Clp; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D019791:Guanidine / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D015534:Trans-Activators
D000596:Amino Acids; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D049452:Green Fluorescent Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D019791:Guanidine