SUBUNIT INTERACTIONS IN THE ESCHERICHIA-COLI PROTEIN TRANSLOCASE - SECE AND SECG ASSOCIATE INDEPENDENTLY WITH SECY

We used hexahistidine-tagged SecE and SecY to study how the core subun its (SecY, SecE and SecG) of Escherichia coli protein translocase inte ract with each other. Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+-NTA agarose affinity binding. Alth ough His(6)-SecE, expressed in wild-type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells. His(6)-SecY brought down both Se cE and SecG, as expected. Interestingly, His(6)-SecY24 was able to bri ng down SecG but not SecE. These results confirm our previous conclusi on that the secY24 alteration impairs the SecY-SecE interaction, and d emonstrate that SecY and SecG can form a complex that does not contain SecE. Likewise, SecY-SecE complex could be isolated from the secG-del eted strain. The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26 degrees C, whereas the Sec Y-SecE complex without SecG dissociated at a slightly lower temperatur e (20-23 degrees C). We conclude that each of SecE and SecG independen tly binds to SecY, the central subunit of protein translocase, althoug h the trimeric complex is more stable than the binary complexes. (C) 1 997 Federation of European Biochemical Societies.