Complementation of two overlapping fragments of SecA, a protein translocation ATPase of Escherichia coli, allows ATP binding to its amino-terminal region.
SecA is a protein translocation ATPase. The secA gene was engineered so as to code for SecA fragments of different sizes, either from the amino terminus or the carboxyl terminus. These SecA fragments, most of which formed aggregates in the cytosol, were overproduced and then purified in the presence of 6 M guanidine hydrochloride. The fragments were renatured by means of dilution and dialysis, and then examined as to their ability to interact with ATP by means of photoaffinity cross-linking with [alpha-32P]ATP. Individual SecA fragments thus renatured were inactive as to ATP binding. However, when two fragments (amino- and carboxyl-terminal ones), which structurally complemented each other and which had an overlapping region, were mixed, cross-linking was observed at the amino-terminal segments. The cross-linking was appreciably enhanced when two such fragments were first mixed together in 6 M guanidine hydrochloride and then renatured. It is concluded that SecA has an ATP-binding domain near its amino-terminal region and that the binding requires a carboxyl-terminal fragment that is large enough to cover the region deleted from the amino-terminal fragment. An amino-terminal fragment, which constituted about 92% of the entire SecA molecule, was active in not only ATP binding but also protein translocation. Based on these findings, the structure-function relationship of SecA is discussed.
Matsuyama
S
S
Institute of Applied Microbiology, University of Tokyo, Japan.
Kimura
E
E
Mizushima
S
S
D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011485:Protein Binding; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping
D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases