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J. Biol. Chem., Vol. 278, Issue 36, 34141-34149, September 5, 2003
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From the
Scuola Normale Superiore and
NEST-Istituto Nazionale per la Fisica della Materia, 56100 Pisa, Italy,
Istituto di Fisiologia Clinica, Consiglio
Nazionale delle Ricerche, 56100 Pisa, Italy, and
¶Molecular Medicine Laboratory, International
Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy
Received for publication, May 25, 2003 , and in revised form, April 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (4–6). 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 (2, 3).
The property of Tat to cross cell membranes is common to a small group of proteins that also includes the herpesvirus structural protein VP22 (7), the homeotic protein of Drosophila melanogaster Antennapedia (8), and the fibroblast growth factor (9) 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
(10) or the cross-link of Tat
(11) 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 
-galactosidase/Tat fusion protein
results in the transcellular transduction into virtually all tissues in mice,
including the passage of the blood-brain barrier
(12). 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
(13,
14) and in the heart
(15), extension of the
cytotoxic activity of herpes simplex virus-1 thymidine kinase for cancer gene
therapy (16), improvement of
the I
B
activity
(17), enhancement of antigen
presentation by dendritic cells
(18,
19), quenching of the tumor
phenotype (20), and
enhancement of viral-mediated gene delivery
(21), 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
(22), phage vectors
(23), liposomes
(24), and plasmid DNA
(25).
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 observed that extracellular Tat specifically interacts with the heparan sulfate chains of cell membrane heparan sulfate proteoglycans (HSPGs) (26) and that this interaction is absolutely required for the uptake process to occur (27). These results together with the observation that the internalization of large molecules fused to Tat is impaired at 4 °C suggest that the interaction of Tat with HSPGs is followed by an active endocytic process.
Endocytosis is a complex mechanism that involves different pathways and a large network of protein-protein and protein-lipid interactions. The first and best-characterized pathway is clathrin-dependent endocytosis, which starts on the plasma membrane with the formation of clathrin-coated invaginations that pinch off to make up clathrin-coated vesicles (28). Less defined are the non-classical, clathrin-independent pathways, among which is caveolae-mediated endocytosis.
Caveolae are flask-shaped, small (50–70 nm) invaginations in the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids that are called lipid rafts (29). Caveolae are involved in signal transduction and the intracellular transportation of lipid raft-associated molecules, whereas proteins internalized by the clathrin pathway, such as transferrin, are excluded from these membrane domains (30, 31). Several bacterial toxins, including cholera toxin (32), and some viruses, including SV40 (33), make use of caveolar endocytosis to enter the cells.
Here we show that extracellular proteins containing full-length Tat or the Tat transduction domain are internalized through an active endocytic pathway originating from cell membrane lipid rafts and involving caveolar endocytosis. These results are of relevance for all those applications that exploit Tat as a vehicle for transcellular protein transduction.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Cultures—HL3T1 cells (a HeLa cell derivative stably transfected with a silent LTR-CAT cassette), a kind gift of B. Felber (34) and HeLa and Cos-1 cells (both obtained from the American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamicin. Jurkat T-cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamicin.
Recombinant Tat Proteins—The plasmid expressing the glutathione S-transferase (GST)-Tat11-EGFP fusion protein was obtained by replacing the Tat86-coding region in the GST-Tat86-EGFP plasmid (27) with the sequence encoding for amino acids 48–58 in the basic domain of Tat. The recombinant GST-Tat86-EGFP and GST-Tat11-EGFP fusion proteins were produced and purified as described (27).
