Mechanism for HIV-1 Tat Insertion into the Endosome Membrane
2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês
10.1074/jbc.m109.023705
ISSN1083-351X
AutoresHocine Yezid, Karidia Konate, Solène Debaisieux, Anne Bonhoure, Bruno Beaumelle,
Tópico(s)Virus-based gene therapy research
ResumoThe human immunodeficiency virus, type 1, transactivating protein Tat is a small protein that is strictly required for viral transcription and multiplication within infected cells. The infected cells actively secrete Tat using an unconventional secretion pathway. Extracellular Tat can affect different cell types and induce severe cell dysfunctions ranging from cell activation to cell death. To elicit most cell responses, Tat needs to reach the cell cytosol. To this end, Tat is endocytosed, and low endosomal pH will then trigger Tat translocation to the cytosol. Although this translocation step is critical for Tat cytosolic delivery, how Tat could interact with the endosome membrane is unknown, and the key residues involved in this interaction require identification. We found that, upon acidification below pH 6.0 (i.e. within the endosomal pH range), Tat inserts into model membranes such as monolayers or lipid vesicles. This insertion process relies on Tat single Trp, Trp-11, which is not needed for transactivation and could be replaced by another aromatic residue for membrane insertion. Nevertheless, Trp-11 is strictly required for translocation. Tat conformational changes induced by low pH involve a sensor made of its first acidic residue (Glu/Asp-2) and the end of its basic domain (residues 55–57). Mutation of one of these elements results in membrane insertion above pH 6.5. Tat basic domain is also required for efficient Tat endocytosis and membrane insertion. Together with the strict conservation of Tat Trp among different virus isolates, our results point to an important role for Tat-membrane interaction in the multiplication of human immunodeficiency virus type 1. The human immunodeficiency virus, type 1, transactivating protein Tat is a small protein that is strictly required for viral transcription and multiplication within infected cells. The infected cells actively secrete Tat using an unconventional secretion pathway. Extracellular Tat can affect different cell types and induce severe cell dysfunctions ranging from cell activation to cell death. To elicit most cell responses, Tat needs to reach the cell cytosol. To this end, Tat is endocytosed, and low endosomal pH will then trigger Tat translocation to the cytosol. Although this translocation step is critical for Tat cytosolic delivery, how Tat could interact with the endosome membrane is unknown, and the key residues involved in this interaction require identification. We found that, upon acidification below pH 6.0 (i.e. within the endosomal pH range), Tat inserts into model membranes such as monolayers or lipid vesicles. This insertion process relies on Tat single Trp, Trp-11, which is not needed for transactivation and could be replaced by another aromatic residue for membrane insertion. Nevertheless, Trp-11 is strictly required for translocation. Tat conformational changes induced by low pH involve a sensor made of its first acidic residue (Glu/Asp-2) and the end of its basic domain (residues 55–57). Mutation of one of these elements results in membrane insertion above pH 6.5. Tat basic domain is also required for efficient Tat endocytosis and membrane insertion. Together with the strict conservation of Tat Trp among different virus isolates, our results point to an important role for Tat-membrane interaction in the multiplication of human immunodeficiency virus type 1. The human immunodeficiency virus, type 1 (HIV-1), 2The abbreviations used are: HIV-1human immunodeficiency virus, type 110-DN10-doxylnonadecaneSUVsmall unilamellar vesiclesPEAPseudomonas exotoxin APTDprotein transduction domainTftransferrinPCphosphatidylcholinePGphosphatidylglycerolLTRlong terminal repeat. 