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.

HeLa Cell Entry by Guanidinium‐Rich β‐Peptides: Importance of Specific Cation–Cell Surface Interactions

“…Recent evidence suggests a role for transbilayer potential in peptide translocation, [25,28,29] although there is debate on this point. [30] Silvius and co-workers showed that the cationic peptide penetratin could cross synthetic phospholipid membranes in the presence of a membrane potential, [28] while similar results were reported by Graslund and colleagues [29] in experiments designed to model the endosomal escape of penetratin. In the absence of a membrane potential, little or no peptide translocation was observed.…”

“…However, Bµrµny-Wallje et al were unable to detect movement of penetratin across synthetic bilayers in the presence or absence of a potential. [30] Wender et al reported a role for membrane potential in the entry of arginine oligomers into living cells. [25] We have exploited unique features of b-amino acid oligo-A C H T U N G T R E N N U N G mers ("b-peptides") to explore the effects of molecular shape stability and of the geometry of guanidinium group display on the entry of cationic peptides into mammalian cells.…”

110 Years of the Meyer–Overton Rule: Predicting Membrane Permeability of Gases and Other Small Compounds

“…In light of this, recent reports excluding endocytosis as an uptake mechanism do not appear convincing. [79] Since passive membrane diffusion of peptides has long been known as impossible, [80,81] what else can possibly be offered as a transport mechanism? Transient water wires across a lipid bilayer.…”

Polymeric Linear Peptide Chimeric Vaccine-Induced Antimalaria Immunity Is Associated with Enhanced In Vitro Antigen Loading

“…CPPs are linear sequences ranging between 10 and 30 amino acids that contain positively charged residues (tryptophan, arginine, or phenylalanine). Peptide uptake is enhanced by the interaction of positively charged residues with negatively charged membranes, resulting in endocytosis (3). In contrast with CPPs, the P. yoelii peptides tested here contain a single tryptophan with an isoelectric point (pI) of 3.96.…”