Fluorescence Microscopy—For treatment with the different fluorescent proteins, HeLa and Cos-1 cells were grown on chamber slides (Labtech International, Woodside, UK) to about 60% confluence; Jurkat cells were treated in suspension cultures. After a 30-min incubation in serum-free Dulbecco's modified Eagle's medium, fresh, serum-free medium supplemented with Hepes 25 mM (pH 7.2) and containing the different fluorescent molecules was added. Final concentrations were 2 µg/ml recombinant Tat86-EGFP and Tat11-EGFP, 10 µg/ml transferrin-TRITC, 10 µg/ml cholera toxin B-subunit, 50 nM Lysotracker Red, and 0.1 mg/ml dextran-TRITC. At the different time points indicated in each figure, cells were washed twice with PBS and fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. Cells were subsequently washed and incubated with glycine 100 mM in PBS for 5 min and mounted in Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA). Lysotracker Red was added 30 min before fixation. For immuno-staining, fixed cells were washed twice with 0.1% Triton X-100 in PBS for 10 min and incubated with antibodies in PBS supplemented with 0.15% glycine and 0.5% bovine serum albumin. Images were obtained by confocal microscopy using an Olympus FV300 microscope. For live cell recording, cells plated on 6-cm glass bottom dishes were placed in a humidified Plexiglas chamber and maintained at 37 °C and 5% CO2 throughout the experiment. For the fast dynamics recording and co-localization experiments cells were imaged using a TCS-SP laser-scanning confocal microscope (Leica Microsystems, Mannheim, Germany).
Treatment with Triton X-100 —Cells were incubated with either Tat86-EGFP or Tat11-EGFP together with labeled transferrin for 1 h, washed, and left in ice-cold 1% Triton X-100 in PBS for 20 min and then fixed with 2% paraformaldehyde.
Drug Treatments—Cells were pretreated with the different
drugs (5 µM cytochalasin D, 20 µM nocodazole, 20
µM taxol, 10 µM brefeldin A, 10 µg/ml heparin,
5 mM methyl--cyclodextrin) for 30 min in serum-free
Dulbecco's modified Eagle's medium, and then recombinant proteins in fresh
serum-free medium containing the same inhibitors were added. Cells were then
processed at different time points (1 h for fluorescence microscopy and 8 h
for flow cytometry and competitive reverse transcriptase-PCR analysis).
Flow Cytometry—Quantification of internalized Tat-EGFP recombinant proteins was performed as already described (27). Briefly, cells were plated in 6-well plates to about 60% confluence and incubated with Tat86-EGFP, Tat11-EGFP, or labeled transferrin for the time points indicated in the figures. Cells were then washed twice with PBS, trypsinized, again washed with PBS, washed with 2 M NaCl to completely take off surface-bound proteins, again washed twice with PBS, and finally, analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences).
Competitive PCR—The day before treatment, 2.5 x
105 HL3T1 cells were plated on 6-cm-diameter dishes. Cells were
incubated with recombinant Tat86-EGFP protein (or Tat11-EGFP control; 2
µg/ml) as described (27).
After incubation, cells were harvested, and RNA was isolated using the RNeasy
mini kit (Qiagen GmbH, Hilden, Germany). After treatment with DNase I
amplification grade (Invitrogen), 1 µg of RNA was reverse-transcribed with
SuperScriptTM II RNase H reverse transcriptase and random hexamers
(Invitrogen) according to the manufacturer's instructions. Competitive PCR was
performed by mixing the cDNA obtained in previous step with a fixed amount of
a competitor DNA molecule containing the primers for amplification of the
-actin RNA (BA1 and BA4) and of the CAT RNA (CATupper and CATlower)
(35). PCR amplification was
performed with 1/25 and 1/250 of the cDNA for the CAT and -actin
amplifications, respectively. For both primers, the PCR profiles were
denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and
extension at 72 °C for 30 s for 30 cycles of amplification. Amplification
products were resolved by agarose gel electrophoresis, stained with ethidium
bromide, and quantified.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Kinetics of Tat Internalization—The kinetics of cellular internalization of the two Tat fusions is shown comparatively in Fig. 1 together with the kinetics of internalization of recombinant TRITC-labeled transferrin. The three proteins were added to the cell culture medium of human HeLa epithelial cells at a concentration of 2 µg/ml, and cellular fluorescence was quantitatively assessed at different time points by flow cytometry after cell treatment with trypsin to remove any protein adsorbed to the cell surface. Analysis of the flow cytometry profiles and of the mean cellular fluorescence values (shown in Fig. 1) clearly indicates that the kinetics of internalization of transferrin and of Tat fusion proteins is remarkably different. Transferrin is internalized rapidly, with >80% of maximum fluorescence already obtained after 15 min of incubation. In contrast, internalization of both the Tat86 and Tat11 proteins is slower and progresses in the first 10 h, after which it reaches a plateau, which persists for at least up to 20 h. There was no difference noticed between Tat86-EGFP and Tat11-EGFP.
