Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.

Abstract

The channel-forming activity of colicin E1 in artificial membranes is known to incease at low pH values and to have a maximum near pH 4 in such membrane vesicles. This pH dependence of activity can be attributed to membrane binding. Maximal binding of colicin E1 and a more slowly binding channel-forming carboxyl-terminal tryptic peptide occurred at acidic pH values, with the effective pK values for binding equal to 4.6 and < 4.0, respectively. The binding did not require imposition of a transmembrane potential. Insertion of the tryptic peptide into the membrane was shown by retention of bound [3H]L-labeled peptide by vesicles after digestion with protease, as well as by retention of the peptide in salt-washed vesicles. The retention after protease treatment was also used to estimate the amount of carboxyl-terminal peptide inserted into the membrane. Approximately 12 of the 21 L present in the carboxyl-terminal peptide were retained after Pronase treatment at pH < 4. Reversibility of the insertion at low pH values was seen after an alkaline shift of pH to 6.0, resulting in a decrease of the protease-inaccessible fraction of the bound protein. A model is presented describing a mechanism in which protonation of 1 or more carboxyl residues is necessary for effective binding and insertion into the membrane by the channel-forming domain of colicin E1. This model may also be relevant to the mechanism of membrane insertion by certain toxins.