Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins

The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery. The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery. The Tat protein of the human immunodeficiency virus type 1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; HSPG, heparan sulfate proteoglycan; TRITC, tetramethylrhodamine-isothiocyanate; GST, glutathione S-transferase; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyl-transferase; MβCD, methyl-β-cyclodextrin. is a powerful transcriptional activator of viral gene expression. At the long terminal repeat (LTR) promoter, the protein binds a cis-acting RNA element (trans-activation-responsive region) present at the 5′-end of each viral transcript (1Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 59: 273-282Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Through this interaction Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (for review, see Refs. 2Marcello A. Zoppe M. Giacca M. IUBMB Life. 2001; 51: 175-181Crossref PubMed Scopus (72) Google Scholar and 3Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Besides its fundamental role in the control of HIV-1 gene expression, more than 10 years ago it was first demonstrated that Tat also possesses the unusual property of entering the cells and translocating to the nucleus when present in the extracellular environment (4Green M. Loewenstein P.M. Cell. 1988; 55: 1179-1188Abstract Full Text PDF PubMed Scopus (1291) Google Scholar, 5Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2353) Google Scholar, 6Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (451) Google Scholar). This unusual characteristic depends upon the integrity of the basic region of the protein, a nine-amino acid, arginine-rich sequence that also corresponds to the nuclear localization signal and the trans-activation-responsive region binding domain of the protein (2Marcello A. Zoppe M. Giacca M. IUBMB Life. 2001; 51: 175-181Crossref PubMed Scopus (72) Google Scholar, 3Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). The property of Tat to cross cell membranes is common to a small group of proteins that also includes the herpesvirus structural protein VP22 (7Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar), the homeotic protein of Drosophila melanogaster Antennapedia (8Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar), and the fibroblast growth factor (9Jans D.A. FASEB J. 1994; 8: 841-847Crossref PubMed Scopus (134) Google Scholar) as well as several toxins of bacterial and plant origin. In most cases, the cellular mechanisms that these proteins use for cellular penetration are still poorly understood and are most probably different. The mounting interest in the cell-penetrating capacity of these proteins is due to their ability to drive the internalization of large protein cargoes that are chemically coupled or fused to them. In particular, the addition of an 11-amino acid stretch from the basic domain of Tat (10Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar) or the cross-link of Tat (11Fawell S. Seery J. Daikh Y. Moore C. Chen L.L. Pepinsky B. Barsoum J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 664-668Crossref PubMed Scopus (1107) Google Scholar) to heterologous molecules, even of a large size, mediates their cellular uptake; this property is currently widely exploited as a biotechnological tool for transcellular protein transduction. In the case of Tat, it has been demonstrated that the intraperitoneal injection of a 120-kDa β-galactosidase/Tat fusion protein results in the transcellular transduction into virtually all tissues in mice, including the passage of the blood-brain barrier (12Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar). Based on these observations, the translocating activity of the Tat basic domain is now being extensively utilized for several therapeutic applications, including protection from apoptosis in the brain (13Asoh S. Ohsawa I. Mori T. Katsura K. Hiraide T. Katayama Y. Kimura M. Ozaki D. Yamagata K. Ohta S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17107-17112Crossref PubMed Scopus (179) Google Scholar, 14Cao G. Pei W. Ge H. Liang Q. Luo Y. Sharp F.R. Lu A. Ran R. Graham S.H. Chen J. J. Neurosci. 2002; 22: 5423-5431Crossref PubMed Google Scholar) and in the heart (15Gustafsson A.B. Sayen M.R. Williams S.D. Crow M.T. Gottlieb R.A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar), extension of the cytotoxic activity of herpes simplex virus-1 thymidine kinase for cancer gene therapy (16Tasciotti E. Zoppe M. Giacca M. Cancer Gene Ther. 2003; 10: 64-74Crossref PubMed Scopus (48) Google Scholar), improvement of the IκBα activity (17Kabouridis P.S. Hasan M. Newson J. Gilroy D.W. Lawrence T. J. Immunol. 2002; 169: 2587-2593Crossref PubMed Scopus (46) Google Scholar), enhancement of antigen presentation by dendritic cells (18Wang H.Y. Fu T. Wang G. Zeng G. Perry-Lalley D.M. Yang J.C. Restifo N.P. Hwu P. Wang R.F. J. Clin. Invest. 2002; 109: 1463-1470Crossref PubMed Scopus (83) Google Scholar, 19Shibagaki N. Udey M.C. J. Immunol. 