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.