Identification of a soluble SecA/SecB complex by means of a subfractionated cell-free export system.

We have reconstituted the cell-free synthesis of the Escherichia coli precursor protein LamB from partially purified subfractions of an E. coli cell extract. PreLamB synthesized in this manner is translocated into salt-extracted plasma membrane vesicles only in the presence of SecA/SecB- or SecB-containing preparations of the E. coli cytosol. The most active preparations obtained upon purification were those containing a soluble SecA/SecB complex. Complex formation between SecA and SecB was verified by co-sedimentation and co-immunoprecipitation. When preLamB was synthesized in the presence of this material, a considerable amount of precursor was recovered from a soluble ternary complex consisting of preLamB, SecA, and SecB. Our results suggest that a soluble SecA/SecB complex participates in the export of preLamB and that this complex is functionally equivalent to a previously described 12 S (7 S) export factor (Müller, M., and Blobel, G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7737-7741; Watanabe, M., and Blobel, G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2728-2732).