2002; 168: 2393-2401Crossref PubMed Scopus (125) Google Scholar), quenching of the tumor phenotype (20Datta K. Sundberg C. Karumanchi S.A. Mukhopadhyay D. Cancer Res. 2001; 61: 1768-1775PubMed Google Scholar), and enhancement of viral-mediated gene delivery (21Gratton J.P. Yu J. Griffith J.W. Babbitt R.W. Scotland R.S. Hickey R. Giordano F.J. Sessa W.C. Nat. Med. 2003; 9: 357-363Crossref PubMed Scopus (162) Google Scholar), to name but a few of the current applications. In addition, the Tat transduction domain has also been shown to mediate cell internalization of large molecules or particles, including magnetic nanoparticles (22Lewin M. Carlesso N. Tung C.H. Tang X.W. Cory D. Scadden D.T. Weissleder R. Nat. Biotechnol. 2000; 18: 410-414Crossref PubMed Scopus (1650) Google Scholar), phage vectors (23Eguchi A. Akuta T. Okuyama H. Senda T. Yokoi H. Inokuchi H. Fujita S. Hayakawa T. Takeda K. Hasegawa M. Nakanishi M. J. Biol. Chem. 2001; 276: 26204-26210Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), liposomes (24Torchilin V.P. Rammohan R. Weissig V. Levchenko T.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8786-8791Crossref PubMed Scopus (707) Google Scholar), and plasmid DNA (25Sandgren S. Cheng F. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Despite this variety of successful biotechnological applications of Tat as a delivery vector across cell membranes, the cellular mechanisms involved in the uptake of wild type HIV-1 Tat and of Tat fusion proteins are still obscure. We have previously that extracellular Tat with the heparan sulfate of cell membrane heparan sulfate M. G. D. E. P. G. Giacca M. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and that this interaction is for the uptake process to M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). These results with the that the internalization of large molecules fused to Tat is at that the interaction of Tat with is by an active endocytic is a that and a large of protein-protein and The first and pathway is endocytosis, on the membrane with the of that to 1997; PubMed Scopus Google Scholar). are the is endocytosis. are small in the membrane that a of membrane in and that are lipid rafts 1998; PubMed Scopus Google Scholar). are involved in signal transduction and the of lipid molecules, proteins internalized by the as are from these membrane K. E. 1997; PubMed Scopus Google Scholar, K. D. Nat. Biol. 2000; PubMed Scopus Google Scholar). bacterial including R.S. R. K. J. J. Biol. 2001; PubMed Scopus Google Scholar), and including J. A. Nat. Biol. 2001; PubMed Scopus Google Scholar), use of caveolar to the Here we show that extracellular proteins Tat or the Tat transduction domain are internalized through an active endocytic pathway from cell membrane lipid rafts and caveolar endocytosis. These results are of for all those applications that exploit Tat as a vehicle for transcellular protein transduction. and antigen 1 and from and all from from cells cell with a a of B. Science. 1988; PubMed Scopus Google Scholar) and and cells from the in with and in with and Tat plasmid the glutathione fusion protein was by the region in the plasmid M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) with the sequence for in the basic domain of The and fusion proteins and as M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). with the fluorescent and cells on to cells in a in with and the fluorescent molecules was and 10 10 and At the in each cells with and with in for 10 at and with in for and in mounting was cells with in for 10 and with in with and by an cell cells on in a and at and the the and cells a with with or with for 1 and in in for and with with the drugs 10 10 for in and proteins in the at for and for and of internalized proteins was as M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cells in to and with or for the in the with PBS, with PBS, with to with PBS, and by a cells on with protein as M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). cells and RNA was the with 1 of RNA was with and to the was by the in with a of a DNA the for of the RNA and and of the RNA and F. F. A. Giacca M. J. 1996; PubMed Google Scholar). was with and of the for the and the at for at for and extension at for for of by with and study the pathway involved in the internalization of extracellular Tat, we each the protein at the the enhanced of the green fluorescent protein at the and in the acid of Tat or its 11-amino acid transduction domain and and We have previously shown that the protein transcriptional activity on the viral LTR and is internalized by cell when to the extracellular of these cells M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of Tat kinetics of cellular internalization of the Tat is shown in 1 with the kinetics of internalization of The proteins to the cell of human cells at a of and cellular was at by cell with to protein to the cell of the and of the cellular in that the kinetics of internalization of and of Tat fusion proteins is different. is internalized with of of In internalization of the and proteins is and in the first 10 it a for at to was and of cells with the proteins in the of that was to in the of internalization by an endocytic This is with a of from