The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48 -60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48 -60). These included such peptides as the D-amino acid-and arginine-substituted Tat-(48 -60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4°C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg) n (n ‫؍‬ 4 -16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n ϳ 8) for the efficient translocation.Recently, methods have been developed for the delivery of exogenous proteins into living cells with the help of membranepermeable carrier peptides such as HIV-1 1 Tat-(48 -60) and Antennapedia-(43-58) (1-11). By genetically or chemically hybridizing these carrier peptides, the efficient intracellular delivery of various oligopeptides and proteins was achieved. One of the most amazing examples is the Tat-␤-galactosidase fusion protein (4), which has a molecular mass as high as 120 kDa.Intraperitoneal injection of the protein resulted in delivery of the protein with ␤-galactosidase activity to various tissues in mice, including the brain. The peptide-mediated approaches would allow the incorporation of peptides containing unnatural amino acids or nonpeptide molecules such as fluorescence probes. These methods would become powerful tools not only for therapeutic purposes as an alternative to gene delivery, but also for the understanding of the mechanisms behind fundamental cellular events, such as signal transduction and gene transcription.Besides the potential of Tat-(48 -60) as a protein carrier, the internalization mechanism of the peptide attracted our interest. For example, Tat-(48 -60) (GRKKRRQRRRPPQ) is a highly basic and hydrophilic peptide, which contains 6 arginine and 2 lysine residues in its 13 amino acid residues. However, the peptide was reported to be translocated through the cell membranes in 5 min at a concentration of 0.1 M (2). Internalization of the peptide was not inhibited even at 4°C. The peptide is less toxic to cells than other basic membrane-interacting agents. The above features suggested that the internalization mechanism of Tat-...

Basic peptides derived from the HIV-1 1 Tat protein (Tat-(48 -60)) and Drosophila Antennapedia protein (Antp-(43-58)) have been reported to have the ability to translocate through the cell membranes and to carry exogenous molecules into the cytoplasm and nucleus (1-13). A 119-kDa protein, ␤-galactosidase, genetically fused with the former peptide segment, was successfully carried into various tissues in mice including the brain via intraperitoneal injection (6). The 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-gal) staining of the tissues indicated that the fusion protein was delivered in its active form. Oligo-DNAs and metal chelates were also brought into cells using the Tat-derived peptide (4, 7). Such a method to deliver bioactive molecules into cells using membrane-permeable peptides has a great potential for therapeutic fields.We have recently demonstrated that not only Tat-(48 -60) and Antp-(43-58) but also various arginine-rich RNA-or DNAbinding peptides such as HIV-1 Rev-(34 -50) and flock house virus (FHV) coat-(35-49) were membrane-permeable and have the ability to bring exogenous protein into cells (14). Even octaarginine (Arg 8 ) gave similar results based on the fluorescence microscopic observation of the fluorescein-labeled peptides (14, 15). These peptides seem to have other similarities in translocation, namely facile internalization within 5 min, little uptake inhibition at 4°C, and localization in the nucleus and cytosol. The above results suggested the possible existence of a ubiquitous mechanism for the internalization of the argininerich peptides. Since there were no sequence similarities among these peptides except that they had several arginine residues, arginine seemed to be the key amino acid for membrane permeability.Despite the great potential of the arginine-rich peptides as carriers of proteins, nucleic acids, and other bioactive compounds, little is known about the mechanism of their internalization. Involvement of the cell surface heparan sulfate (HS) and low density lipoprotein receptor-related protein (LRP) was suggested in the translocation of the full-length Tat protein (16,17). The addition of HS and the inhibitor of LRP to the culture medium produced a significant decrease in the cellular uptake of the protein. However, the uptake of the full-length Tat protein suffered a certain decrease at 4°C (16), and some energydependent endocytosis pathway seemed to play a significant role in the internalization of the Tat protein. These results suggested that the mechanisms of internalization of the Tat-(48 -60) peptide and the full-length Tat protein may not be completely parallel. Actually, the importance of the "core" domain (Tat-(37-48)) of the Tat protein has been claimed for the LRP-dependent internalization pathway (16).We have pointed out that many arginine-rich peptides showed very similar characteristics in translocation with HIV-1 Tat-(48 -60) (14). In addition to the translocation mechanisms of these arginine-rich peptides, it is unclear whether these peptides share...