A microscopic analysis of cells treated with the three proteins (inset in the graphs of Fig. 1) indicated that fluorescence was localized to discrete compartments in the cytoplasm, suggestive of internalization by an endocytic process. This observation is consistent with a number of reports from our laboratory as well as from other laboratories which showed that the internalization of large protein cargoes fused or conjugated to Tat is an energy-dependent process, that it does not occur at 4 °C, and that is sensitive to drugs blocking endocytosis (36); see also Figs. 5 and 6. The cellular internalization of Tat by endocytosis was further corroborated by the observations that both extracellular Tat11-EGFP and Tat86-EGFPs were found to be internalized in discrete vesicles when mixed together with labeled Mr 10,000 dextran, a fluid phase marker, before the addition to the cell medium (shown in Fig. 2A for Tat11-EGFP).
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Tat Is Not Internalized by Endocytosis in Clathrin-coated Endosomes—Which is the endocytic pathway involved in Tat internalization? Transferrin is known to be internalized from clathrin-coated invaginations on the plasma membrane that eventually detaches to form clathrin-coated vesicles (28). Maturation and trafficking of a vast proportion of these endosomes eventually leads to their fusion with cell lysosomes. To assess whether this might also be the case of Tat endosomes, we mixed Tat11-EGFP to TRITC-labeled transferrin and added the two proteins to the culture medium of HeLa cells. As shown in Fig. 2B, both proteins appeared in discrete intracellular endosomes at different time points after treatment; however, these compartments were clearly distinguishable. Additionally, at earlier time points (10 and 30 min), although transferrin-containing endosomes were dispersed all through the cytoplasm and enriched in the perinuclear recycling compartment, Tat endosomes were still localized to the cell periphery, consistent with the different kinetics of endocytosis shown in Fig. 1. The involvement of clathrin-coated endosomes in Tat internalization was further ruled out by the observation that internalized Tat does not co-localize with the early endosome antigen 1 (EEA1), a protein associated with early endosomes (Fig. 2C). Finally, endocytosis of clathrin-coated vesicles is known to involve progressive endosome maturation and eventual fusion with the cell lysosome. Therefore, we tested the possible co-localization of internalized fluorescent Tat with Lysotracker, a marker for cell lysosomes. As shown in Fig. 2D, at all the analyzed time points, Tat and lysosome fluorescence appeared in vesicles that were clearly distinct. These results are shown in Fig. 2, A–D, for the Tat11-EGFP molecule; those obtained with the Tat86-EGFP protein were superimposable, as shown in Fig. 2, E–G, for selected time points. Taken together, these results clearly indicate that Tat is not internalized by endocytosis in clathrincoated endosomes.
Tat Endocytosis Occurs from Cell Membrane Lipid Rafts— Similar to other microbial toxins (37), the active fraction of cholera toxin is the one internalized by clathrin-independent endocytosis from lipid rafts (38), cell membrane microdomains enriched in cholesterol and sphingolipid. To assess whether this might be the case also for our Tat fusions, we tested their co-localization with the labeled B-subunit of cholera toxin. As shown in Fig. 3, A and B, respectively, more than 80% of both Tat11-EGFP- and Tat86-EGFP endosomes also contained the cholera toxin.
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One group of cell membranes that have lipid raft properties are caveolae, typical flask-shaped membrane invaginations defined by a distinctive membrane coat composed of the cholesterol-binding protein caveolin-1 (39). Therefore, we tested whether Tat endosomes were also positive for the presence of caveolin-1. As shown in Fig. 3C, in Cos-1 cells, which are known to express caveolin-1, a large proportion of endosomes proved positive for both caveolin-1 and Tat11-EGFP.
