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.
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
J H
JH
Seol
K M
KM
Woo
M S
MS
Kang
D B
DB
Ha
C H
CH
Chung
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