title: A critical reassessment of penetratin translocation across lipid membranes.
abstract: Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Stockholm, Sweden.
authors.forename: Elsa
authors.initials: E
authors.lastname: Bárány-Wallje
authors.forename: Sandro
authors.initials: S
authors.lastname: Keller
authors.forename: Steffen
authors.initials: S
authors.lastname: Serowy
authors.forename: Sebastian
authors.initials: S
authors.lastname: Geibel
authors.forename: Peter
authors.initials: P
authors.lastname: Pohl
authors.forename: Michael
authors.initials: M
authors.lastname: Bienert
authors.forename: Margitta
authors.initials: M
authors.lastname: Dathe
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry*; D057846:Cell-Penetrating Peptides; D003956:Dialysis / Q000379:methods; D004058:Diffusion; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008564:Membrane Potentials; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009038:Motion; D010539:Permeability; D010743:Phospholipids / Q000737:chemistry*; D021381:Protein Transport
chemical_list: D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D008051:Lipid Bilayers; D008081:Liposomes; D010743:Phospholipids; C414409:penetratin