2The abbreviations used are: HIV-1human immunodeficiency virus, type 110-DN10-doxylnonadecaneSUVsmall unilamellar vesiclesPEAPseudomonas exotoxin APTDprotein transduction domainTftransferrinPCphosphatidylcholinePGphosphatidylglycerolLTRlong terminal repeat. transactivating protein Tat is a small basic protein of 86–102 residues, depending on the viral isolate (1Huigen M.C. Kamp W. Nottet H.S. Eur. J. Clin. Invest. 2004; 34: 57-66Crossref PubMed Scopus (93) Google Scholar). An essential function of this protein is to participate in the transcription of viral genes. In the absence of Tat, transcription from the HIV-1 long terminal repeat only produces short RNA, whereas the expression of Tat results in the production of long RNA and a marked increase in gene expression (2Strebel K. AIDS. 2003; 17 (Suppl. 4): S25-S34Crossref PubMed Scopus (54) Google Scholar). Tat is also involved in other steps enabling virus production and is strictly required for HIV-1 multiplication within infected cells (2Strebel K. AIDS. 2003; 17 (Suppl. 4): S25-S34Crossref PubMed Scopus (54) Google Scholar). human immunodeficiency virus, type 1 10-doxylnonadecane small unilamellar vesicles Pseudomonas exotoxin A protein transduction domain transferrin phosphatidylcholine phosphatidylglycerol long terminal repeat. human immunodeficiency virus, type 1 10-doxylnonadecane small unilamellar vesicles Pseudomonas exotoxin A protein transduction domain transferrin phosphatidylcholine phosphatidylglycerol long terminal repeat. A number of studies suggest that Tat function is not restricted to infected cells (1Huigen M.C. Kamp W. Nottet H.S. Eur. J. Clin. Invest. 2004; 34: 57-66Crossref PubMed Scopus (93) Google Scholar, 3Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Crossref PubMed Scopus (800) Google Scholar, 4Rubartelli A. Poggi A. Sitia R. Zocchi M.R. Immunol. Today. 1998; 19: 543-545Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Gallo R.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8324-8326Crossref PubMed Scopus (146) Google Scholar, 6King J.E. Eugenin E.A. Buckner C.M. Berman J.W. Microbes Infect. 2006; 8: 1347-1357Crossref PubMed Scopus (175) Google Scholar, 7Wu R.F. Ma Z. Myers D.P. Terada L.S. J. Biol. Chem. 2007; 282: 37412-37419Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Indeed, the infected cells secrete Tat (3Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Crossref PubMed Scopus (800) Google Scholar) using a secretion process that is termed unconventional, because Tat does not have a signal sequence. Accordingly, Tat secretion is insensitive to the fungal metabolite brefeldin A, indicating that Tat bypasses the endoplasmic reticulum to exit the cell (8Chang H.C. Samaniego F. Nair B.C. Buonaguro L. Ensoli B. AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (391) Google Scholar). This is the case for most proteins secreted using nonclassical secretion mechanisms (9Nickel W. Traffic. 2005; 6: 607-614Crossref PubMed Scopus (280) Google Scholar). Although some of these pathways have been characterized (10Temmerman K. Ebert A.D. Müller H.M. Sinning I. Tews I. Nickel W. Traffic. 2008; 9: 1204-1217Crossref PubMed Scopus (86) Google Scholar), this is not the case for Tat. Tat concentrations measured in the sera of patients infected with HIV-1 were in the nanomolar range (11Xiao H. Neuveut C. Tiffany H.L. Benkirane M. Rich E.A. Murphy P.M. Jeang K.T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11466-11471Crossref PubMed Scopus (324) Google Scholar). These values are most likely underestimated because Tat binds very efficiently to several cell types, such as endothelial cells (12Albini A. Soldi R. Giunciuglio D. Giraudo E. Benelli R. Primo L. Noonan D. Salio M. Camussi G. Rockl W. Bussolino F. Nat. Med. 1996; 2: 1371-1375Crossref PubMed Scopus (348) Google Scholar). Circulating Tat does not seem to result from the lysis of infected cells that instead appear to actively secrete large amounts of Tat into the bloodstream (3Ensoli B. Barillari G. Salahuddin S.Z. Gallo R.C. Wong-Staal F. Nature. 1990; 345: 84-86Crossref PubMed Scopus (800) Google Scholar). Extracellular Tat can exert different effects on cell functions and generate a wide array of cell responses ranging from cell activation (T-cells and endothelial cells) to stimulation of cytokine secretion (T-cells and monocytes) and cell death (neurons and endothelial and T-cells) (1Huigen M.C. Kamp W. Nottet H.S. Eur. J. Clin. Invest. 2004; 34: 57-66Crossref PubMed Scopus (93) Google Scholar, 4Rubartelli A. Poggi A. Sitia R. Zocchi M.R. Immunol. Today. 1998; 19: 543-545Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Gallo R.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8324-8326Crossref PubMed Scopus (146) Google Scholar, 6King J.E. Eugenin E.A. Buckner C.M. Berman J.W. Microbes Infect. 2006; 8: 1347-1357Crossref PubMed Scopus (175) Google Scholar, 7Wu R.F. Ma Z. Myers D.P. Terada L.S. J. Biol. Chem. 2007; 282: 37412-37419Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Accumulating evidence suggests that extracellular Tat is involved in the evolution to AIDS (1Huigen M.C. Kamp W. Nottet H.S. Eur. J. Clin. Invest. 2004; 34: 57-66Crossref PubMed Scopus (93) Google Scholar, 4Rubartelli A. Poggi A. Sitia R. Zocchi M.R. Immunol. Today. 1998; 19: 543-545Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Gallo R.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 8324-8326Crossref PubMed Scopus (146) Google Scholar, 6King J.E. Eugenin E.A. Buckner C.M. Berman J.W. Microbes Infect. 2006; 8: 1347-1357Crossref PubMed Scopus (175) Google Scholar), and Tat is considered as an important component in developing anti-HIV-1 vaccines (13Ensoli B. Fiorelli V. Ensoli F. Cafaro A. Titti F. Buttò S. Monini P. Magnani M. Caputo A. Garaci E. AIDS. 2006; 20: 2245-2261Crossref PubMed Scopus (68) Google Scholar). To elicit cell responses, at least on T-cells and monocytes, Tat needs to reach the cytosol (14Ott M. Emiliani S. Van Lint C. Herbein G. Lovett J. Chirmule N. McCloskey T. Pahwa S. Verdin E. Science. 1997; 275: 1481-1485Crossref PubMed Scopus (187) Google Scholar, 15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar, 16Rayne F. Vendeville A. Bonhoure A. Beaumelle B. J. Virol. 2004; 78: 12054-12057Crossref PubMed Scopus (20) Google Scholar). To this end, Tat first binds to several cellular receptors such as CD26 (17Gutheil W.G. Subramanyam M. Flentke G.R. Sanford D.G. Munoz E. Huber B.T. Bachovchin W.W. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6594-6598Crossref PubMed Scopus (131) Google Scholar), CXCR4 (11Xiao H. Neuveut C. Tiffany H.L. Benkirane M. Rich E.A. Murphy P.M. Jeang K.T. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 11466-11471Crossref PubMed Scopus (324) Google Scholar), heparan sulfate proteoglycans (18Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar), and the low density lipoprotein receptor-related protein (19Liu Y. Jones M. Hingtgen C.M. Bu G. Laribee N. Tanzi R.E. Moir R.D. Nath A. He J.J. Nat. Med. 2000; 6: 1380-1387Crossref PubMed Scopus (330) Google Scholar). Tat is then endocytosed (20Frankel A.D. Pabo C.O. Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2329) Google Scholar, 21Mann D.A. Frankel A.D. EMBO J. 1991; 10: 1733-1739Crossref PubMed Scopus (446) Google Scholar), essentially by the clathrin-coated pit pathway (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar). Once in the endosome, Tat crosses the membrane to enter the cytosol. This translocation step is triggered by endosomal low pH (pH 5.3–5.5) and is facilitated by the cytosolic chaperone Hsp90 (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar). Hence, Tat enters cells using a "conventional" endosomal translocating toxin strategy, just like diphtheria toxin, which is the best characterized example of this type of toxin (22Falnes P.O. Sandvig K. Curr. Opin. Cell Biol. 2000; 12: 407-413Crossref PubMed Scopus (244) Google Scholar). The study of the translocation process of endosome-translocating toxins was facilitated by their ability to insert into model membranes upon acidification (23Rosconi