Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 37, pp. 27503–27517, September 14, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Cellular Internalization of Green Fluorescent Protein Fused
with Herpes Simplex Virus Protein VP22 via a Lipid
Raft-mediated Endocytic Pathway Independentof Caveolae and Rho Family GTPases but
Dependent on Dynamin and Arf6*□
S

Received for publication, May 9, 2007, and in revised form, July 2, 2007 Published, JBC Papers in Press, July 20, 2007, DOI 10.1074/jbc.M703810200

Kenji Nishi1 and Kaoru Saigo2
From the Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan

Short peptides and protein domains capable of transducing
cargo across the plasma membrane are called protein transduction domains (PTDs)3 (reviewed in Refs. 1 and 2). A short basic
peptide from the TAT protein, a transcription activator ofhuman immunodeficiency virus type 1 (3–5), and the third
helix of the Drosophila Antennapedia homeodomain (6) are
widely used as short peptide-type PTDs. Recent experiments* This work was supported in part by a special coordination fund for promoting science and technology and by grants from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (to K. S.). The costs ofpublication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–3.
1
To whom correspondence may be addressed. Tel.: 81-3-5841-3044; Fax:
81-3-5841-3044; E-mail: kennishi@biochem.s.u-tokyo.ac.jp.
2
To whom correspondence may be addressed. Tel.: 81-3-5841-3044; Fax:
81-3-5841-3044; E-mail: saigo@biochem.s.u-tokyo.ac.jp.
3
The abbreviations used are: PTDs, protein transduction domains; GFP, green
fluorescent protein; M␤CD, methyl-␤-cyclodextrin; HA, hemagglutinin;
DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-bufferedsaline; RNAi, RNA interference; siRNAs, small interfering RNAs; GAG,
glycosaminoglycan.

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

showed that these short peptide-type PTDs and the proteinsassociated with them are most likely to be incorporated into
cells via some endocytic mechanism (7, 8).
Endocytosis is a complex mechanism involving numerous
protein-protein and protein-lipid interactions (9) and may
occur as clathrin-dependent endocytosis, macropinocytosis,
caveola-mediated endocytosis, and lipid raft-dependent/caveolaindependent endocytosis (9, 10). TAT and TAT-PTD fusion proteins are capable of interacting with negatively charged plasma
membrane (11, 12), but their endocytic mechanism differs considerably depending on cell type or protein moieties associated with
TAT-PTD (12–18). In some cases, they are internalized via macropinocytosis (13, 14), whereas in other cases, they appear to beincorporated into cells via caveola-dependent endocytosis (15, 16)
or clathrin-dependent endocytosis (12, 18). Endosomal escape of
TAT-PTD fusion proteins may require endosome acidificationand cytosolic Hsp90 activity (18–20).
VP22 is a major component of the herpes simplex virus type1 tegment and is situated between the capsid and envelope (21).
This component has been shown to be secreted from cells
expressing it through a Golgi-independent mechanism (22).
The C-terminal half of VP22, residues 159 –301 (termed
VP22.C1), is highly homologous in sequence to counterparts ofother ␣-herpes virus VP22 proteins and is considered to be
associated with intercellular trafficking ability (23, 24).
Although others have reported that a 34-amino acid C-terminal
region of VP22.C1 is necessary and sufficient for cellular internalization (25), our results indicate that the entire 142-aminoacid region is involved in cellular uptake.4
VP22.C1 has been noted to form complexes with fluoresceinlabeled oligonucleotides and generate particles of 0.3–1 ␮m indiameter (24). These particles are efficiently taken up into cultured cells or mice and release active antisense oligonucleotide
reagents into the cytoplasm in response to light activation (24,
26, 27). The uptake of VP22.C1-fused green fluorescent protein(GFP), hereafter referred to as VP22-GFP, into an entire tumor
has been observed following peritumoral injection into humanpancreatic tumors in SCID mice (28). VP22.C1-Rab9 has beenreported to be internalized into cells and to exhibit biologicalactivity (29). Thus, as with short peptide-type TAT-PTD, the4

K. Nishi and K. Saigo, unpublished data.

JOURNAL OF BIOLOGICAL CHEMISTRY

27503

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

VP22 is a structural protein of the herpes simplex virus andhas been reported to possess unusual trafficking properties.
Here we examined the mechanism of cellular uptake of VP22
using a fusion protein between the C-terminal half of VP22 andgreen fluorescent protein (GFP). Adsorption of VP22-GFP ontoa cell surface required heparan sulfate proteoglycans and basic
amino acids, in particular, Arg-164 of VP22. Inhibitor treatment, RNA interference, expression of dominant-negative
mutant genes, and confocal microscopy all indicated that VP22GFP enters cells through an endocytic pathway independent ofclathrin and caveolae but dependent on dynamin and Arf6 activity. As with CD59 (a lipid raft marker), cell-surface VP22-GFP
signals were resistant to Triton X-100 treatment but only partially overlapped cell-surface CD59 signals. Furthermore, unlike
other lipid raft-mediated endocytic pathways, no Rho family
GTPase was required for VP22-GFP internalization. Internalized VP22 initially entered early endosomes and then moved tolysosomes and possibly recycling endosomes.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis
142-amino acid VP22.C1 protein may possibly serve as a vector
for delivering cargo into cells. As in the case of TAT-PTD and
its fusion proteins, VP22.C1 fusion proteins may also enter acell via endocytosis because they exhibit cytoplasmic vesicular
distribution (8). To date, the cellular internalization mechanism of VP22 fusion proteins is but little understood.
In this work, we studied the mechanism of cellular internalization of VP22 using VP22-GFP as a model system. VP22-GFP
was internalized through interactions with cell-surface heparansulfate proteoglycans and subsequent lipid raft-mediated endocytosis independent of clathrin, caveolae, and Rho family
GTPases but dependent on dynamin and Arf6 (ADP-ribosylation factor 6). Also, as with transferrin internalized via clathrindependent endocytosis, nearly all internalized VP22-GFP signals were incorporated into the early endosomes andco-localized with transferrin.

27504 JOURNAL OF BIOLOGICAL CHEMISTRY

5

The sequences of the primers used for mutagenesis are available uponrequest.

VOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

EXPERIMENTAL PROCEDURES
Drugs and Antibodies—Alexa Fluor 647-labeled transferrin,
phalloidin-labeled rhodamine, and Hoechst 33342 were purchased from Molecular Probes. Heparin (porcine intestinal
mucosa), chlorpromazine hydrochloride, methyl-␤-cyclodextrin (M␤CD), genistein, and Clostridium difficile toxin B wereobtained from Sigma. Cytochalasin D, okadaic acid, PP2, andsodium orthovanadate were from Calbiochem, and chloroquine diphosphate salt was from Wako. Chondroitin sulfate A(whale cartilage), chondroitin sulfate B (pig skin), chondroitin
sulfate C (shark cartilage), keratan sulfate (bovine cornea), heparitinase, and chondroitinase ABC were from Seikagaku Corp.
The antibodies used were as follows: anti-heparan sulfate
monoclonal antibody (clone NAH46; Seikagaku Corp.); rabbit
anti-hemagglutinin (HA) tag polyclonal IgG and goat antimouse IgM rhodamine (Upstate); mouse anti-c-Myc monoclonal IgG (Oncogene); anti-Myc tag polyclonal antibody (Cell
Signaling Technology); mouse anti-human clathrin heavy
chain monoclonal antibody (Affinity BioReagents); anti-CD71
antibody (transferrin receptor; Santa Cruz Biotechnology, Inc.);
rabbit anti-EEA1 polyclonal IgG (Upstate); mouse anti-EEA1
monoclonal antibody and anti-human CD107a (LAMP1) antibody (Pharmingen); anti-human CD59 monoclonal antibody(Cedarlane Laboratories); rabbit anti-caveolin-1 polyclonal
antibody and mouse anti-GM130 monoclonal antibody (BDTransduction Laboratories); Cy3- or Cy5-conjugated goat antimouse IgG (Amersham Biosciences); and fluorescein isothiocyanate-conjugated goat anti-mouse, Cy5-conjugated goatanti-rabbit, and Cy3-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratories).
Plasmids and VP22 Mutagenesis—pVP22-GFP, an expression plasmid for VP22-GFP production in Escherichia coli cells,
was constructed as follows. A 0.6-kb EcoRI/NotI fragment of
pEGFP-N3 (Clontech) was inserted into the EcoRI/NotI site of
the pVP22/myc-His plasmid (Invitrogen). A fragment encoding
the C-terminal half of VP22 (amino acids 159 –301) and fulllength enhanced GFP was amplified from the plasmid by PCR;
digested with NheI and NotI; and inserted into the NheI/NotI
site of pET28a (Novagen), which is capable of producing
recombinant protein with an N-terminal polyhistidine tag. The
following primers were used: 5⬘-AAATTTGCTAGCACG-GCGCCAACCCGATCCAA-3⬘ and 5⬘-CGGGTTAGATCTCAATGGTGATGGTGATGATGAC-3⬘. PCR-based systematic mutagenesis was carried out, and a series of pVP22-GFP
derivatives encoding mutants of VP22-GFP were generated.5
pGFP, a GFP expression plasmid, was constructed by insertingthe 0.6-kb EcoRI/NotI fragment of pEGFP-N2 (Clontech) into
the EcoRI/NotI site of pET28a.
The pcDNA3-HA-dynamin-2a-WT and pcDNA3-HAdynamin-2a-K44A constructs (30) were obtained from Kazuhisa
Nakayama (Kyoto University). The pcDNA3-myc-Rab5-WT and
pcDNA3-myc-Rab5-Q79L constructs (31) were kindly provided
by Harald Hirling (Faculté des Science de la Vie). pRK5-myc-Rac1T17N was obtained from Gary Bokoch (Scripps Research Institute). The pXS-HA-Arf6-Q67L plasmid (32) was kindly provided
by Julie G. Donaldson (National Institutes of Health).
Protein Purification—VP22-GFP and GFP were produced
using E. coli BL21 Star (DE3) cells (Invitrogen) as host. Cells
were first grown overnight in L-broth with kanamycin at 37 °C.
The overnight culture was diluted 10 times with the same
medium, and shaking was continued at room temperature. Isopropyl ␤-D-thiogalactopyranoside was added to a final concentration of 1 mM at A600 ⫽ 0.8 –1.0. After an additional 12 h ofshaking at room temperature, the cell cultures were cooled onice, and the cells were collected by centrifugation at 4 °C. The
pellets were frozen and stored at ⫺20 °C until used. Cells were
thawed on ice, resuspended in lysis buffer (50 mM sodium phosphate (pH 8.0) containing 10 mM imidazole and 300 mM NaCl),and disrupted by sonication. The lysate was centrifuged at
12,000 ⫻ g for 1 h at 4 °C. The supernatant was loaded onto anickel-nitrilotriacetic acid column (Qiagen Inc.) equilibrated
with lysis buffer. The column was washed with lysis buffer andthen wash buffer (50 mM sodium phosphate (pH 8.0) containing
20 mM imidazole and 300 mM NaCl) and finally eluted withelution buffer (50 mM sodium phosphate (pH 8.0) containing
250 mM imidazole and 300 mM NaCl). Purified protein was
immediately used or frozen in 10% glycerol and stored at⫺80 °C until used.
Cell Culture and Transfection—Wild-type CHO-K1 andHeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum at
37 °C in 5% CO2. Glycosaminoglycan-deficient (pgsA-745) andheparan sulfate-deficient (pgsD-677) mutant CHO-K1 cells(American Type Culture Collection) were cultured in Ham’s
F12K medium supplemented with 10% fetal calf serum at 37 °C
in 5% CO2. Transfection was carried out using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s instructions.
Usually, cells were analyzed within the period of 16 – 48 h following transfection.
Visualization of Internalized Proteins by Confocal Microscopy—Cells were cultured in 12-well culture plates (Sumitomo
Bakelite Co., Ltd.) on a glass coverslip. When cells were treatedwith drug or protein (VP22-GFP, GFP, or transferrin), the culture medium was replaced with fresh serum-free medium at theprotein and/or drug concentrations indicated. The cells wereincubated for 1 h and then washed twice with ice-cold phos-