as well as from that the internalization of large protein cargoes fused or to Tat is an that it at and that is to drugs M. M. Giacca M. B. E. J. 2002; PubMed Scopus Google see also and The cellular internalization of Tat by was by the that extracellular and to internalized in when with a the addition to the cell in for caveolar Tat was to the cell of cells and at the the of the gene by RNA was to a DNA for the gene and for the cellular and The show for the the of each the the of the is The on the of the of LTR at the the the of on the The as of in for each shown are the and S.D. of and are internalized through of endocytosis. colocalization of and 1 of cells with and for 10 and 1 that the proteins are internalized in the The localization was also in the cells with and and with the by in and and present in with with the proteins for the Tat by in is the endocytic pathway involved in Tat is to internalized from on the membrane that to 1997; PubMed Scopus Google Scholar). and of a of these to their fusion with cell this also the case of Tat endosomes, we to and the proteins to the of shown in proteins in at these at and all through the and in the Tat still to the cell with the kinetics of shown in The of in Tat internalization was by the that internalized Tat with the antigen 1 a protein with of is to and fusion with the cell we the of internalized fluorescent Tat with a for cell shown in at all the Tat and in that These results are shown in for the those with the protein as shown in for these results that Tat is internalized by in Tat from to toxins K. Biol. 2000; PubMed Scopus Google Scholar), the active of is the internalized by from lipid rafts J. Biol. 1998; PubMed Scopus Google Scholar), cell membrane in and this the case also for Tat we their with the of shown in and more than of and also the group of cell membranes that have lipid are membrane by a membrane of the protein J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we Tat also for the of shown in in are to a large of for and rafts are by their in nonionic we the of on cells that previously internalized Tat and The the endosomes, those or in and to the that the internalization of extracellular Tat from cell membrane lipid we cells with a that from cell membranes, lipid show in and for and in cells with MβCD, of Tat was and a was the cell internalized fluorescent In internalization of was by the an that Tat uptake caveolar endocytosis, we internalization of Tat, and in cells by was to or to and to the of of was at for each with for a of In cells to Tat and the localization of the Tat this in to the of In cells with Tat and proteins the localization of a of internalization of is to in also in J. Biol. 1998; PubMed Scopus Google Scholar). These results are in with the that the at caveolar is than that of I. C. A. A. W. J. Sci. 1997; PubMed Google Scholar). The internalization of fluorescent Tat in the of cell was also by and with the internalization of shown in Tat uptake in cells was at for an active and by with of cell with M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). The also the internalization of as drugs that cellular and on Tat or cell with Tat that the at the cell in cells with this and was due to protein with cell internalized and by a of cell Tat a that is with the of in caveolar M. B. Biol. Cell. 2000; PubMed Scopus Google Scholar). on Tat cell for each of these interest is the of cell in cells with the of large fluorescent at the cell shown in and by results also with we the pathway of extracellular Tat internalization also for the as a for HIV-1 has also been shown to a for extracellular Tat H. C. M. Rich P.M. Jeang Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, S. D.M. G. M. R. M. E. G. A. Res. 2000; PubMed Scopus Google Scholar). by and with internalization by with or at to with Tat uptake also in these the of lipid rafts in the internalization to the of Tat to the have previously demonstrated that the fusion protein is active in of the viral LTR when to the cell of a cell from cells an HIV-1 Science. 1988; PubMed Scopus Google Scholar). we exploited this property to that the of protein that was internalized through caveolar from lipid rafts was the that was to the nucleus and was active in this we a on to the of upon with This is on the of the cellular with a DNA the for the transcript and for the cellular a is shown in of cells with for a of more than 10 with the fluorescent protein internalization when cells with MβCD, or at of the LTR was In with on fluorescent protein a in the of was in control cells with These that the drugs or caveolar from lipid rafts also by Tat, that this of internalization to the of transcriptionally active protein in the cell of its the cell membrane as a barrier that the passage of This barrier also a to the use of active that are the it has been that several toxins of bacterial and plant are of translocating across cellular membranes to their in the or in the In most cases, the of these proteins for the delivery of heterologous proteins has been by their large size, and the that a acid stretch from the basic domain of the HIV-1 Tat translocating capacity even to large