M.P. London E. J. Biol. Chem. 2002; 277: 16517-16527Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 24Méré J. Morlon-Guyot J. Bonhoure A. Chiche L. Beaumelle B. J. Biol. Chem. 2005; 280: 21194-21201Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Whether Tat is also able to do so has yet to be demonstrated. Moreover, although the implication of each Tat residue in transcriptional activity has been thoroughly determined (25Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), the molecular determinants that enable Tat to enter the cytosol of mammalian cells has yet to be identified. The Tat basic domain (residues 49–57) is likely involved in cell binding because the corresponding peptide recognizes cell surface heparan sulfate proteoglycans (26Soane L. Fiskum G. J. Neurochem. 2005; 95: 230-243Crossref PubMed Scopus (23) Google Scholar, 27Ziegler A. Seelig J. Biophys. J. 2004; 86: 254-263Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), just like native Tat (18Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). From its capacity to introduce attached proteins into the cell cytosol, this peptide has been termed the protein transduction domain (PTD), and Tat-PTD is a popular tool to deliver cargos intracellularly (28Lindgren M. Hällbrink M. Prochiantz A. Langel U. Trends Pharmacol. Sci. 2000; 21: 99-103Abstract Full Text Full Text PDF PubMed Scopus (792) Google Scholar). Tat-PTD has been shown to enter cells using either macropinocytosis (29Wadia J.S. Stan R.V. Dowdy S.F. Nat. Med. 2004; 10: 310-315Crossref PubMed Scopus (1413) Google Scholar) or clathrin-mediated endocytosis (30Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1479) Google Scholar). The fact that Tat and Tat-PTD share a common receptor and can both undergo clathrin-dependent uptake indicates that these early steps of Tat entry into cells might involve the PTD. Whether this is the case for later events such as transmembrane transport is unknown. A key step in Tat translocation is Tat insertion into the endosome membrane. It is not clear how such a small protein as Tat, which is devoid of a hydrophobic α-helix (31Bayer P. Kraft M. Ejchart A. Westendorp M. Frank R. Rösch P. J. Mol. Biol. 1995; 247: 529-535Crossref PubMed Scopus (188) Google Scholar), could undergo membrane insertion. Nevertheless, we previously showed that insertion of Pseudomonas exotoxin A (PEA) into the endosome membrane relies on a key tryptophan residue that becomes exposed at endosomal pH (pH 5.3–5.5) and then triggers membrane insertion (24Méré J. Morlon-Guyot J. Bonhoure A. Chiche L. Beaumelle B. J. Biol. Chem. 2005; 280: 21194-21201Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Other proteins such as annexin-V (32Campos B. Mo Y.D. Mealy T.R. Li C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar) and protein kinase C-α (33Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) also undergo such regulated Trp-mediated membrane insertion, although in that case the stimulus triggering insertion is a rise in Ca2+ concentration and not acidification. Interestingly, Tat has a single Trp that, despite the high HIV-1 mutation rate (34Walker B.D. Burton D.R. Science. 2008; 320: 760-764Crossref PubMed Scopus (296) Google Scholar), is one of the best conserved Tat residues (35Pantano S. Carloni P. Proteins. 2005; 58: 638-643Crossref PubMed Scopus (16) Google Scholar). According to a three-dimensional structure based on two-dimensional NMR experiments, Trp-11 is located in the center of the molecule, sandwiched between the core and glutamine-rich domains (31Bayer P. Kraft M. Ejchart A. Westendorp M. Frank R. Rösch P. J. Mol. Biol. 1995; 247: 529-535Crossref PubMed Scopus (188) Google Scholar), and is therefore potentially involved in Tat insertion into the endosome membrane. All known proteins whose membrane insertion is based on a Trp side chain have a molecular device that induces Trp exposure and thereby controls insertion (24Méré J. Morlon-Guyot J. Bonhoure A. Chiche L. Beaumelle B. J. Biol. Chem. 2005; 280: 21194-21201Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 32Campos B. Mo Y.D. Mealy T.R. Li C.W. Swairjo M.A. Balch C. Head J.F. Retzinger G. Dedman J.R. Seaton B.A. Biochemistry. 