A critical reassessment of penetratin translocation across lipid membranes

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.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.     

Mechanism for translocation of fluoroquinolones across lipid membranes

Classical atom-scale molecular dynamics simulations, constrained free energy calculations, and quantum mechanical (QM) calculations are employed to study the diffusive translocation of ciprofloxacin (CPFX) across lipid membranes. CPFX is considered here as a representative of the fluoroquinolone antibiotics class. Neutral and zwitterionic CPFX coexist at physiological pH, with the latter being predominant. Simulations reveal that only the neutral form permeates the bilayer, and it does so through a novel mechanism that involves dissolution of concerted stacks of zwitterionic ciprofloxacins. Subsequent QM analysis of the observed molecular stacking shows the important role of partial charge neutralization in the stacks, highlighting how the zwitterionic form of the drug is neutralized for translocation. The findings propose a translocation mechanism in which zwitterionic CPFX molecules approach the membrane in stacks, but they diffuse through the membrane as neutral CPFX monomers due to intermolecular transfer of protons favored by partial solvation loss. The mechanism is expected to be of importance in the permeation and translocation of a variety of ampholitic drugs with stacking tendencies.     

Measuring translocation of fluorescent lipid derivatives across yeast Golgi membranes

Phospholipid asymmetry is a fundamental feature of the plasma membrane of most eukaryotic cells and its regulation is linked to diverse physiological processes such as apoptosis and blood clotting [P. Williamson, R.A. Schlegel, Biochim. Biophys. Acta 1585 (2002) 53-63; R.F. Zwaal, A.J. Schroit, Blood 89 (1997) 1121-1132]. In addition, the phospholipid translocases (flippases) that are thought to establish asymmetry are also implicated in vesicle-mediated protein transport throughout the secretory and endocytic pathways [T.R. Graham, Trends Cell Biol. 14 (2004) 670-677]. However, the biochemical properties of phospholipid translocases in membranes of the Golgi complex and endosomes have received much less attention than translocases in the plasma membrane. We describe here a method for purifying yeast Golgi membranes and assaying an ATP-dependent phospholipid translocase activity in these membranes using fluorescent lipid analogues. This assay detects ATP-dependent translocation of labeled phosphatidylserine across late Golgi membranes, which requires the activity of a P-type ATPase called Drs2p [P. Natarajan, J. Wang, Z. Hua, T.R. Graham, Proc. Natl. Acad. Sci. USA 101 (2004) 10614-10619].     

Vesicle membrane interactions of penetratin analogues

Reports on serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides, also denoted protein transduction domains, have demonstrated the need for a reevaluation of the current understanding of peptide-mediated cellular delivery of large, hydrophilic molecules. In a recent study on the internalization in unfixed cells of penetratin and its analogues in which tryptophans are substituted for phenylalanines (Pen2W2F), lysines for arginines (PenArg), and arginines for lysines (PenLys), we revealed large dissimilarities in cell interactions among the peptides [Thorén et al. (2003) Biochem. Biophys. Res. Commun. 307, 100-107]. We here investigated possible correlations with their respective affinities for the lipid membranes of large unilamellar vesicles. The variations found in membrane affinity correlated qualitatively with differences in hydrophobicity among the peptides but were by far too small to account for the striking differences in cell membrane binding. Interestingly, we found that the inclusion of a small fraction of lipids conjugated to poly(ethylene glycol) (PEG) in the vesicles both stabilized the vesicle dispersion against peptide-induced aggregation and, furthermore, enhanced the binding of the peptides to the membrane. By use of PEG-conjugated lipids, it could be shown that vesicle aggregation drives an alpha-helix to beta-sheet conformational transition for these peptides. A similar transition was discovered at submicellar concentrations of sodium dodecyl sulfate in aqueous solution for all peptides except PenLys. Finally, significant changes of the contributions to CD spectra from aromatic residues due to their insertion into the membrane were observed.     

Requirements for the translocation of diphtheria toxin fragment A across lipid membranes

The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Protein translocation across chloroplast envelope membranes

Nuclear-encoded chloroplast proteins are imported from the cytosol into the chloroplast stroma by a common translocation machinery. Several components of the import apparatus, including GTP-binding proteins and Hsp70 proteins, have recently been identified and characterized. This review discusses the role of these proteins in chloroplast protein import.     

Protein translocation across mitochondrial membranes.

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes. In particular we will focus on recently identified components of the mitochondrial import apparatus.     

Mechanistic insights into the translocation of full length HIV-1 Tat across lipid membranes

The mechanism of how full length Tat (aa 1-86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1-86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35-86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles' interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane.     

The antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation

The potential use of polypeptides and oligonucleotides for therapeutical purposes has been questioned because of their inherently poor cellular uptake. However, the 16-mer oligopeptide penetratin, derived from the homeodomain of Antennapedia, has been reported to enter cells readily via a non-endocytotic and receptor- and transporter-independent pathway, even when conjugated to large hydrophilic molecules. We here present the first study where penetratin is shown to traverse a pure lipid bilayer. The results support the idea that the uptake mechanism involves only the interaction of the peptide with the membrane lipids. Furthermore, we conclude that the translocation does not involve pore formation.     

Protein translocation across and integration into membranes

This review concentrates mainly on the translocation of proteins across the endoplasmic reticulum membrane and cytoplasmic membrane in bacteria. It will start with a short historical review and will pinpoint the crucial questions in the field. Special emphasis will be given to the present knowledge on the molecular details of the first steps, i.e., on the function of the signal recognition particle and its receptor. The knowledge on the signal peptidase and the ribosome receptor(s) will also be summarized. The various models for the translocation of proteins across and the integration of proteins into membranes will be critically discussed. In particular, the function of signal, stop-transfer, and insertion sequences will be dealt with and molecular differences discussed. The cotranslational mode of membrane transfer will be compared with the post-translational transport found for mitochondria and chloroplasts. This review will conclude with open questions and an outlook.     

Fluorescence coupling across lipid bilayer membranes

Summary An energy transfer between donor and acceptor fluorophores across single lipid bilayer membranes is demonstrated. Anilino-naphthalene sulfonate is used as the donor chromophore: its fluorescence is enhanced by the presence of lipid and thus indicates association with the purely lipid membranes of our preparation of vesicles in suspension. Light emit ted by the donor molecules excites fluorescence of acriflavine, a suitable acceptor enclosed inside the vesicles. Absorption and fluorescence spectra of this system, in its components and as a whole, are presented in evidence for an energy transfer.Supported by a grant from the Medical Research Council of Canada. The results of this work were presented, in part, at the 17th Annual Meeting of the Biophysical Society, February 27–March 2, 1973, Columbus, Ohio.Scholar of the Medical Research Council of Canada.