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

tion. A projection image was constructed using successive optical sections of 0.25 ␮m each. A region of interest was drawn
around each cell, and the average intensity was calculated using
the software that came with the confocal microscope.
Immunofluorescence—Cells were cultured in 12-well culture
plates on a glass coverslip, incubated with protein at the specified concentrations in serum-free DMEM for the indicatedtimes, washed twice with ice-cold PBS, and fixed with 4%paraformaldehyde at room temperature for 15 min. After being
washed twice with ice-cold PBS, the cells were permeabilizedwith PBS containing 0.2% Triton X-100 and washed twice with
ice-cold PBS. Nonspecific binding sites were blocked with PBS
containing 5–10% goat whole serum (IBL Medical ProductsCo.) at room temperature for 30 min. Following incubation
with the primary antibody (1:100 – 400 dilution) in PBS containing 1–5% goat whole serum for 1 h at room temperature orovernight at 4 °C, the cells were washed twice with PBS andtreated with the secondary antibody (1:100 – 400 dilution) in
PBS containing 1–5% goat whole serum for 1 h at room temperature or overnight at 4 °C. This was followed by two washes
with PBS and visualization by confocal microscopy as describedabove.
RNA Interference (RNAi)—Two sets of human clathrin heavy
chain small interfering RNA (siRNAs) were selected using
siDirect (33): CHC1 (clathrin heavy chain 1), GCUUCAGUACCCUGACUAUGG (passenger strand) and AUAGUCAGGGUACUGAAGCCA (guide strand); and CHC2, CCUGGUACGUCGAAAGGAUCC (passenger strand) and AUCCUUUCGACGUACCAGGUA (guide strand). Firefly luciferase siRNA wasused as a control: CGUACGCGGAAUACUUCGAAA (passenger strand) and UCGAAGUAUUCCGCGUACGUG (guidestrand). RNA oligonucleotides were synthesized by Proligo, anddouble-stranded siRNA was prepared as described previously(34). HeLa cells in 12-well plates were transfected three times at
24 –36-h intervals with 50 nM siRNA. Firefly luciferase siRNAtreated and CHC siRNA-treated cells were trypsinized, mixed
at a ratio of 1:1, and seeded in 12-well plates 20 h prior toobservation of the effects of clathrin heavy chain depletion in
the same field. DNA transfection was conducted with a third
siRNA transfection.
DNA-mediated RNAi was also carried out to knock down thegenes encoding RhoA, Cdc42, and Arf6. A firefly luciferase
short hairpin RNA expression construct was used as a control.
Details of vector construction will be described elsewhere.6
HeLa cells in 12-well plates were transfected with a suitable
construct, and transfectants were selected with puromycin (2␮g/ml) 24 h after transfection. Cells were analyzed 96 h after
transfection.
Drug Treatment—Cells were washed twice with serum-free
DMEM and incubated in serum-free DMEM containing a given
drug at the indicated concentrations for 30 min except for
C. difficile toxin B. Following protein addition to the medium,cells were incubated for 1 or 4 h. Protein uptake and cell-surface
binding were visualized and measured as described above.
Triton X-100 Treatment—After being put on ice with 0.25
␮M VP22-GFP, 2 ␮g/ml anti-CD59 antibody, or 1 ␮g/ml anti6

K. Ui-Tei, K. Nishi, and K. Saigo, unpublished data.

JOURNAL OF BIOLOGICAL CHEMISTRY

27505

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

phate-buffered saline (PBS). After being fixed with 2– 4%
paraformaldehyde in PBS for 15 min at room temperatureunless indicated otherwise, the cells were washed twice with
ice-cold PBS and mounted in VECTASHIELD mounting
medium (Vector Laboratories). This was followed by cell visualization with an Olympus Fluoview FV1000 confocal microscopy system. Successive 0.25-␮m optical sections were takenusing a ⫻60 objective lens. In some cases, after cells had been
incubated on ice, the medium was replaced with fresh medium
containing protein(s) at the indicated concentrations. The cells
were then placed on ice for 1 h, washed twice with serum-free
medium, placed in prewarmed serum-free medium, incubatedfor a specified period of time, and then visualized as describedabove. Transferrin internalization was examined by exposing
cells to culture medium containing 0.25 ␮M Alexa Fluor 647labeled transferrin and observing stained cells under a confocal
microscope.
Quantification of Internalized VP22-GFP—Cells were seededon each well of 12-well culture plates in culture medium containing 10% fetal calf serum. Culturing was continued until preconfluency at 37 °C. The medium was replaced with fresh
serum-free medium containing 1 ␮M VP22-GFP. Cells wereincubated for the indicated times or for 1 h, washed once with
ice-cold PBS, and trypsinized for 15 min at 37 °C to eliminate
non-internalized cell-surface protein. This was followed bycentrifugation in 1.5-ml tubes, and the cells were washed threetimes with ice-cold PBS, lysed with PBS containing 0.5% Triton
X-100, and disrupted by sonication. The cell lysate was centrifuged. The protein content of the supernatant was measured
using a Bio-Rad protein assay kit with bovine serum albumin as
the standard. Fluorescence intensity was measured with a Hitachi FL-2500 spectrofluorometer at a excitation wavelength of
488 nm and a emission wavelength of 511 nm. Replacement of
0.5% Triton X-100 with 0.1% SDS gave no appreciable difference in measurement, indicating that almost all internalized
VP22-GFP is solubilized by 0.5% Triton X-100 treatment.
Low Temperature Treatment and ATP Depletion—For low
temperature treatment, after being incubated at 4 °C for 1 h, thecells were further incubated in the presence of VP22-GFP for
1 h at 4 °C and subjected to trypsin digestion or fixation. For
removal of cellular ATP, the cells were incubated with ATP
depletion medium (glucose-free DMEM with 10 mM sodium
azide and 6 mM 2-deoxy-D-glucose) containing 10% fetal calf
serum for 1.5 h, followed by incubation in the presence of theindicated concentrations of VP22-GFP for 1 h in serum-free
ATP depletion medium. Cellular protein uptake was visualized
or measured as described above.
Quantification of Cell-surface VP22-GFP and Anti-heparanSulfate Antibody Binding Signals—Cells were cultured in
12-well culture plates on a glass coverslip. Cells were washedtwice with serum-free DMEM and incubated on ice for 1 h.
After addition of the indicated concentrations of VP22-GFP or
2 ␮g/ml anti-heparan sulfate antibody in serum-free DMEM,
cells were further incubated on ice for 1 h, washed twice with
ice-cold PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and visualized by confocal microscopy. For
anti-heparan sulfate antibody staining, the secondary antibody
treatment was also carried out before paraformaldehyde fixa-

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

RESULTS
Cellular Uptake of VP22-GFP Occurs through Endocytosis—
To clarify the mechanism for the cellular uptake of VP22,
VP22-GFP, a fusion protein between GFP and VP22.C1 (Fig.
1A), was prepared from E. coli cells and added to the culture
medium with HeLa cells. GFP signal distribution was examinedat 1 h of incubation at 37 °C using a confocal microscope (Fig.
1B). Significant VP22-GFP signals were observed not only on

FIGURE 1. Cellular VP22 uptake via endocytosis. A, structure of VP22-GFP.
The 6-amino acid histidine tag, VP22.C1, and GFP are shown in yellow, red, andgreen, respectively. AAs, amino acids. B, VP22-GFP signals in HeLa cells with(lower panel) or without (upper and middle panels) trypsin treatment. HeLa
cells were exposed to 0.5 ␮M VP22-GFP at 37 °C for 1 h, fixed with paraformaldehyde, and observed under a confocal microscope. Cells were trypsinized

27506 JOURNAL OF BIOLOGICAL CHEMISTRY

before paraformaldehyde fixation. VP22-GFP signals (green) in the top andintermediate optical sections (see the lower margin) and a differential interference contrast image were merged. Arrows indicate internalized VP22-GFP
signals. C, HeLa cells exposed to VP22-GFP at 4 °C. Note the absence of internalized VP22-GFP signals. D, time course of intracellular accumulation of
VP22-GFP in HeLa cells at 37 °C. Cells were exposed to 1 ␮M VP22-GFP. The
amount of intracellular VP22-GFP signals was measured spectrofluorometrically after trypsin treatment. Two measurements were averaged. E, effects oflow temperature and ATP depletion on cellular uptake of VP22-GFP in HeLa
and CHO-K1 cells. HeLa and CHO-K1 cells were exposed to 1 ␮M VP22-GFP at
4 °C or under ATP depletion conditions. Three independent spectrofluorometric measurements obtained after trypsin treatment were averaged. Error
bars indicate S.D. The cellular uptake of VP22-GFP found in cells cultured
under normal conditions at 37 °C is also shown (control).

VOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010transferrin receptor antibody on ice for 1 h, cells were washedtwice with serum-free DMEM, stained with the secondary antibody for 1 h in serum-free DMEM, washed twice with ice-cold
PBS, treated with 1% Triton X-100 in PBS for 7 min, and washedtwice again with ice-cold PBS on ice. They were then fixed with
4% paraformaldehyde and visualized.
Cell Treatment with Glycosaminoglycan (GAG) Lyases—Enzymatic treatment with GAG lyases was carried out as reported
previously (35). Briefly, preconfluent cells were washed oncewith PBS, followed by incubation with GAG lyases in PBS containing 0.1% bovine serum albumin, 0.2% gelatin, and 0.1% glucose for 2 h at 37 °C. Four washings with PBS or serum-free
DMEM were carried out. Following addition of the indicatedconcentrations of VP22-GFP to the medium, cells were incubated for 1 h. VP22-GFP uptake and binding were visualized
and measured as described above.
Real-time PCR—The total RNA from transfected cells was
isolated using an RNeasy mini kit (Qiagen Inc.). The RNA wastreated with RQ1 DNase (Promega Corp.), purified using the
RNeasy mini kit, and reverse-transcribed using SuperScript III(Invitrogen). Before the PCR, a mixture of the synthesized
cDNA and SYBR Green PCR Master Mix (Applied Biosystems)
was incubated at 95 °C for 10 min. PCR was then carried out at
95 °C for 15 s and at 60 °C for 1 min for 40 cycles. All reactions
were carried out in triplicate. The levels of PCR products weremonitored with an ABI PRISM 7000 sequence detection system
and analyzed with ABI PRISM 7000 SDS software (Applied Biosystems). For each sample, the amount of target mRNA wasnormalized using endogenous ␤-actin mRNA. The following
primers were used: Arf6, 5⬘-ATCAATGACCGGGAGATGAG-3⬘ (forward) and 5⬘-AGGGCTGCACATACCAGTTC-3⬘(reverse); Cdc42, 5⬘-CGATGGTGCTGTTGGTAAAA-3⬘ (forward) and 5⬘-TCCTCTTGCCCTGCAGTATC-3⬘ (reverse);
RhoA, 5⬘-CGGTCTGGTCTTCAGCTACC-3⬘ (forward) and5⬘-CCATCACCAACAATCACCAG-3⬘ reverse; and ␤-actin,
5⬘-CACACTGTGCCCATCTACGA-3⬘ (forward) and 5⬘-GCCATCTCTTGCTCGAAGTC-3⬘ (reverse).

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

pgsD-667 cells have been shown not to produce heparan sulfate
but instead 3– 4 times as many chondroitin sulfate molecules as
wild-type cells (38). Fig. 2C shows that cell-surface and internalized VP22-GFP signals underwent significant reduction in
pgsA-745 and pgsD-667 cells; thus, as with HeLa cells, heparansulfate (but not chondroitin sulfates) is required for VP22-GFP
binding to the cell surface and internalization in CHO-K1 cells.
To further confirm the close relation between cell-surface
VP22-GFP adsorption and heparan sulfate, we examined
whether the distribution of VP22-GFP signals on the cell surface is in some way related to that of heparan sulfate signals (Fig.
2D). Anti-heparan sulfate antibody staining revealed that the
heparan sulfate on the surface of HeLa cells was punctate.
Nearly all punctate signals of heparan sulfate were found toco-localize with VP22-GFP signals that had distributed in apunctate fashion and vice versa (Fig. 2D). However, it should be
noted that heparan sulfate signal intensity was not always proportional to VP22-GFP signal intensity, possibly suggesting
involvement of other factors in cell-surface VP22-GFP
adsorption.
Involvement of Basic Amino Acids in the VP22.C1 N-terminal
Region in Cell-surface Adsorption and Intracellular Uptake of
VP22-GFP—As with TAT-PTD, VP22.C1 is rich in basic aminoacids (Fig. 2E) (23). Mutational analysis indicated that 8 basic
amino acids in TAT-PTD are equally requisite for cellular
uptake activity (39). To determine which basic amino acids of
VP22.C1 are required for interactions between heparan sulfate
and VP22-GFP, the basic amino acids of VP22.C1 were systematically replaced with alanine (neutral amino acid) or glutamic
acid (acidic amino acid), and we examined spectrofluorometrically whether mutant VP22-GFP proteins were effectively
incorporated into cells (Fig. 2F). The R164A and R164E mutant
proteins were found to lose 90% of the intracellular incorporation activity of the wild-type protein, indicating the importanceof arginine at position 164 for VP22-GFP internalization. At
positions 174, 192, and 199, only glutamic acid substitutes
exhibited significant reduction in internalization activity, suggesting that the absence of negative charges at these positions isrequired for effective intracellular uptake of VP22-GFP. Fig. 2Fshows that most, if not all, other basic amino acids of VP22.C1,
which are associated with moderate mutational effects, mayalso be involved in VP22-GFP internalization to some extent.
Reduction in VP22-GFP internalization activity appeared to
be virtually proportional to that in cell-surface binding activity.
Indeed, cell-surface VP22-GFP signals were almost completely
abolished or significantly reduced in R164E, R164A, and R192E,
three strong internalization-defective mutants, whereas cellsurface VP22-GFP signals in R192A and R207A, associatedwith moderate internalization defects, were moderately
reduced (Fig. 2, compare F and G).
Requirement of Dynamin in Cellular Internalization of
VP22-GFP—Dynamin is a GTPase that is essential for various
endocytic pathways (10). Cellular expression of the GTPasedeficient, dominant-negative mutant of dynamin (dynaminK44A) has no effect on macropinocytosis or certain lipid
raft-mediated endocytic pathways, including that induced
by Arf6-Q67L (32), but almost entirely blocks clathrin-mediated endocytosis and other lipid raft-mediated endocytic pathJOURNAL OF BIOLOGICAL CHEMISTRY

27507

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

the surface of the cells (Fig. 1B, top section (t.s.)) but also inside(intermediate sections (i.s.)). Fifteen minutes of trypsin treatment at 37 °C eliminated almost completely the cell-surface
signals without affecting intracellular signal intensity (Fig. 1B,
compare middle and lower panels). Cellular VP22-GFP uptake
appeared to depend on the VP22 moiety because no intracellular GFP signal was detected upon elimination of the entire
VP22.C1 moiety from the fusion protein (data not shown). Change
in intracellular GFP signal intensity was examined spectrofluorometrically using trypsin-treated HeLa cells (Fig. 1D). The intracellular VP22-GFP signal increased gradually for ⬎6 h.
Fig. 1B shows that the cytoplasmic distribution of VP22-GFP
in HeLa cells is vesicular, supporting the notion that VP22-GFP
is internalized through endocytosis (8). Endocytosis-dependent
cellular internalization would not occur either at 4 °C or in theabsence of ATP (7). As shown in Fig. 1 (C and E), no or hardly
any intracellular accumulation of VP22-GFP could be detected
in HeLa cells exposed to VP22-GFP at 4 °C. In contrast to cellular uptake, VP22-GFP adsorption onto the cell surface
appeared to occur quite normally even at 4 °C. The cellular ATPpool becomes empty upon preincubation of cells with sodium
azide and deoxyglucose (36). Only slight cellular uptake of
VP22-GFP without significant reduction in surface signals wasapparent in both ATP-depleted HeLa and CHO-K1 cells (Fig.
1E). VP22-GFP may thus be concluded to be internalized
through endocytosis in HeLa and CHO-K1 cells.
Requirement of Heparan Sulfate for Cell-surface Binding andCellular Uptake of VP22-GFP—Cellular internalization of
TAT-PTD requires proteoglycans (11, 12). In the case of mammalian cell-surface GAGs, heparan sulfate and chondroitin sulfates A–C are the main components. We thus investigatedwhether GAGs are required for cell-surface adsorption and cellular uptake of VP22-GFP. After HeLa cells were treated withheparitinase or chondroitinase ABC, cell-surface adsorption
and cellular uptake of VP22-GFP signals were assessed. Heparitinase digests heparan sulfate, whereas chondroitinase ABC
digests chondroitin sulfates A–C but not heparan sulfate. Heparitinase treatment eliminated not only cell-surface VP22-GFP
signals but cellularly internalized VP22-GFP signals as well (Fig.
2A). In contrast, virtually no reduction in VP22-GFP signals oneither the surface of or inside cells could be detected by chondroitinase ABC treatment (Fig. 2A). GAGs were added to theculture medium, and only heparin (analog of heparan sulfate)
addition was found to induce a significant reduction in intracellularly accumulated VP22-GFP signals (Fig. 2B). Thus, in
HeLa cells, heparan sulfate (but not chondroitin sulfates) is
required for the surface adsorption and intracellular accumulation of VP22-GFP.
pgsA-745 and pgsD-677 cells are mutant GAG strains of
CHO-K1 cells (37, 38). pgsA-745 is deficient in xylosyltransferase, the key enzyme required for GAG attachment to coreprotein. Only 1% of GAGs expressed in wild-type cells havebeen reported to be expressed in pgsA-745 cells (37). In thisstudy, very few if any heparan sulfate signals could be detectedby anti-heparan sulfate antibody staining in both pgsA-745 andpgsD-677 cells (supplemental Fig. 1). pgsD-667 is deficient in
N-acetylglucosaminyltransferase and glucuronyltransferase,both of which are essential for heparan sulfate polymerization.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

27508 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

FIGURE 3. Requirement of dynamin for VP22-GFP endocytosis. A, HeLa
cells transfected with pcDNA3-HA-dynamin-2a-K44A, an expression plasmid
encoding an HA-tagged dominant-negative form of dynamin (K44A). Cells
were exposed to VP22-GFP (0.5 ␮M) and Alexa Fluor 647-labeled transferrin(0.25 ␮M) 24 h after transfection. Thick arrows show anti-HA antibody-stained
K44A-expressing cells, and thin arrows show normal cells exhibiting intracellular accumulation of VP22-GFP and transferrin signals but lacking dynaminactivity. B, normal accumulation of heparan sulfate on the surface of cells
lacking dynamin activity. Cell-surface heparan sulfate was detected by antiheparan sulfate (anti-HS) antibody. Note that heparan sulfate signals were
not reduced in cells expressing dynamin-K44A. C, proportion of cells associated with internalized signals of VP22-GFP or Alexa Fluor 647-labeled transferrin (Tf). Measurements from two independent experiments were averaged(each case, n ⱖ 30). Error bars indicate S.D. D, cell-surface VP22-GFP signals in
K44A-expressing cells. HeLa cells transfected with pcDNA3-HA-dynamin2a-WT or pcDNA3-HA-dynamin-2a-K44A were incubated for 1 h on ice with fresh
serum-free medium containing 0.25 ␮M VP22-GFP. Surface signals were determined by confocal microscopy (n ⱖ 33). Error bars indicate S.D. WT, wild-type
dynamin-2.

rin-depleted cells (45, 46). For observation of clathrin-depleted
and non-depleted cells in the same field, a 1:1 mixture of CHC1
siRNA-treated cells and firefly luciferase siRNA (control siRNA
unrelated in sequence to the CHC gene)-treated cells was

FIGURE 2. Requirement of cell-surface heparan sulfate and VP22.C1 basic amino acids for cellular internalization of VP22-GFP. A, effects of heparitinaseor chondroitinase treatment on cell-surface binding and intracellular accumulation of VP22-GFP. HeLa cells were treated with 25 milliunits/ml heparitinase orchondroitinase ABC and exposed to 0.25 ␮M (cell-surface binding) or 1 ␮M (intracellular accumulation) VP22-GFP for 1 h. Black bars, cell-surface VP22-GFP
signals measured using a confocal microscope (n ⱖ 23) (see “Experimental Procedures”); white bars, intracellular GFP signals measured spectrofluorometicallyafter trypsin treatment. Three independent measurements were averaged. Error bars indicate S.D. B, inhibition of intracellular VP22-GFP accumulation by GAGs.
Cells were exposed to 1 ␮M VP22-GFP in medium containing 1–25 ␮g/ml heparin (analog of heparan sulfate) and 25 ␮g/ml chondroitin sulfate (CS) A, B, or C
or keratan sulfate (KS) for 1 h, and intracellular GFP signals were measured spectrofluorometically after trypsin treatment. Three independent measurements
were averaged. C, relative cell-surface binding (black bars) and intracellular accumulation (white bars) of VP22-GFP in wild-type (WT) and mutant (pgsA-745 andpgsD-677) CHO-K1 cells. Cell-surface binding (0.5 ␮M VP22-GFP; n ⱖ 34) and intracellular accumulation (1 ␮M VP22-GFP; six measurements) of VP22-GFP signals
were determined as described for A. D, co-localization of VP22-GFP and heparan sulfate signals on the HeLa cell surface. HeLa cells were incubated for 1 h on
ice with fresh serum-free medium containing 0.25 ␮M VP22-GFP and 2 ␮g/ml anti-heparan sulfate (anti-HS) antibody. The secondary antibody treatment was
carried out before paraformaldehyde fixation. A cell-surface section is shown. Scale bar ⫽ 5 ␮m. E, amino acid sequence of VP22.C1. Ser159 of VP22 corresponds
to the N terminus of VP22.C1. Basic amino acids are shown in red. F and G, effects of the substitution of basic amino acids of VP22.C1 on intracellularaccumulation (1 ␮M VP22-GFP; F) and cell-surface binding (0.25 ␮M VP22-GFP; G) of VP22-GFP. Black bars, intracellular accumulation in alanine-substituted
mutants; white bars, intracellular accumulation in glutamic acid-substituted mutants. Intracellular accumulation of VP22-GFP signals was determined spectrofluorometrically after trypsinization. Three measurements were averaged. R254E, R287E, R295E, R297E, and R298E are not tested. Cell-surface signals wereobserved under a confocal microscope (projection figures; G). Note that cell-surface VP22-GFP signals were significantly reduced in R164A-, R164E-, andR192E-exposed cells. R192A and R207A gave moderately reduced signals, whereas K249E gave virtually no reduction in signal intensity.

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

JOURNAL OF BIOLOGICAL CHEMISTRY

27509

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

ways such as caveola-mediated endocytosis (10, 40 – 43). Using
transferrin as a dynamin-dependent endocytosis marker, we
examined VP22-GFP internalization in HeLa cells transfectedwith wild-type dynamin-2 or dynamin-2-K44A. Dynaminoverexpressing cells were identified by anti-HA antibody.
Overexpression of wild-type dynamin-2 resulted in virtually nochange in VP22-GFP and transferrin internalization (data notshown). In contrast the internalization of VP22-GFP and transferrin was noted to be significantly reduced in nearly 60 and
80% of cells overexpressing dynamin-2-K44A, respectively (Fig.
3, A and C). No apparent dynamin-2-K44A-dependent change
in cell-surface heparan sulfate signals was observed (Fig. 3B).
Dynamin-2 is thus essential for the normal cellular internalization of VP22-GFP, and also, VP22-GFP may not be internalized
through macropinocytosis and lipid raft-mediated endocytosis
induced by Arf6-Q67L but instead by clathrin-mediated endocytosis or dynamin-dependent lipid raft-mediated endocytosis,
such as that mediated by caveolae. Note that heparan sulfate(Fig. 3B) as well as cell-surface VP22-GFP (Fig. 3D) signals were
not affected by the absence of dynamin activity, indicating thatdynamin is involved in cellular uptake, but not in VP22-GFP
adsorption.
VP22-GFP Endocytosis Is Independent of Clathrin—By far,clathrin-mediated endocytosis has been best characterized andhas been shown to be inhibited specifically by chlorpromazine(44). Cell treatment with chlorpromazine induces clathrincoated pit misassembly at the plasma membrane, so clathrinmediated endocytosis such as transferrin uptake is prevented toa significant degree in chlorpromazine-treated cells. To confirm any possible involvement of clathrin-mediated endocytosis in the cellular internalization of VP22, we examined theeffects of chlorpromazine treatment on VP22-GFP internalization in HeLa cells (Fig. 4, A and B).
Chlorpromazine treatment (10 ␮g/ml, 30 min) significantly
reduced transferrin internalization in 60% of the cells (Fig. 4A,
arrows), whereas VP22-GFP internalization was actuallyenhanced in virtually all cells exhibiting a significant reduction
in transferrin uptake (Fig. 4A). Clathrin-mediated endocytosis
thus apparently is not involved in VP22-GFP internalization in
HeLa cells.
For further clarification of the above, the CHC gene was
knocked down using RNAi because clathrin-mediated endocytosis was reported previously to be severely inhibited in clath-Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

plated. In clathrin-negative cells, transferrin uptake was almost
completely inhibited, whereas VP22-GFP uptake was essentially the same as that in clathrin-positive normal cells (Fig. 4, C
and D); but unlike the case of chlorpromazine treatment, noapparent enhancement of VP22-GFP uptake could be detected(Fig. 4, compare A and C). Essentially the same was noted with
RNAi using a different CHC siRNA (CHC2) (data not shown),
indicating that the siRNA-induced change is not due to theoff-target effects (47) but instead to the knockdown of clathrin
gene activity. The cellular uptake of VP22-GFP may thus beconcluded to be independent of clathrin-mediated endocytosis.

27510 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

FIGURE 4. Clathrin-independent cellular uptake of VP22-GFP. A, stimulation of intracellular VP22-GFP accumulation by chlorpromazine treatment.
Lower panels, HeLa cells treated with 10 ␮g/ml chlorpromazine for 30 min
were exposed to 0.5 ␮M VP22-GFP and 0.25 ␮M Alexa Fluor 647-labeled transferrin at 37 °C for 1 h. Arrows show cells with reduced transferrin uptake. In
these cells, intracellular VP22-GFP signals significantly increased, indicatingthat clathrin is not involved in cellular uptake of VP22-GFP. B, quantitative
data for chlorpromazine-dependent intracellular VP22-GFP signal accumulation. After chlorpromazine treatment, cells were exposed to 1 ␮M VP22-GFP
for 1 h. Measurements from three experiments were averaged. Error bars indicate S.D. C, effect of CHC1 RNAi. RNAi for the clathrin heavy chain was carriedout using CHC1 siRNA. Thick arrows show clathrin-negative cells in whichinternalized transferrin (but not VP22-GFP) signals were abolished, and thinarrows show wild-type cells with clathrin production. D, proportion of cells
associated with internalized signals of VP22-GFP or Alexa Fluor 647-labeledtransferrin (Tf) in anti-clathrin antibody-positive (Clathrin⫹) or -negative(Clathrin⫺) cells. Measurements from two independent experiments wereaveraged (each case, n ⱖ 50). Errors bars indicate S.D.

