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A Critical Reassessment of Penetratin Translocation Across Lipid Membranes

Elsa Bárány-Wallje ^* , Sandro Keller , Steffen Serowy  , Sebastian Geibel , Peter Pohl  , Michael Bienert  and Margitta Dathe 

^* Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Stockholm, Sweden; Research Institute of Molecular Pharmacology FMP, Berlin, Germany; Institute for Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany; and Institute for Biophysics, Johannes Kepler University, Linz, Austria

Correspondence: Address reprint requests to Sandro Keller, Research Institute of Molecular Pharmacology FMP, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. Tel.: 49-30-94793-368; Fax: 49-30-94793-159; E-mail: mail{at}sandrokeller.com.

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.

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