protein cargoes has been with and has the of a of applications in and in The in this that the cell uptake of large protein cargoes fused to Tat or to its 11-amino acid transduction domain through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. This is supported by the slow kinetics of the that Tat are to the nonionic the colocalization of Tat fusion proteins with in cells that this protein but with markers of endocytosis, and the of Tat internalization to drugs that lipid as or disturb of to the as D. this to the of internalization of large fused to Tat including a phage the Tat transduction domain on its (23Eguchi A. Akuta T. Okuyama H. Senda T. Yokoi H. Inokuchi H. Fujita S. Hayakawa T. Takeda K. Hasegawa M. Nakanishi M. J. Biol. Chem. 2001; 276: 26204-26210Abstract Full Text Full Text PDF PubMed Scopus (275) Google is small the Tat transduction domain also the cells by has been to show that basic including the Tat basic cross cell membranes by a that is to and T. S. M. S. K. Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, E. P. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). protein was with a fluorescent the Tat basic domain by the molecules to the cell it was that the the cells at the protein M. M. Giacca M. B. E. J. 2002; PubMed Scopus Google Scholar). to that the that the Tat basic domain cross the cell membrane the of due to or of protein J.P. K. E. C. B. B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). an active process also for the cellular uptake of as also by and Frankel D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (451) Google Scholar). The of caveolar as the cellular pathway of Tat fusion proteins now this to more We have that Tat internalization binding of the protein to cell the uptake process in cells in and by cell with or by with extracellular M. G. D. E. P. G. Giacca M. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of the of cell is the a domain and are to the of the membrane G. FASEB J. PubMed Scopus Google Scholar). proteins are in lipid rafts on the cell R.S. R. K. J. J. Biol. 2001; PubMed Scopus Google Scholar, M. H. EMBO J. 2000; PubMed Scopus Google Scholar). In addition, is to internalized and the G. B. A. PubMed Scopus Google Scholar), with the internalization process in F. K. J. K. Belting M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). a is that the internalization of Tat its interaction with the of this In this it is of interest to that the internalization process in a that binds extracellular Tat H. C. M. Rich P.M. Jeang Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, S. D.M. G. M. R. M. E. G. A. Res. 2000; PubMed Scopus Google Scholar) and of lipid rafts W. W.C. J. 2002; PubMed Scopus Google Scholar, D. J. Immunol. 2002; 168: PubMed Scopus Google Scholar, D. J. 2002; PubMed Scopus Google Scholar), is also by from This that this is involved in Tat internalization or that its to lipid rafts is for the internalization process to to HIV-1 extracellular including bacterial toxins and are internalized caveolar endocytosis. that the of this pathway are slow when with I. C. A. A. W. J. Sci. 1997; PubMed Google Scholar, P. K. M. B. Biol. Cell. 2002; PubMed Scopus Google Scholar). This is in with the in cells that are as with when in a with these on cells has shown that an of molecules of the from the cell membrane the region with an of and that this process the integrity of the and F. in Scholar). shown in with the of in large at the cell This is in with the that are with the M. B. Biol. Cell. 2000; PubMed Scopus Google Scholar) and that with to of to the membrane P. K. M. B. Biol. Cell. 2002; PubMed Scopus Google Scholar, T. A. K. J. Sci. PubMed Google Scholar). In of on Tat is internalized by the and it in the nucleus in a transcriptionally active that the pathway to caveolar of the protein from the to the of the protein in the This is the all the drugs and that also the transcriptional activity of the This that is an pathway for extracellular Tat In addition, it also that the internalized Tat has to the to to the The from the endocytic is a process that is to through a cellular In this it is in the with the Tat in this at we in the the protein the LTR and by with for M. M. M. Giacca M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). probably this that the fusion proteins membrane as in this an property of is to in an active at A. V. P. R. T. C. S. J. Cell. 2001; Scopus Google Scholar). This also that the use of the as the by to study the of Tat from the cells process of a cell as S. Gene Ther. 2002; 9: PubMed Scopus Google Scholar), and plant toxins as and to the through the and the A. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google with these of with J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.C. Res. 1996; PubMed Scopus Google Scholar). interest in this is the that with of the protein into the as by it its nuclear as shown by the LTR This that the for this Despite this we have to Tat in the or in the that the process in a of the internalized molecules, as is also the case for A. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). at of proteins through the this We are to V. for and for