Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: relevance to the mechanism of action of colicins and certain toxins.
The channel-forming activity of colicin E1 in artificial membranes is known to increase at low pH values and to have a maximum near pH 4 in such membrane vesicles. The present work demonstrates that 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 less than 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]leucine-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 leucines present in the carboxyl-terminal peptide were retained after Pronase treatment at pH less than 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 one 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.
Davidson
V L
VL
Brunden
K R
KR
Cramer
W A
WA
D003087:Colicins; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D008081:Liposomes; D008564:Membrane Potentials; D008565:Membrane Proteins; D010446:Peptide Fragments; D013329:Structure-Activity Relationship
D003087:Colicins; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D010446:Peptide Fragments