The carboxyl-terminal region of SecE interacts with SecY and is functional in the reconstitution of protein translocation activity in Escherichia coli.
Genes encoding the C- and N-terminal regions of SecE were constructed and placed under the control of the tac promoter on plasmids. The C-terminal region of SecE (SecE-C) was sufficient for suppression of the secEcs phenotype, confirming the results of Schatz et al. (Schatz, P. J., Bieker, K. L., Ottemann, K. M., Silhavy, T. J., and Beckwith, J. (1991) EMBO J. 10, 1749-1757). SecE-C allowed the overproduction of SecY, and its overproduction was achieved when the tac-secY gene, on a plasmid, was induced, indicating that the C-terminal region is the site of interaction of SecE with SecY and that the interaction makes the two Sec proteins stable. SecE-C was purified and used with SecY for the reconstitution of protein translocation activity. SecE-C was active in the functional reconstitution. The SecE-C/SecY-dependent protein translocation absolutely required SecA and ATP as the native translocation reaction did. Quantitative analysis revealed that SecE-C was 50% as active as intact SecE. The N-terminal region of SecE (SecE-N) also suppressed in vivo the defect caused by the secEcs mutation. SecE-N was, however, inactive in the overproduction of SecY. A possible oligomeric structure of SecE is discussed.
Nishiyama
K
K
Institute of Applied Microbiology, University of Tokyo, Japan.
Mizushima
S
S
Tokuda
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D011510:Proteolipids / Q000378:metabolism; D000069816:SEC Translocation Channels; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011510:Proteolipids; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; C020485:proteoliposomes; D000255:Adenosine Triphosphate