The 65-kDa protein derived from the internal translational initiation site of the clpA gene inhibits the ATP-dependent protease Ti in Escherichia coli.
The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
J H
JH
Seol
S J
SJ
Yoo
K I
KI
Kim
M S
MS
Kang
D B
DB
Ha
C H
CH
Chung
D000251:Adenosine Triphosphatases; D001483:Base Sequence; D017931:DNA Primers; D049071:Endopeptidase Clp; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases