SYNERGISTIC INSERTION OF 2 HYDROPHOBIC REGIONS DRIVES SEC-INDEPENDENTMEMBRANE-PROTEIN ASSEMBLY

We have studied the membrane insertion of two proteins from the inner membrane of Escherichia coli, both with two transmembrane segments con nected by a short periplasmic loop: the M13 procoat protein and a muta nt ''inverted'' leader peptidase. Neither molecule depends on the Sec machinery for insertion. We show that the introduction of a charged re sidue in the second transmembrane segment completely blocks insertion of both proteins. In contrast, a Sec-dependent procoat mutant, where t he periplasmic region has been lengthened, inserts into the membrane e ven in the presence of a charged residue in the second hydrophobic dom ain. In addition, a large deletion within the second transmembrane dom ain of the leader peptidase mutant allows membrane translocation, but only under conditions where the SecA protein is functional. Furthermor e, we show that the first hydrophobic domain is required for insertion of the short periplasmic loop of the ''inverted'' leader peptidase. T hese results suggest that Sec-independent insertion occurs by a synerg istic entry of the two neighboring hydrophobic domains into the lipid bilayer.