Lipid rafts are operationally defined by their insolubility in nonionic
detergents. Thus, we tested the effect of Triton X-100 on cells that had
previously (1 h earlier) internalized both Tat and transferrin. The detergent
completely solubilized the transferrin endosomes, whereas those containing
either Tat11-EGFP or Tat86-EGFP remained unaffected (shown in
Fig. 3, D and
E, respectively). Finally, to further corroborate the
conclusion that the internalization of extracellular Tat occurs from cell
membrane lipid rafts, we treated cells with methyl--cyclodextrin
(MCD), a drug that extracts cholesterol from cell membranes, thus
disrupting lipid rafts. As show in Fig. 3,
F and G, for Tat86-EGFP and Tat11-EGFP,
respectively, in cells treated with MCD, endocytosis of Tat was clearly
impaired, and a peculiar fluorescence pattern was visualized marking the cell
membrane. No internalized fluorescent endosomes were evident. In contrast,
internalization of transferrin was not affected by the drug.
As an additional indication that Tat uptake involves caveolar endocytosis, we comparatively analyzed internalization of Tat, transferrin, and cholera toxin B in living cells by time lapse confocal microscopy. Tat86-EGFP was mixed to either transferrin-TRITC or to CtxB-Alexa 594 and added to the culture medium of HeLa cells. After 30 min, fluorescence of endosomes was visualized at 10-s intervals. Fig. 4 shows one confocal plane for each experiment, with images taken for a total of 40 s. In cells exposed to Tat and transferrin (panel A), the localization of the endosomes containing Tat appeared unchanged during this time frame, in sharp contrast to the relative mobility of transferrin-containing endosomes. In cells treated with Tat and cholera toxin B (panel B), endosomes containing both proteins again were relatively immobile, whereas the localization of a subset of endosomes containing only cholera toxin B changed over time; internalization of cholera toxin B is known to occur, in part, also in clathrin-coated endosomes (38). These results are in agreement with the notion that the velocity at which caveolar endocytosis proceeds is remarkably slower than that of clathrin-dependent endocytosis (40).
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The internalization of fluorescent Tat in the presence of different cell
treatments was also quantitatively addressed by flow cytometry and compared
with the internalization of labeled transferrin. As shown in
Fig. 5A, Tat uptake in
HeLa cells was blocked at 4 °C (as expected for an active endocytosis
process) and by treatment with heparin (a soluble competitor of cell
membrane-associated HSPGs), consistent with previous findings
(27). The former treatment
also inhibited the internalization of transferrin, as expected. Two drugs that
affect cellular microtubules (taxol, which stabilizes microtubules, and
nocodazole, which disrupts polymerized microtubules) had no apparent effect on
either Tat or transferrin internalization. Notably, cell treatment with
MCD drastically reduced Tat uptake, thus indicating that the
fluorescence apparent at the cell periphery in cells treated with this drug
(Fig. 3, F and
G) was due to protein interacting with cell surface
HSPGs, which eventually were not internalized and were removed by trypsin
treatment before flow cytometry. Interestingly, cytochalasin D, a drug causing
depolymerization of cell microfilaments, significantly impaired Tat (but not
transferrin) internalization, a finding that is consistent with the
involvement of microfilaments in caveolar trafficking
(41). Brefeldin A, which
impairs Golgi trafficking, had no apparent effect on Tat uptake.
Fig. 5B shows
representative cell images for each of these treatments. Of interest is the
pattern of cell fluorescence in cells treated with cytochalasin D, showing the
formation of large fluorescent structures at the cell periphery. All data
shown in Fig. 4, A and
B, were obtained by using Tat86-EGFP. Superimposable
results were also observed with Tat11-EGFP (not shown).
Finally, we tested whether the observed pathway of extracellular Tat
internalization also holds true for CD4+ T-cells expressing the CXCR4
chemokine receptor, which besides acting as a co-receptor for HIV-1 infection,
has also been shown to be a biologically relevant receptor for extracellular
Tat (42,
43). As assessed by flow
cytometric analysis (Fig.
5A), both Tat86-EGFP and Tat11-EGFP readily entered CD4+
CXCR4+ Jurkat T-lymphocytes, with internalization inhibited by treatment with
soluble heparin or at 4 °C. Similar to HeLa cells, treatment with
MCD significantly blocked Tat uptake also in these cells, again
indicating the involvement of lipid rafts in the internalization process.