1998; 37: 8004-8010Crossref PubMed Scopus (47) Google Scholar, 33Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Because the Tat membrane insertion process only takes place within endosomes (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar), if Tat actually belongs to this group of proteins it likely possesses a low-pH sensor able to trigger Trp exposure upon endosomal delivery. Here we showed that Tat inserts into model membranes upon acidification, and we identified key determinants of this insertion process. We found that Tat uses a low-pH sensor involving Asp/Glu-2 and the basic domain to regulate exposure of its single Trp that allows Tat to initiate membrane insertion. Chemicals were from Sigma, and phospholipids were from Avanti Polar Lipids. 10-DN was kindly provided by Erwin London (Stony Brook University, New York). Transferrin (Tf) was labeled with Cy5 using the protocol provided by the manufacturer of the labeling kit (GE Healthcare). Monoclonal antibodies were from Advanced Biotechnology Inc. (anti-Tat) and the Iowa Developmental Studies Hybridoma Bank (anti-Lamp-1). Secondary antibodies were obtained as indicated (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar). Jurkat T-cells (clone E6–1) were from ATCC and transfected using electroporation (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar). We used a Tat (BH10 isolate) of 86 residues. An elongated version of 101 residues, obtained by mutating the stop codon into Ser (25Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), was also used for some assays with similar results. Site-directed mutagenesis of Tat in pET-11d vector (for expression in Escherichia coli) and pBi-GL (for expression in mammalian cells) was performed using QuikChange kits (Stratagene) and appropriate primers. All mutant DNA coding sequences were entirely sequenced (Genome Express, Meylan, France). Recombinant Tat or Tat mutants were expressed and purified from E. coli as described (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar), and identification was confirmed by mass spectrometry (Proteomic Facilities of the IFR3, Montpellier, France). An SS35 spectrofluorometer and ultramicro-quartz 200-μl cuvettes (PerkinElmer Life Sciences) were used. Slit widths were 7 nm for both excitation and emission. Fluorescence intensity was measured at 351 nm using excitation at 279 nm. The background intensity measured in the absence of Tat was subtracted. To prepare small unilamellar vesicles (SUV), lipids (dioleoylphosphatidylcholine (PC)/dioleoylphosphatidylglycerol (PG) 75/25) were dried under N2 and further dried under high vacuum overnight. The dried lipids were then suspended in 100 mm NaCl, 50 mm citrate, pH 7.2 (citrate buffer), and sonicated using a bath sonicator for 1 h and then a microtip sonicator for 3 min. The resulting SUV had a diameter of 100–200 nm. To prepare brominated vesicles, half of the PC was replaced by 1,2-(9,10-dibromo)stearoyl phosphatidylcholine (24Méré J. Morlon-Guyot J. Bonhoure A. Chiche L. Beaumelle B. J. Biol. Chem. 2005; 280: 21194-21201Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). When indicated the 10-DN quencher was added at 10 mol % before drying the lipids (36Caputo G.A. London E. Biochemistry. 2003; 42: 3265-3274Crossref PubMed Scopus (85) Google Scholar). Experiments were performed using 2 μm Tat in citrate buffer, pH 7.0, unless otherwise stated. SUV were used at a concentration of 400 μm lipids. Quencher and SUV concentrations were high enough to produce a minimal dilution that was taken into account for calculation. The inner filter effect caused by the highest acrylamide concentrations was negligible and was not corrected (37Jiang J.X. London E. J. Biol. Chem. 1990; 265: 8636-8641Abstract Full Text PDF PubMed Google Scholar). Tat insertion into a phospholipid monolayer of PC/PG (75/25) was measured at 23 °C by monitoring the change in surface pressure (π) at constant surface area using a 10-ml square Teflon trough and a small diameter wire probe fitted on a Kibron microtrough S instrument (38Stahelin R.V. Long F. Peter B.J. Murray D. De Camilli P. McMahon H.T. Cho W. J. Biol. Chem. 2003; 278: 28993-28999Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The lipid monolayer was spread onto the citrate buffer subphase until the desired initial surface pressure (π0) was reached. After 15 min, Tat (26 nm) was injected into the subphase. Following pressure stabilization (15–20 min), the pH was lowered to pH 5.3 by injecting concentrated HCl over a 1-min period. The increase in surface pressure (Δπ) was monitored for 30 min while stirring the subphase at 60 rpm. Typically, the Δπ value reached a maximum after 20 min. The resulting Δπ was plotted versus π0, allowing us to determine the critical surface pressure (πC) as the x-intercept (38Stahelin R.V. Long F. Peter B.J. Murray D. De Camilli P. McMahon H.T. Cho W. J. Biol. Chem. 2003; 278: 28993-28999Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). For conversion, 1 dyne = 1 mn/m. The statistical significance of the data was examined using the correlation coefficient test. To examine the capacity of exogenous Tat to enter cells and transactivate the LTR, Jurkat T-cells (9.106) were cotransfected with 7 μg of pGL3-LTR, which expresses firefly luciferase under control of the Tat-activated HIV-1-LTR promoter, and 1 μg of pRL-TK (Promega), which codes for Renilla luciferase under control of the herpes simplex virus thymidine kinase promoter and is used to normalize results. After 18 h, 200 nm recombinant Tat (or Tat mutant) and 100 μm chloroquine were added. One day later, cells were lysed for dual luciferase assays (Promega), and transactivation activity was calculated using the firefly/Renilla activity ratio (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar). To assess whether Tat mutants preserved a native and reactive structure, Jurkat cells were cotransfected with the luciferase plasmids together with a Tat expression vector (3 or 10 μg, as indicated). After 48 h, cells were lysed, and transactivation activity was determined as described above. Jurkat T-cells were incubated for 6 h with 50 nm Tat. Tf-Cy5 (100 nm) was added for the last 40 min of labeling. Cells were then washed, fixed with 3.7% paraformaldehyde, processed for Tat and Lamp-1 detection as described (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar), and finally viewed under a Leica SPE confocal microscope. To quantify Tat colocalization with transferrin and Lamp-1 that are early and late endosome markers, respectively, we used images from 20 to 30 cells and the Metamorph software (Universal Imaging). To assess Tat endocytosis efficiency, the same images were used. Tf and Lamp-1 images were summed using Adobe Photoshop, and Tat internalization efficiency (%) was calculated using Metamorph as the percentage of Tat pixels present within a Lamp-1 or transferrin-positive compartment. Visual examination of processed images confirmed that Tat staining outside these structures localized to the plasma membrane. The anti-Tat monoclonal antibody we used for immunofluorescence binds to the first nine residues of Tat. It poorly recognized Tat-E2A and Tat-D5A, and endocytosis of these mutants could therefore not be reliably quantified. Structural studies of HIV-1 Tat performed by two-dimensional NMR suggested that the Tat single Trp is located within a valley and that Trp fluorescence should be highly sensitive to conformational modifications (31Bayer P. Kraft M. Ejchart A. Westendorp M. Frank R. Rösch P. J. Mol. Biol. 1995; 247: 529-535Crossref PubMed Scopus (188) Google Scholar). To assess whether Tat conformation changes upon solvent acidification, we monitored the effect of pH on Tat Trp fluorescence, first in the absence of membrane. Although fluorescence intensity decreased almost linearly with pH, there was nevertheless a small