Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.
The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.
Seol
J H
JH
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
Woo
K M
KM
Kang
M S
MS
Ha
D B
DB
Chung
C H
CH
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017354:Point Mutation; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases