Direct evidence that the proton motive force inhibits membrane translocation of positively charged residues within membrane proteins.

The M13 phage procoat protein requires both its signal sequence and its membrane anchor sequence in the mature part of the protein for membrane insertion. Translocation of its short acidic periplasmic loop is stimulated by the proton motive force (pmf) and does not require the Sec components. We now find that the pmf becomes increasingly important for the translocation of negatively charged residues within procoat when the hydrophobicity of the signal or membrane anchor is incrementally reduced. In contrast, we find that the pmf inhibits translocation of the periplasmic loop when it contains one or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compromised. No pmf effect is observed for translocation of an uncharged periplasmic loop even when the hydrophobicity is reduced. We also show that the Delta Psi component of the pmf is necessary and sufficient for insertion of representative constructs and that the translocation effects of charged residues are primarily due to the DeltaPsi component of the pmf and not the pH component.