Caveolar Endocytosis Leads to the Translocation of Functional Tat to
the Cell Nucleus—We have previously demonstrated that the
GST-Tat86-EGFP fusion protein is fully active in transactivation of the viral
LTR when exogenously added to the cell culture medium of HL3T1 cells, a cell
clone derived from HeLa cells containing an integrated, silent HIV-1 LTR-CAT
construct (34). Therefore, we
exploited this property to verify that the fraction of protein that was
internalized through caveolar endocytosis from lipid rafts was indeed the same
one that was eventually translocated to the nucleus and was active in
transactivation. To this aim, we developed a quantitative PCR procedure based
on competitive PCR to measure the levels of CAT mRNA upon treatment with
exogenous Tat86-EGFP. This procedure is based on the co-amplification of the
cellular cDNA with a multicompetitor DNA bearing the primer recognition
sequences for the CAT transcript and for the reference cellular -actin
mRNA; a representative competitive PCR experiment is shown in
Fig. 6A. Treatment of
HL3T1 cells with Tat86-EGFP for different time periods determined a remarkable
increase of LTR-driven transcription, which reached more than 100-fold
transactivation after 10 h. Consistent with the fluorescent protein
internalization data, when cells were treated with MCD, heparin, or were
kept at 4 °C during internalization, transactivation of the LTR was almost
completely abolished (Fig.
6B). In contrast, treatment with nocodazole had no
effect. Notably, brefeldin A, which had no apparent effect on fluorescent
protein internalization, caused a remarkable decrease in the levels of
transactivation. As expected, no transactivation was detected in control cells
incubated with Tat11-EGFP.
These observations clearly indicate that the same drugs or treatments impairing caveolar endocytosis from lipid rafts also block transactivation by exogenously added Tat, thus indicating that this route of internalization eventually leads to the presence of transcriptionally active protein in the cell nucleus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The work described in this manuscript indicates that the cell uptake of large protein cargoes fused to both full-length Tat or to its 11-amino acid transduction domain occurs through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. This conclusion is supported by (i) the relatively slow kinetics of internalization, (ii) the finding that Tat endosomes are resistant to the nonionic detergent Triton X-100, (iii) the colocalization of Tat fusion proteins with caveolin-1 in cells that express this protein but not with markers of clathrin-mediated endocytosis, and (iv) the selective sensitivity of Tat internalization to drugs that impair lipid raft formation, such as cyclodextrin, or disturb anchorage of caveolae to the actin cytoskeleton, such as cytochalasin D.
Although this undoubtedly appears to be the route of internalization of
large particles fused to Tat (also including a recombinant 
phage
particle displaying the Tat transduction domain on its surface
(23)), much less clear is
whether small peptides encompassing the Tat transduction domain might also
enter the cells by alternative pathways. Evidence has been provided to show
that short basic peptides, including the Tat basic domain, can directly cross
cell membranes by a mechanism that is insensitive to typical endocytosis and
metabolic inhibitors (44,
45). When our Tat86-EGFP
protein was compared with a fluorescent peptide encompassing the Tat basic
domain by simultaneously adding the two molecules to the cell culture medium,
it was observed that the peptide entered the cells at 4 °C, whereas the
larger recombinant protein did not
(36). However, recent
experimental evidence seems to indicate that the notion that the Tat basic
domain can directly cross the cell membrane might be the consequence of
artifacts due to post-fixation diffusion or poor removal of extracellularly
bound protein (46). Thus, an
active endocytosis process might also be required for the cellular uptake of
short, polybasic peptides, as also originally proposed by Mann and Frankel
(6). The identification of
caveolar endocytosis as the cellular entry pathway of Tat fusion proteins will
now permit this issue to be readdressed more specifically.
We have recently observed that Tat internalization requires binding of the protein to cell surface HSPGs, since the uptake process does not occur in cells selectively impaired in HSPG biosynthesis and can be abolished by cell treatment with heparinase III or by competition with soluble, extracellular heparin (26, 27). One of the two families of cell surface HSPGs is the glypicans, which lack a membrane-spanning domain and are anchored to the external surface of the plasma membrane via glycosylphosphatidylinositol (47). Indeed, glycosylphosphatidylinositol-anchored proteins are highly enriched in lipid rafts on the cell surface (32, 48). In addition, glypican-1 is known to be internalized and recycled via the Golgi (49), with the internalization process occurring in caveolin-1-containing endosomes (50). Thus, a testable prediction is that the internalization of Tat might occur after its interaction with the sugar moiety of this HSPG. In this respect, it is of interest to note that the internalization process in CD4+ T-lymphocytes expressing CXCR4, a chemokine receptor that specifically binds extracellular Tat (42, 43) and mainly resides outside of lipid rafts (51–53), is also severely inhibited by cholesterol depletion, indistinguishable from CXCR4-negative cells. This observation indicates that either this receptor is not significantly involved in Tat internalization or that its recruitment to lipid rafts is required anyhow for the internalization process to occur, similar to HIV-1 entry.
