SecY is an indispensable component of the protein secretory machinery of Escherichia coli.
Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.
Nishiyama
K
K
Institute of Applied Microbiology, University of Tokyo, Japan.
Kabuyama
Y
Y
Akimaru
J
J
Matsuyama
S
S
Tokuda
H
H
Mizushima
S
S
D000595:Amino Acid Sequence; D000906:Antibodies; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008970:Molecular Weight; D000069816:SEC Translocation Channels; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease