Ion gradient-induced membrane translocation of model peptides.
The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.
de Kroon
A I
AI
Centre for Biomembranes and Lipid Enzymology, University of Utrecht, The Netherlands.
Vogt
B
B
van't Hof
R
R
de Kruijff
B
B
de Gier
J
J
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D008566:Membranes; D008954:Models, Biological; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010713:Phosphatidylcholines; D011487:Protein Conformation
D008081:Liposomes; D010455:Peptides; D010713:Phosphatidylcholines