Several extracellular macromolecules, including bacterial toxins and
viruses, are internalized via caveolar endocytosis. Different studies
demonstrate that the dynamics of this pathway are relatively slow when
compared with clathrin-dependent endocytosis
(40,
54). This conclusion is in
perfect agreement with the observation made in living cells that
Tat-containing endosomes are relatively immobile as compared with transferrin
endosomes when analyzed in a 10-s time scale. Consistent with these
conclusions, complementary biophysical work performed on living, unfixed cells
has shown that endosomes containing an average of 
300 molecules of the
Tat86-EGFP molecule proceed from the cell membrane toward the perinuclear
region with an average velocity of 3 µm/h and that this translocation
process requires the integrity of the actin cytoskeleton
(55). As shown in
Fig. 5, treatment with
cytochalasin D determined the aggregation of Tat-EGFP endosomes in large
clusters at the cell periphery. This observation is in perfect agreement with
the notion that caveolae are connected with the actin cytoskeleton
(41) and that treatment with
cytochalasin D leads to clustering of caveolae connected to the plasma
membrane (54,
56). In contrast, disruption
of microtubules had no effect on Tat-EGFP entry.
Extracellular Tat is internalized by the cells, and eventually it ends up in the nucleus in a transcriptionally active form. One formal possibility existed that the pathway leading to caveolar endocytosis of the protein might be different from the one leading to the presence of the protein in the nucleus. This is clearly not the case, since all the drugs and treatments that inhibited endocytosis also impaired the transcriptional activity of the protein. This observation excludes that there is an alternative pathway for extracellular Tat uptake. In addition, it also implies that the internalized Tat eventually has to egress the endosomes to be transported to the nucleus.
The exit from the endocytic compartment is a molecular process that is likely to require translocation through a cellular membrane. In this respect, it is worth noting that, in the experiments with the Tat fusions presented in this work, at no time did we observe EGFP fluorescence in the nucleus. Still the protein transactivated the LTR and could be evidenced by staining with anti-Tat antibodies (see, for example, Ref. 27). Most probably this observation implies that the Tat-EGFP fusion proteins unfold during membrane translocation, as commonly occurs in this process; once unfolded, an intrinsic property of GFP is to re-fold in an optically active form only at very low efficiency (57). This consideration also implies that the use of the EGFP reporter tag as the sole method by which to study the release of Tat from the cells (another process possibly requiring crossing of a cell membrane), as recently described (58), might possibly bring misleading conclusions.
Bacterial and plant toxins such as cholera toxin and ricin translocate to the cytosol after retrograde transport through the Golgi apparatus and the endoplasmic reticulum (59, 60); consistent with these findings, disruption of Golgi trafficking with brefeldin A inhibits toxin action (61, 62). Of possible interest in this respect is the observation that treatment with brefeldin A did not affect entry of the Tat-EGFP recombinant protein into the cells, as observed by flow cytometry, whereas it did impair its nuclear translocation, as shown by the LTR transactivation data. This observation indirectly implies that the Golgi transport might be required for this process. Despite this suggestion, we have so far failed to visualize Tat in the Golgi or in the endoplasmic reticulum, possibly indicating that the translocation process only occurs in a minority of the internalized molecules, as is also the case for ricin (59). Experiments aimed at identifying specific molecular modification of proteins transiting through the Golgi will directly permit addressing this issue.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Molecular Medicine Laboratory, ICGEB, Padriciano, 99, 34012 Trieste, Italy. Tel.: 39-40-3757324; Fax: 39-40226555; E-mail: giacca{at}icgeb.org.
1 The 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; MCD, methyl--cyclodextrin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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