plateau between pH 5.8 and pH 5.5 indicating that a conformational change might take place within this pH range (Fig. 1A). Conformational modifications can also be detected by a shift in the wavelength of maximum emission (λmax). We thus monitored whether acidification induced modifications of the ratio of emission intensity at 330 nm to that at 350 nm, a method that is recognized as more accurate than direct measurements of minute λmax shifts (37Jiang J.X. London E. J. Biol. Chem. 1990; 265: 8636-8641Abstract Full Text PDF PubMed Google Scholar). The 330/350 ratio increased from pH 7 to pH 4 (Fig. 1B), indicating a blue shift in Trp fluorescence. Hence, the Tat Trp environment changes upon acidification. We used fluorescence quenchers to examine whether this environment modification was associated with structural changes leading to enhanced Trp accessibility. Whereas the large ions I− and Cs+ failed to reveal a difference in Trp access (Fig. 2, A and B), acrylamide quenching was more efficient at low pH (Fig. 2C). Hence, acidification enabled this small and neutral molecule to reach Tat Trp more easily. This indicates that Tat structure in this region opens when pH drops from pH 7.0 to pH 5.3, and that Tat Trp has easier access to the solvent at pH 5.3. It should be noted that the latter value is within the endosome pH range (40Rodríguez M. Torrent G. Bosch M. Rayne F. Dubremetz J.F. Ribó M. Benito A. Vilanova M. Beaumelle B. J. Cell Sci. 2007; 120: 1405-1411Crossref PubMed Scopus (52) Google Scholar), and that this molecular reorganization is therefore likely biologically relevant.FIGURE 2Effect of soluble quenchers on the Tat Trp fluorescence. 2 μm Tat in citrate buffer, at pH 7.0 or pH 5.3, received the indicated concentration of quencher. F0/F is the ratio of fluorescence intensity in the absence of quencher (F0) to the intensity in the presence of quencher (F), against the quencher concentration. A, quenching by I−; B, quenching by Cs2+; C, quenching by acrylamide. Representative results of triplicate experiments are presented. Similar data were obtained using a phosphate buffer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tat undergoes acid-triggered translocation across the endosomal membrane (15Vendeville A. Rayne F. Bonhoure A. Bettache N. Montcourrier P. Beaumelle B. Mol. Biol. Cell. 2004; 15: 2347-2360Crossref PubMed Scopus (171) Google Scholar), but it has yet to be shown whether it could insert into model membranes at low pH. To examine this issue, we first monitored the capacity of Tat to penetrate membrane monolayers of dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (75/25). They were spread at constant area and at an initial surface pressure (π0) onto a subphase at neutral pH. The change in surface pressure (Δπ) was monitored after Tat injection into the subphase. Extrapolation of the Δπ versus π0 plot yields πC, the highest π0 pressure of a monolayer that a protein can penetrate (38Stahelin R.V. Long F. Peter B.J. Murray D. De Camilli P. McMahon H.T. Cho W. J. Biol. Chem. 2003; 278: 28993-28999Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Fig. 3A shows that, at neutral pH, Tat does not penetrate monolayers, regardless of π0. Nevertheless, acidification to pH 5.3 following Tat injection induced membrane penetration with πC in the 30–35 mn/m range. Because the surface pressure of biological membranes and large unilamellar vesicles has also been estimated at 30–35 mn/m, this result indicates that Tat can penetrate model and biological membranes at pH 5.3, hence at endosomal pH (40Rodríguez M. Torrent G. Bosch M. Rayne F. Dubremetz J.F. Ribó M. Benito A. Vilanova M. Beaumelle B. J. Cell Sci. 2007; 120: 1405-1411Crossref PubMed Scopus (52) Google Scholar). If Tat is able to insert into model membranes upon acidification, and if Trp-11 is significantly involved in this insertion process, it should directly contact lipids and therefore be accessible to membrane-embedded quenchers such a
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