Lipid Raft Dependence of Cellular Uptake of VP22-GFP—Dynamin-2 is required for certain lipid raft-mediated endocytic
pathways such as caveola-mediated endocytosis. We thus
examined whether VP22-GFP uptake is dependent on lipid
raft-mediated endocytosis. Lipid rafts are detergent (Triton
X-100)-resistant domains rich in cholesterol and sphingolipids(48), and CD59 serves as a marker for a subgroup of lipid rafts(32). Cell-surface signals of not only CD59 but also VP22-GFP
were found to be resistant to Triton X-100 treatment (Fig. 5A),
but virtually all transferrin receptor signals, internalized in alipid raft-independent manner, were completely eliminatedsubsequent to Triton X-100 treatment (Fig. 5A). Double staining showed that cell-surface VP22-GFP signals partially overlapped (if at all) CD59 signals in Triton X-100-treated HeLa
cells (Fig. 5B), suggesting that VP22 may be internalized by
endocytosis in a different way compared with CD59. Consistent
with this, most intracellular VP22-GFP signals were found not
to be co-localized with internalized CD59 signals at early stages(Fig. 5C and supplemental Fig. 2A), although at late stages inparticular, in most cells expressing Arf6-Q67L, VP22-GFP signals were co-localized with large CD59-positive Arf6-Q67Linduced vesicles (Fig. 5D) (32). Cell-surface heparan sulfatebinding proteins such as lipoprotein lipase were shown
previously to be resistant to Triton X-100 treatment (49), indicating the presence of heparan sulfate on the surface of Triton
X-100-treated cells.
M␤CD disrupts lipid rafts (50). Genistein is a tyrosine kinase
inhibitor that prevents lipid raft-mediated endocytosis such ascaveola-dependent endocytosis (50 –52). Consistent with thenotion that VP22-GFP is internalized through lipid raft-mediated endocytosis, intracellular VP22-GFP signals in HeLa cells
were significantly reduced subsequent to M␤CD (Fig. 5E) or
genistein (Fig. 5F) treatment. M␤CD treatment did not change
the intensity of cell-surface heparan sulfate signals, whereas aconsiderable heparan sulfate signal was abolished by genisteintreatment (Fig. 5H), suggesting that genistein may prevent
VP22-GFP internalization via reduction in cell-surface heparansulfate.
The actin cytoskeleton is required for lipid raft-mediatedendocytosis such as that mediated by caveolae (52). Upon treatment of HeLa cells with cytochalasin D, an actin-depolymerizing reagent, actin filament disruption occurred, with consequent large membrane bleb (Fig. 5G) or protrusion (53)formation on the cell surface. Virtually all cell-surface VP22GFP signals appeared to be co-localized with membrane blebs
induced by cytochalasin D (Fig. 5G). A similar phenomenon has
been reported to occur in the case of the Helicobacter pylori
vaculating cytotoxin VacA (53). In cytochalasin D-treated cells
manifesting these membrane blebs, the cellular uptake of
VP22-GFP was significantly reduced (Fig. 5, F and G) without
reduction in cell-surface heparan sulfate signals (Fig. 5H). It
may thus follow that VP22-GFP is internalized via lipid raftmediated endocytosis.
Caveolae Are Not Involved in VP22-GFP Internalization—To
determine whether caveolae are involved in VP22-GFP internalization, we examined the possible requirement of caveolae
for the cellular internalization of VP22-GFP. In cells other thanthose derived from muscle, caveolin-1 is a major structural

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

JOURNAL OF BIOLOGICAL CHEMISTRY

27511

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

FIGURE 5. Requirement of lipid rafts for VP22 endocytosis. A, HeLa cells exposed to VP22-GFP, anti-CD59 antibody, or anti-transferrin receptor (Tf-R)
antibody were treated with Triton X-100. Projection images of successive 0.25-␮m optical sections are shown. The cell-surface signals of VP22-GFP wereresistant to Triton X-100 treatment, whereas those of the transferrin receptor were completely eliminated. PBS indicates the control. B–D, relationship between
cell-surface (B) and intracellular (C and D) signals of VP22-GFP and CD59. B, after HeLa cells exposed to 0.25 ␮M VP22-GFP were treated with Triton X-100, cells
were stained with anti-CD59 antibody. A cell-surface section is shown. Scale bar ⫽ 5 ␮m. C, cells were exposed to 0.5 ␮M VP22-GFP and 2 ␮g/ml anti-CD59
antibody on ice for 1 h, washed, incubated at 37 °C for 15 min, fixed with 4% paraformaldehyde, and stained with secondary antibody. In the Merged panels,
VP22-GFP and CD59 signals are shown in green and red, respectively. The boxed regions in the upper panels are enlarged in the lower panels. Scale bars ⫽ 5 ␮m.
Cell-surface signals and early (5–15 min) intracellular signals of VP22-GFP overlapped only partially with the corresponding signals of CD59. D, HeLa cells
transfected with the pXS-HA-Arf6-Q67L plasmid were allowed to internalize VP22-GFP and anti-CD59 antibody at 37 °C for 60 min, fixed with 4% paraformaldehyde, and stained with anti-HA antibody. At a late stage (60 min) in Arf6-Q67L-expressing cells, VP22-GFP signals were almost completely co-localized with
CD59 signals. Arrows show Arf6-Q67L-induced vacuolar structures. E, HeLa cells treated with 10 mM M␤CD for 30 min were exposed to 0.5 ␮M VP22-GFP at 37 °C
for 1 h and fixed with 4% paraformaldehyde. M␤CD appeared to prevent VP22-GFP signals from intracellularly accumulating. M␤CD-treated cells exhibited aconsiderable instability to trypsinization. F, shown are the effects of genistein, cytochalasin D, okadaic acid (OA), vanadate, and PP2 on intracellular VP22-GFP
accumulation. HeLa cells were treated with 100 ␮g/ml genistein, 10 ␮M cytochalasin D, 1 ␮M okadaic acid, 1 mM vanadate, or 10 ␮M PP2 for 30 min and then cells
exposed to 1 ␮M VP22-GFP at 37 °C for 1 h. Measurements from three to six experiments were averaged. Error bars indicate S.D. G, shown are the morphologicalchanges induced by cytochalasin D treatment. HeLa cells treated with 10 ␮M cytochalasin D for 30 min were exposed to VP22-GFP (0.5 ␮M). VP22-GFP andphalloidin-labeled rhodamine signals are shown in green and red, respectively. Arrowheads indicate VP22-GFP-accumulated membrane blebs. H, shown is the
drug treatment dependence on cell-surface heparan sulfate and VP22-GFP signals. Except for genistein treatment, no appreciable reduction in cell-surface
heparan sulfate signals was detected microscopically. In the case of genistein treatment, ⬃60% of the cell-surface heparan sulfate signals were lost. Antiheparan sulfate (anti-HS) antibody and cell-surface VP22-GFP signals were determined by confocal microscopy (n ⱖ 31). Error bars indicate S.D. I, shown is therelationship between VP22-GFP and caveolin-1 signals. Cells were exposed to 0.5 ␮M VP22-GFP on ice for 1 h, washed, incubated at 37 °C for 15 min, fixed with
4% paraformaldehyde, and stained with anti-caveolin-1 antibody. In the Merged panels, VP22-GFP and caveolin-1 signals are shown in green and red, respectively. The boxed regions in the upper panels are enlarged in the lower panels. No overlap between intracellular VP22-GFP and caveolin-1 signals was observed.
Scale bars ⫽ 5 ␮m.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

27512 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

and incubated again at 37 °C for 15
min. Hardly any internalized VP22GFP signals were localized withcaveolin-1 signals (Fig. 5I and supplemental Fig. 2B), indicating that
VP22-GFP is internalized independently of caveolae.
Caveola-mediated endocytosis isenhanced by okadaic acid, an inhibitor of several protein serine/threonine phosphatases, and vanadate, aninhibitor specific for tyrosine phosphatases (56). In HeLa cells, okadaicacid treatment enhanced cellular
VP22-GFP uptake, but there was noapparent okadaic acid-dependent
stimulation of VP22-GFP internalization in CHO-K1 cells (data notshown). Vanadate brought about
no enhancement of VP22-GFP
internalization but instead significantly reduced the uptake of
VP22-GFP in either cell type (Fig.
5F). PP2 is an Src kinase-specificinhibitor and prevents caveolamediated endocytosis (57). Fig. 5Fshows that VP22-GFP internalization was completely resistant to PP2
treatment. No appreciable change
in cell-surface heparan sulfate signals was found upon treatment withokadaic acid, vanadate, or PP2 (Fig.
5H). It follows conclusively fromthese findings that VP22-GFP internalization takes place through a
caveola-independent type of lipid
FIGURE 6. Possible roles of small GTPases in VP22-GFP internalization. A, effect of toxin B on intracellular
VP22 accumulation. HeLa cells treated with 0.5 ␮g/ml C. difficile toxin B at 37 °C for 4 h were exposed to 0.5 ␮M raft-mediated endocytosis.
VP22-GFP at 37 °C for 1 h, fixed with 4% paraformaldehyde, and stained with phalloidin-labeled rhodamine.
Rho Family GTPases Are Not
Phalloidin images are those for the bottom of the cells. B, quantitative analysis of toxin B effects. Little effect of
toxin B on intracellular VP22 accumulation was observed. Three spectrofluorometric measurements were Required for Cellular Internalizaaveraged. Error bars indicate S.D. C and D, effect of RhoA RNAi on intracellular VP22 accumulation. RhoA mRNA tion of VP22-GFP—Small GTPases
was significantly reduced (C), but no reduction in VP22 internalization was evident (D). Firefly luciferase (FL) are grouped into five families, Ras,
RNAi served as the control. E and F, effect of Cdc42 RNAi on VP22 uptake. Cdc42 mRNA was almost completely
eliminated (E), but little reduction in VP22 internalization was detected (F). G–I, effect of overexpression of Rho, Rab, Arf, and Ran (58), some ofRac1-T17N, a dominant-negative form of Rac1. HeLa cells were transfected with pRK5-myc-Rac1-T17N. Twen- which have been shown recently to
ty-four hours after transfection, cells were exposed to 0.5 ␮M VP22-GFP at 37 °C for 1 h, fixed with 4% paraformaldehyde, and stained with anti-Myc antibody. In H, cells were also stained with anti-heparan sulfate (anti-HS) be differentially involved in lipid
antibody. Cells moderately and highly expressing Rac1-T17N are shown in G and H, respectively. Thick arrows raft-mediated endocytosis (10). Rhoshow Rac1-T17N expressing cells, and thin arrows show cells lacking Rac1-T17N expression. Strong internalized family GTPases include Cdc42,
signals of VP22-GFP and heparan sulfate were found in cells expressing a high level of Rac1-T17N. In these cells,
cell-surface signals of VP22 and heparan sulfate were occasionally reduced. Quantitative data are shown in I. RhoA, and Rac1. RhoA and Rac1 are
Cell-surface VP22-GFP signals were measured by confocal microscopy (n ⱖ 71). J–L, effect of Arf6 RNAi on required for lipid raft-mediatedintracellular VP22 and transferrin accumulation. Not only Arf6 mRNA (J) but also VP22 and transferrin internalendocytosis of interleukin-2 recepization (K and L) were significantly eliminated subsequent to Arf6 RNAi. HeLa cells transfected with pSUPERretro-puro-FL or pSUPER-retro-puro-Arf6 were exposed to 0.5 ␮M VP22-GFP and 0.25 ␮M Alexa Fluor 647- tors (42), whereas Cdc42 is involved
labeled transferrin, fixed with 2% paraformaldehyde, and stained with anti-heparan sulfate antibody. in that of glycosylphosphatidylinoCell-surface heparan sulfate signals appeared to be reduced to some extent in Arf6 knockdown cells.
sitol-anchored proteins (59). C. difficile toxin B specifically inactivates
component of caveolae, and consequently, ligands of caveola- Rho family GTPases through monoglucosylation (60). To
mediated endocytosis enter caveolin-1-positive subcellular examine the possible involvement of Rho family GTPases incompartments (54, 55). To determine the subcellular distribu- VP22-GFP internalization, HeLa cells were treated with 0.2–1
tion of VP22-GFP at early stages of cellular uptake, HeLa cells ␮g/ml toxin B, which resulted in typical morphological changes
were incubated in the presence of VP22-GFP at 0 °C, washed, such as cell rounding and actin filament disruption. As shownSupplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

At 60 min of internalization, a considerable number of VP22GFP molecules appeared to have moved from early endosomes
to lysosomes for degradation. Overlapping between VP22-GFP
and EEA1 signals at 60 min became less prominent than that at
15 min (Fig. 7, E and I; and supplemental Fig. 3A); instead, some
VP22-GFP signals appeared to overlap LAMP1 signals (Fig. 7, F
and I; and supplemental Fig. 3B), suggesting that VP22-GFP ispartially transferred to the lysosome at later stages. There was
no overlap between VP22-GFP and GM130 signals (Fig. 7, G
and I; and supplemental Fig. 3C).
Transferrin (internalized through clathrin-mediated endocytosis) accumulates in early endosomes initially and in recycling endosomes at later stages (63). We thus compared thesubcellular location of VP22-GFP and transferrin signals at
15 and 60 min (Fig. 7, D, H, and I; and supplemental Figs. 2Cand 3D). Nearly all VP22-GFP signals were found to haveco-localized with transferrin signals at both 15 and 60 min ofinternalization. Thus, the above findings indicate that VP22GFP and transferrin, internalized with different mechanisms, accumulate in the same early endosomes immediately
following internalization and in recycling endosomes at laterstages.
To further confirm the close relationship between internalized VP22-GFP and transferrin in early endosomes, a constitutively active Rab5 mutant (Rab5-Q79L) known to stimulate
early endosomal fusion to form ring-shaped large endosomes(64) was used. In HeLa cells overexpressing wild-type rab5, agene encoding wild-type Rab5 tagged with Myc, the cellular
distribution of VP22-GFP and transferrin was virtually thesame as that in non-transfected HeLa cells (data not shown).
But in HeLa cells overexpressing Myc-tagged Rab5-Q79L,
VP22-GFP and transferrin were co-localized with ring-shaped
endosomes, as visualized with anti-Myc antibody (Fig. 7J andsupplemental Fig. 2D). To rule out the possibility that VP22GFP enters Rab5-Q79L-positive structures via clathrin-mediated endocytosis, the clathrin heavy chain was abolished byRNAi (Fig. 7K). In clathrin-depleted cells, VP22-GFP enteredring-shaped endosomes, and this internalization was inhibitedby genistein, an inhibitor of lipid raft-mediated endocytosis(data not shown). Rab5 and VP22-GFP rings and VP22-GFP
and clathrin rings can be seen to differ slightly in position in Fig.
7K, thus possibly reflecting pathway-dependent substructuresof early endosomes.
Stabilization of Internalized VP22-GFP Signals in Chloroquine-treated Cells—The biological activity of TAT-Cre has
been shown to be significantly increased by chloroquine
treatment (13), possibly suggesting that chloroquine
enhances endosomal release of these proteins. We thus
examined whether chloroquine treatment induces cytoplasmic non-vesicular VP22-GFP signals in HeLa and CHO-K1
cells. Cell were treated with 50 ␮M chloroquine, and possible
changes in intracellular VP22-GFP signals were examined
spectrofluorometrically and microscopically (Fig. 7, L and
M). In contrast to our expectations, no apparent increase incytoplasmic non-vesicular VP22-GFP signals was detectedeven after long chloroquine treatment. Chloroquine treatment instead significantly increased intracellular vesicular
VP22-GFP signals in both cell lines. We interpret these
JOURNAL OF BIOLOGICAL CHEMISTRY

27513

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

in Fig. 6 (A and B), toxin B treatment brought about significant
changes in cell morphology and the actin cytoskeleton, butthere was no appreciable reduction in internalized VP22-GFP
signals in toxin B-treated cells, suggesting that Rho family
GTPases are not involved in VP22-GFP internalization.
To further confirm this point, HeLa cells were subjected to
DNA-mediated RNAi to knock down the genes encoding
RhoA (Fig. 6, C and D) and Cdc42 (E and F). Rac1 activity was
depleted by overexpressing a dominant-negative form of
Rac1 (Rac1-T17N) (Fig. 6, G–I). RhoA and Cdc42 mRNAs
were significantly eliminated by RNAi (Fig. 6, C and E), but
no appreciable reduction in VP22-GFP internalization was
detected (D and F). Rac1-T17N expression levels varied
depending on the cells. About 70% of the cells expressed
Rac1-T17N moderately and exhibited a slight increment ininternalized VP22-GFP signals without any substantial lossof surface signals (Fig. 6G), supporting the notion that Rac1
is not essential for cellular VP22 internalization. In theremaining cells, not only cell-surface VP22-GFP signals but
also cell-surface heparan sulfate signals were frequently
observed to be considerably reduced, and instead, the cellular internalized signals of both VP22-GFP and heparan sulfate were increased (Fig. 6, H and I). We interpreted thesefindings as suggesting that high levels of Rac1-T17N expression might result in rapid cellular uptake of cell-surface complexes of heparan sulfate and VP22-GFP. We thus concluded
that Rho family small GTPases are not essential for normal
VP22-GFP internalization.
Arf6 is a member of the Arf family and is essential for certain
lipid raft- or clathrin-mediated endocytosis (10, 61). To show
Arf6 involvement in VP22-GFP internalization, short hairpin
RNA specific for arf6 was used to knock down arf6 activity. In
Fig. 6 (K and L), internalized VP22-GFP and transferrin signals
were significantly reduced upon elimination of arf6 activity by
RNAi (Fig. 6J). Arf6 is thus assumed to be involved in cellular
VP22-GFP internalization. Because cell-surface heparan sulfate
signals were occasionally observed to be reduced moderately in
Arf6 knockdown cells (Fig. 6K), Arf6 may be partly involved inrecruitment of heparan sulfate to the cell surface. Transferrin
internalization was inhibited by arf6 depletion in the HeLa cells
used here; this is inconsistent with previous findings for humanembryonic kidney 293 and HeLa cells (61, 62) and could suggest
differences in cell lines.
Co-localization of VP22-GFP and Transferrin in Early
Endosomes—To determine the subcellular localization of internalized VP22-GFP signals, clarification was sought as towhether VP22-GFP signals are co-localized with cell compartment markers. HeLa cells were exposed to VP22-GFP at 0 °C,
and VP22-GFP that was not bound to cells was removed bywashing at 0 °C. VP22-GFP internalization was then examinedby shifting cells to 37 °C. Most, if not all, cytoplasmic VP22-GFP
signals at 15 min of internalization were found to have co-localized with EEA1, an early endosomal marker (Fig. 7A andsupplemental Fig. 3A), but not with LAMP1, a lysosomal
marker (Fig. 7B and supplemental Fig. 3B), or GM130, a Golgi
marker (Fig. 7C and supplemental Fig. 3C). Internalized VP22GFP signals are thus initially incorporated into early endosomes marked with EEA1.

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis
results as suggesting that the predicted endosome-cytoplasm transfer
of VP22-GFP may occur in a mechanism different from that of TAT-Cre.

27514 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

FIGURE 7. Incorporation of internalized VP22 signals into early endosomes. A–H, HeLa cells were exposed
to 0.5 ␮M VP22-GFP and 0.25 ␮M Alexa Fluor 647-labeled transferrin at 0 °C; incubated for 15 min (A–D) or 60
min (E–H) at 37 °C; fixed with 4% paraformaldehyde; and stained with anti-EEA1 (A and E), anti-LAMP1 (B and F),
and anti-GM130 (C and G) antibodies. Alexa Fluor 647-labeled transferrin signals are shown in D and H. In the
Merged panels, VP22-GFP is shown in green, whereas EEA1, LAMP1, GM130, and transferrin are shown in red.
The boxed regions in the last panels are enlarged in the first three panels. Scale bars ⫽ 10 ␮m. I, shown is the timecourse of the fraction of intracellular VP22-GFP co-localized with cell compartment markers. HeLa cells were
exposed to 0.5 ␮M VP22-GFP at 0 °C and then incubated for 5, 15, or 60 min at 37 °C. After trypsin treatment at
37 °C for 5 min, cells were stained with anti-EEA1, anti-LAMP1, anti-GM130, and anti-transferrin receptor (TfR)
antibodies and visualized by confocal microscopy. Co-localization was determined using the software thatcame with the confocal microscope (n ⫽ 12). Error bars indicate S.D. J, HeLa cells transfected with
pcDNA3-myc-Rab5-Q79L, a plasmid encoding Myc-tagged Rab5-Q79L, were exposed to 0.5 ␮M VP22-GFP and
0.25 ␮M Alexa Fluor 647-labeled transferrin at 37 °C for 1 h; fixed with 4% paraformaldehyde; and stained with
anti-Myc antibody. In the Merged panel, VP22-GFP, Myc-Rab5-Q67L, and transferrin are shown in green, red, andblue, respectively. Scale bar ⫽ 10 ␮m. K, HeLa cells transfected with CHC1 siRNA and plasmids expressing
Myc-Rab5-Q79L were incubated at 37 °C for 1 h with fresh serum-free medium containing 0.5 ␮M VP22-GFP,
fixed with 4% paraformaldehyde, stained with anti-clathrin and anti-Myc antibodies. In the Merged panels,
VP22-GFP and Rab5-Q79L are shown in green and red, respectively. Scale bars ⫽ 5 ␮m. L and M, the effects ofchloroquine treatment on intracellular accumulation of VP22-GFP are shown. In L, CHO-K1 cells treated with 50
␮M chloroquine for 30 min were exposed to VP22-GFP (1 ␮M), incubated for 1 or 4 h, and treated with trypsin.
VP22-GFP signals (green) and a differential interference contrast image were merged. In M, HeLa and CHO-K1
cells treated with 50 ␮M chloroquine for 30 min were exposed to VP22-GFP (1 ␮M) and incubated for 1 h, andintracellular accumulation of VP22-GFP signals was determined spectrofluorometrically. Three independentmeasurements were averaged. Error bars indicate S.D.

DISCUSSION
In this work, we have shown thatcell-surface binding and cellularinternalization of VP22 requireinteractions between cell-surface
heparan sulfate and basic aminoacids in the N-terminal region of
VP22.C1 (C-terminal half of VP22),
which includes Arg-164, and that
VP22 is internalized through a lipid
raft-mediated endocytic pathway
independent of clathrin, caveolae,
and Rho family GTPases but
dependent on dynamin and Arf6.
We have also shown that internalized VP22 initially enters earlyendosomes and then moves to lysosomes and possibly recycling
endosomes. Cellular uptake of VP22
appears to be unrelated to lipid raftmediated endocytosis induced in
Arf6-Q67L-expressing cells (32).
Differences in Cell-surface Adsorption between VP22-GFP and
TAT-PTD—Fig. 2 shows that VP22GFP bound to the cell surface
mainly via heparan sulfate. The contribution of chondroitin sulfate tothis binding is apparently quite
small, if at all, because virtually noadsorption of VP22-GFP signals
could be detected in pgsD-677, a
CHO-K1 cell line capable of producing 3– 4 times as many chondroitin sulfate molecules as wildtype cells without production of
heparan sulfate. The intensity ofinternalized mutant VP22 signals
was basically in proportion to that ofsignals adsorbed on the cell surface(Fig. 2, F and G), thus possibly indicating that cell-surface adsorption is
the first rate-limiting step in cellularinternalization of VP22-GFP.
The role of GAG in cell-surface
adsorption and internalization of
the TAT peptide may differ fromthat of VP22-GFP. The TAT peptide that enters pgsD-677 cells is
40% as much as that of wild-type
cells, and TAT-Cre internalization
is significantly inhibited by chondroitin sulfates B and C (12, 13).

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis
TABLE 1
List of caveola-independent lipid raft-mediated endocytic pathways⫹, dependent or regulated; ⫺, independent; CHO, Chinese hamster ovary; GPI-APs, glycosylphosphatidylinositol-anchored proteins; M2mAChR, M2 muscarinic acetylcholine receptor; CPE, carboxypeptidase E; SV40, simian virus 40; CavDC, caveolin-1-deficient cells; CTB, cholera toxin subunit B; Cav⫺/⫺ MEFs, caveolin-1-null mouseembryonic fibroblasts; IL-2R, interleukin-2 receptor.
Rac1

RhoA

Cdc42

Arf6

⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺

⫹
⫹
⫹

⫺
⫺
⫺

⫺
⫺
⫺

⫹
⫹
⫹

⫺

YT, L␣␤␥
CHO
CHO

⫹
⫹
⫹

⫹
⫹
⫹

⫹

⫹
⫹
⫹

⫺
⫺

HeLa

⫹

⫺

⫺

⫺

⫺

DynaminGroup I
VacA
Dextran
GPI-APs
M2mAChR
CD59
CPE
SV40
CTB

HeLa, AGS
CHO
CHO, COS
HeLa
HeLa
Neuro2A
CavDC
Cav⫺/⫺ MEFs

Group II
IL-2R
IL-2R
AlbuminGroup III
VP22-GFP

However, Fig. 2B shows that inhibition of VP22-GFP internalization was hardly inhibited at all by chondroitin sulfate addition to the culture medium.
In TAT-PTD, all 8 basic amino acids are equally required forcellular uptake activity (39). Our mutational analysis of VP22
(Fig. 2F) indicated that the roles of basic amino acids (23 of 142
amino acids) of VP22.C1 in cellular VP22-GFP internalizationdiffer significantly depending on amino acid position. Arg-164
in VP22 is almost indispensable for VP22-GFP internalization
activity, whereas other amino acids may be only moderately orslightly necessary. Thus, a long PTD such VP22.C1 may differ in
the GAG interaction mode from short peptide-type PTDs suchas TAT-PTD. In the former, the amino acid sequence itself
appears to be much more important than the presence orabsence of any positive charge. No or little similarity is foundbetween the VP22 amino acid sequence around position 164
and the consensus amino acid sequences for heparin/heparansulfate-binding domains so far identified (65).
Not only specific receptors but also heparan sulfate coreceptors have been shown to be required for cell-surface binding ofmorphogens such as Hedgehog, bone morphogenetic protein,
Wnt, and fibroblast growth factors (66). Our results in Fig. 2D
also support the notion that cell-surface VP22-GFP binding
requires not only heparan sulfate but also VP22-specificreceptors.
VP22-GFP May Be Internalized through a Unique Endocytic Pathway—Our results show that, in HeLa cells, VP22 isinternalized through clathrin/caveola/Rho family GTPaseindependent but dynamin/Arf6-dependent lipid raft-mediated endocytosis. Various clathrin- and caveola-independent and lipid raft-dependent endocytic pathways have beenreported recently (10) and may be classed into two groups
based on dynamin dependence (Table 1). In several cell
types, cellular internalization of glycosylphosphatidylinositol-anchored proteins, simian virus 40, and cholera toxin
subunit B and Arf6-Q67L-dependent internalization (32) are
independent of dynamin (group I) (59, 67, 68), but dynaminis required for cellular internalization of the interleukin-2
receptor and albumin (group II) (42, 57). The former may beregulated by Cdc42 or Arf6, and the latter by RhoA and Rac1
SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

⫹
⫹
⫹
⫺
⫺

⫹

Tyrosinekinases

Ref.

⫺
⫹

69
57
59
70
32
71
67
68

⫹

⫹
⫹

42
57
57

⫺

This work(32, 42, 57, 59, 69 –71). Our results show that VP22-GFP
internalization occurs via lipid raft-mediated endocytosis
dependent on dynamin and Arf6 but not Rho family GTPases
such as RhoA, Rac1, and Cdc42. Thus, as summarized inTable 1, the VP22-GFP endocytic mechanism may differ significantly from those previously identified. We presume thatthe endocytic pathway for VP22-GFP internalization may
represent the third lipid raft-mediated endocytic pathway(group III in Table 1).
VP22.C1 and short peptide-type PTDs may not have thesame internalization mechanism. Indeed, short peptide-type
PTDs such as TAT, the N-terminal peptide of prion protein,
PDX-1, and the octaarginine peptide, in addition to their fusion
proteins, are internalized via macropinocytosis (13, 14, 72–74).
The TAT peptide itself is internalized via clathrin-dependentendocytosis (12), and TAT-GFP is internalized via caveola-mediated endocytosis in HeLa cells (15, 16).
Internalized VP22-GFP was also shown to be initially incorporated into EEA1-positive early endosomes (Fig. 7A) withtransferrin incorporated via clathrin-mediated endocytosis (Dand H). Internalized TAT-GFP is not incorporated into EEA1positive early endosomes but into caveolin-1-positive structures (15, 16). This difference in the endocytic mechanism of
TAT and VP22 might arise from the difference in their requirement for GAGs.
While this manuscript was being revised, Payne et al. (75)reported that cell-surface proteoglycans and proteoglycanbinding ligands such as cationic polymers, lipids, and polypeptides are internalized via clathrin- and caveolin-independentand flotillin- and dynamin-dependent endocytosis. This system
is resistant to treatment with cholesterol-binding drugs such as
filipin and nystatin and accordingly appears to be independentof lipid rafts (75). In contrast, the clathrin/caveola-independent
but dynamin-dependent endocytic pathway found in VP22
internalization in this work is mediated by lipid rafts (Fig. 5),
suggesting that these two systems are different from each other.
In conclusion, we have shown that the mechanism of cellular
uptake for VP22 is significantly different from that for short
peptide-driven PTDs and that VP22 internalization is carriedout via a type of lipid raft-mediated endocytosis independent of
JOURNAL OF BIOLOGICAL CHEMISTRY

27515

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

Rho family
GTPases

Cells

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis
clathrin, caveolae, and Rho family GTPases but dependent on
dynamin and Arf6.
Acknowledgments—We thank K. Nakayama, J. Pessin, G. Bokoch,
and J. G. Donaldson for plasmids and K. Ui-Tei for helpful discussion.
REFERENCES

