Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli.
SecA interacts with presecretory proteins through recognition of the positive charge at the amino terminus of the signal peptide (Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S. (1989) J. Biol. Chem. 265, 8164-8169). A large variety of amino-terminal and carboxyl-terminal fragments of SecA were prepared in 6 M guanidine hydrochloride. SecA analogues were then reconstituted from them and examined for their ability to cross-link with [35S]proOmpF-Lpp, a presecretory protein, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The reconstituted SecA analogues were active in the cross-linking with proOmpF-Lpp when the SecA fragments used were large enough to structurally complement each other. The cross-linking was signal peptide-dependent and suppressed in the presence of other presecretory proteins. The cross-linking was enhanced in the presence of ATP. The SecA fragments that cross-linked with proOmpF-Lpp were then analyzed on sodium dodecyl sulfate-polyacrylamide gels. The cross-linking preferentially took place on fragments possessing the amino terminus of SecA. Weak cross-linking was also observed with carboxyl-terminal fragments when they were large enough. The smallest amino-terminal and carboxyl-terminal fragments with which the cross-linking was observed were 39 and 72 kDa, respectively. From these results, the region responsible for the cross-linking with presecretory proteins was deduced to be located between amino acid residues 267 and 340 from the amino terminus of SecA. These results are discussed in relation to the structure and function of SecA.
Kimura
E
E
Institute of Applied Microbiology, University of Tokyo, Japan.
Akita
M
M
Matsuyama
S
S
Mizushima
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C057184:outer membrane protein A precursor (E coli); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins