Substrate recognition by the ClpA chaperone component of ClpAP protease.
J Biol Chem
; 275(45): 35361-7, 2000 Nov 10.
Article
em En
| MEDLINE
| ID: mdl-10952988
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Serina Endopeptidases
/
Transativadores
/
Trifosfato de Adenosina
/
Adenosina Trifosfatases
/
DNA Helicases
/
Chaperonas Moleculares
/
Proteínas de Escherichia coli
/
Proteínas de Ligação a DNA
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Estados Unidos