27516 JOURNAL OF BIOLOGICAL CHEMISTRYVOLUME 282 • NUMBER 37 • SEPTEMBER 14, 2007

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

1. Wadia, J. S., and Dowdy, S. F. (2002) Curr. Opin. Biotechnol. 13, 52–56
2. Lindsay, M. A. (2002) Curr. Opin. Pharmacol. 2, 587–594
3. Green, M., and Loewenstein, P. (1988) Cell 55, 1179 –1188
4. Frankel, A., and Pado, C. (1988) Cell 55, 1189 –1193
5. Vivès, E., Brodin, P., and Lebleu, B. (1997) J. Biol. Chem. 272,
16010 –16017
6. Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) J. Biol.
Chem. 269, 10444 –10450
7. Richard, J. P., Melikov, K., Vivès, E., Ramos, C., Verbeure, B., Gait, M. J.,
Chernomordik, L. V., and Lebleu, B. (2003) J. Biol. Chem. 278, 585–590
8. Lundberg, M., Wikstrom, S., and Johansson, M. (2003) Mol. Ther. 8,
143–150
9. Conner, S. D., and Schmid, S. L. (2003) Nature 422, 37– 44
10. Kirkham, M., and Parton, R. G. (2005) Biochim. Biophys. Acta 1745,
273–286
11. Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. (2001) J. Biol. Chem.
276, 3254 –3261
12. Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) J. Biol. Chem. 280, 15300 –15306
13. Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Nat. Med. 10, 310 –315
14. Kaplan, I. M., Wadia, J. S., and Dowdy, S. F. (2005) J. Controlled Release
102, 247–253
15. Fittipaldi, A., Ferrari, A., Zoppe, M., Arcangeli, C., Pellegrini, V., Beltram,F., and Giacca, M. (2003) J. Biol. Chem. 278, 34141–34149
16. Ferrari, A., Pellegrini, V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. (2003) Mol. Ther. 8, 284 –294
17. Jones, S. W., Christison, R., Bundell, K., Voyce, C. J., Brockbank, S. M.,
Newham, P., and Lindsay, M. A. (2005) Br. J. Pharmacol. 145,
1093–1102
18. Vendeville, A., Rayne, F., Bonhoure, A., Bettache, N., Montcourrier, P.,
and Beaumelle, B. (2004) Mol. Biol. Cell 15, 2347–2360
19. Potocky, T. B., Menon, A. K., and Gellman, S. H. (2003) J. Biol. Chem. 278,
50188 –50194
20. Fischer, R., Kohler, K., Fotin-Mleczek, M., and Brock, R. (2004) J. Biol.
Chem. 279, 12625–12635
21. Elliott, G. D., and Meredith, D. M. (1992) J. Gen. Virol. 73, 723–726
22. Elliott, G., and O’Hare, P. (1997) Cell 88, 223–233
23. Kueltzo, L. A., Normand, N., O’Hare, P., and Middaugh, C. R. (2000) J. Biol.
Chem. 275, 33213–33221
24. Normand, N., van Leeuwen, H., and O’Hare, P. (2001) J. Biol. Chem. 276,
15042–15050
25. Stroh, C., Held, J., Samraj, A. K., and Schulze-Osthoff, K. (2003) Oncogene
22, 5367–5373
26. Brewis, N. D., Phelan, A., Normand, N., Choolun, E., and O’Hare, P. (2003)
Mol. Ther. 7, 262–270
27. Zavaglia, D., Normand, N., Brewis, N., O’Hare, P., Favrot, M. C., and Coll,
J. (2003) Mol. Ther. 8, 840 – 845
28. Boenicke, L., Chu, K., Pauls, R., Tams, C., Kruse, M. L., Kurdow, R.,
Schniewind, B., Bohle, A., Kremer, B., and Kalthoff, H. (2003) J. Mol. Med.
81, 205–213
29. Narita, K., Choudhury, A., Dobrenis, K., Sharma, D. K., Holicky, E. L.,
Marks, D. L., Walkley, S. U., and Pagano, R. E. (2005) FASEB J. 19,
1558 –1560
30. Kasai, K., Shin, H. W., Shinotsuka, C., Murakami, K., and Nakayama, K.
(1999) J. Biochem. (Tokyo) 125, 780 –789
31. Steiner, P., Floyd Sarria, J. C., Glauser, L., Magnin, S., Catsicas, S., andHirling, H. (2002) J. Cell Biol. 157, 1197–1209
32. Naslavsky, N., Weigert, R., and Donaldson, J. G. (2004) Mol. Biol. Cell 15,3542–3552

33. Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S., and Saigo, K. (2004) Nucleic Acids Res. 32, W124 –W129
34. Ui-Tei, K., Zenno, S., Miyata, Y., and Saigo, K. (2000) FEBS Lett. 479,
79 – 82
35. Summerford, C., and Samulski, R. J. (1998) J. Virol. 72, 1438 –1445
36. Tang, Y., and DeFranco, D. B. (1996) Mol. Cell. Biol. 16, 1989 –2001
37. Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985) Proc. Natl. Acad. Sci.
U. S. A. 82, 3197–3201
38. Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz,
S., Massague, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci.
U. S. A. 89, 2267–2271
39. Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L.,
and Rothbard, J. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13003–13008
40. van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553–563
41. Oh, P., McIntosh, D. P., and Schnitzer, J. E. (1998) J. Cell Biol. 141,
101–11442. Lamaze, C., Dujeancourt, A., Baba, T., Lo, C. G., Benmerah, A., and Dautry-Varsat, A. (2001) Mol. Cell 7, 661– 671
43. Sauvonnet, N., Dujeancourt, A., and Dautry-Varsat, A. (2005) J. Cell Biol.
168, 155–163
44. Wang, L. H., Rothberg, K. G., and Anderson, R. G. (1993) J. Cell Biol. 123,
1107–1117
45. Motley, A., Bright, N. A., Seaman, M. N., Margaret S., and Robinson, M. S.
(2003) J. Cell Biol. 162, 909 –918
46. Hinrichsen, L., Harborth, J., Andrees, L., Weber, K., and Ungewickell, E. J.
(2003) J. Biol. Chem. 278, 45160 – 45170
47. Jackson, A. L., and Linsley, P. S. (2004) Trends Genet. 20, 521–524
48. Simons, K., and Ikonen, E. (1997) Nature 387, 569 –572
49. Martinho, R. G., Castel, S., Urena, J., Fernandez-Borja, M., Makiya, R.,
Olivecrona, G., Reina, M., Alonso, A., and Vilaro, S. (1996) Mol. Biol. Cell
7, 1771–1788
50. Nabi, I. R., and Le, P. U. (2003) J. Cell Biol. 161, 673– 677
51. Pelkmans, L., and Helenius, A. (2002) Traffic 3, 311–320
52. Robert, G. P., and Richards, A. A. (2003) Traffic 4, 724 –738
53. Gauthier, N. C., Ricci, V., Gounon, P., Doye, A., Tauc, M., Poujeol, P., andBoquet, P. (2004) J. Biol. Chem. 279, 9481–9489
54. Pelkmans, L., Kartenbeck, J., and Helenius, A. (2001) Nat. Cell Biol. 3,
473– 483
55. Nichols, B. J. (2002) Nat. Cell Biol. 4, 374 –378
56. Pelkmans, L., Puntener, D., and Helenius, A. (2002) Science 296, 535–539
57. Cheng, Z. J., Singh, R. D., Sharma, D. K., Holicky, E. L., Hanada, K., Marks,
D. L., and Pagano, R. E. (2006) Mol. Biol. Cell 17, 3197–3210
58. Wennerberg, K., Rossman, K. L., and Der, C. J. (2005) J. Cell Sci. 118,
843– 846
59. Sabharanjak, S., Sharma, P., Parton, R. G., and Mayor, S. (2002) Dev. Cell 2,411– 423
60. Aktories, K., and Just, I. (1995) Trends Cell Biol. 5, 441– 443
61. Houndolo, T., Boulay, P. L., and Claing, A. (2005) J. Biol. Chem. 280,
5598 –5604
62. Hashimoto, S., Hashimoto, A., Yamada, A., Kojima, C., Yamamoto, H.,
Tsutsumi, T., Higashi, M., Mizoguchi, A., Yagi, R., and Sabe, H. (2004)
J. Biol. Chem. 279, 37677–37684
63. Ghosh, R. N., and Maxfield, F. R. (1995) J. Cell Biol. 128, 549 –561
64. Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lutcke, A., Gruenberg,J., and Zerial, M. (1994) EMBO J. 13, 1287–1296
65. Capila, I., and Linhardt, R. J. (2002) Angew. Chem. Int. Ed. Engl. 41,
390 – 412
66. Filmus, J., and Selleck, S. B. (2001) J. Clin. Investig. 108, 497–501
67. Damm, E. M., Pelkmans, L., Kartenbeck, J., Mezzacasa, A., Kurzchalia, T.,
and Helenius, A. (2005) J. Cell Biol. 168, 477– 488
68. Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma,
D. K., Pagano, R. E., Hancock, J. F., Mayor, S., and Parton R. G. (2005) J. Cell
Biol. 168, 465– 476
69. Gauthier, N. C., Monzo, P., Kaddai, V., Doye, A., Ricci, V., and Boquet, P.
(2005) Mol. Biol. Cell 16, 4852– 4866
70. Delaney, K. A., Murph, M. M., Brown, L. M., and Radhakrishna, H. (2002)
J. Biol. Chem. 277, 33439 –33446

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2007/07/24/M703810200.DC1.html

VP22 Internalization via Lipid Raft-mediated Endocytosis
71. Arnaoutova, I., Jackson, C. L., Al-Awar, O. S., Donaldson, J. G., and Loh,Y. P. (2003) Mol. Biol. Cell 14, 4448 – 4457
72. Magzoub, M., Sandgren, S., Lundberg, P., Oglecka, K., Lilja, J., Wittrup, A.,
Göran Eriksson, L. E., Langel, U., Belting, M., and Gräslund, A. (2006)
Biochem. Biophys. Res. Commun. 348, 379 –385
73. Noguchi, H., Matsumoto, S., Okitsu, T., Iwanaga, Y., Yonekawa, Y.,
Nagata, H., Matsushita, M., Wei, F. Y., Matsui, H., Minami, K., Seino,S., Masui, Y., Futaki, S., and Tanaka, K. (2005) Cell Transplant. 14,
637– 645
74. Nakase, I., Niwa, M., Takeuchi, T., Sonomura, K., Kawabata, N., Koike, Y.,
Takehashi, M., Tanaka, S., Ueda, K., Simpson, J. C., Jones, A. T., Sugiura,Y., and Futaki, S. (2004) Mol. Ther. 10, 1011–1022
75. Payne, C. K., Jones, S. A., Chen, C., and Zhuang, X. (2007) Traffic 8,
389 – 401

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010

SEPTEMBER 14, 2007 • VOLUME 282 • NUMBER 37

JOURNAL OF BIOLOGICAL CHEMISTRY

27517