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 mem brane anchor sequence in the mature part of the protein for membrane insert ion. Translocation of its short acidic periplasmic loop is stimulated by th e 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 s ignal or membrane anchor is incrementally reduced. In contrast, we find tha t the pmf inhibits translocation of the periplasmic loop when it contains o ne or two positively charged residues. This inhibitory effect of the pmf is stronger when the hydrophobicity of the inserting procoat protein is compr omised. No pmf effect is observed for translocation of an uncharged peripla smic loop even when the hydrophobicity is reduced. We also show that the De lta Psi component of the pmf is necessary and sufficient for insertion of r epresentative constructs and that the translocation effects of charged resi dues are primarily due to the Delta Psi component of the pmf and not the pH component.