Molecular Biology of the Cell
Vol. 21, 1001–1010, March 15, 2010
Combinational Soluble N-Ethylmaleimide-sensitive Factor
Attachment Protein Receptor Proteins VAMP8 and Vti1b
Mediate Fusion of Antimicrobial and Canonical
Autophagosomes with Lysosomes
Nobumichi Furuta,* Naonobu Fujita,†Takeshi Noda,†Tamotsu Yoshimori,†
and Atsuo Amano*
*Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871,
Japan; and†Department of Cellular Regulation, Division of Cellular and Molecular Biology, Research Institute
for Microbial Diseases, Osaka University, Suita-Osaka 565-0871, Japan
Submitted August 14, 2009; Revised January 6, 2010; Accepted January 12, 2010
Monitoring Editor: Thomas F.J. Martin
Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade
intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise
maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the
molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of
endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome
fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7,
VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluo-
rescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the
colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not
altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indi-
cating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed
colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In
contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings
strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and
canonical autophagosomes with lysosomes, an essential event for autophagic degradation.
Autophagy is an intracellular degradation system for cyto-
plasmic materials, such as proteins and organelles, which
are directed to lysosomes by a membrane-mediated process
(Seglen and Bohley, 1992; Yoshimori, 2004). This system
functions as a survival mechanism during short-term star-
vation by degrading some nonessential components to ob-
tain nutrients for biosynthetic reactions in mammalian cells.
In addition, autophagy is essential for cellular survival, dif-
ferentiation, development, homeostasis, and host defense
(Ravikumar et al., 2002; Komatsu et al., 2006; Hara et al., 2006;
Kamimoto et al., 2006). After signaling for autophagy, a flat
membrane sac structure, called the isolation membrane,
elongates to surround a portion of the cytoplasm and even-
tually forms a closed double-membrane structured vacuole,
which is termed an autophagosome (Yoshimori, 2004). Au-
tophagosomes then undergo a stepwise maturation process
that consists of a fusion event with lysosomes, which allows
the autophagic vacuole to acquire lysosomal proteases and
the vacuolar-type proton ATPase. Then, the interior of the
autophagosome becomes acidified and the cytoplasmic ma-
terials are subjected to degradation. At this final stage after
fusion with lysosomes, autophagosomes are referred to as
autolysosomes (Yoshimori, 2004). This fusion event, essen-
tial for degradation, has been studied by morphological
analysis using electron microscopy, whereas such molecular
analyses of the underlying mechanism have progressed
more slowly (Eskelinen, 2005; Eskelinen and Saftig, 2009).
Autophagy is emerging as a central component of var-
ious immunological functions, such as innate and adap-
tive immune activation, as well as antimicrobial host de-
infections (Deretic, 2009). Among them, its most principal
manifestation is capturing and digesting intracellular for-
eign microbial invaders. Group A Streptococcus (GAS) is an
extracellular pathogen that causes human diseases based on
its ability to bind to extracellular matrix proteins and pro-
duce a wide range of toxins. GAS can invade nonphagocytic
cells (e.g., epithelial cells, keratinocytes), although once in-
This article was published online ahead of print in MBC in Press
on January 20, 2010.
Address correspondence to: Atsuo Amano (email@example.com-
Abbreviations used: GcAV, GAS-containing autophagosome-like
vacuoles; SNAREs, soluble N-ethylmaleimide-sensitive factor at-
tachment protein receptor; GAS, group A Streptococcus; mRFP, mo-
nomeric red fluorescent protein; LAMP, lysosomal membrane pro-
tein; MOI, multiplicity of infection.
© 2010 by The American Society for Cell Biology1001
side a cell it is unable to proliferate and degraded by mech-
anisms that until recently were unidentified (Cunningham,
2000). We showed previously that the bacterium invades
nonphagocytic human cells via endocytosis, then escapes
from endosomes to the cytoplasm (Nakagawa et al., 2004),
after which the pathogen is entrapped within antimicrobial
autophagosomes, termed GAS-containing autophagosome-
like vacuoles (GcAVs). Initially, the structures of GcAVs do
not include lysosomal membrane protein (LAMP)1 similar
to autophagosomes, although they are subsequently associ-
ated with and colocalize with that protein. Finally, GAS is
degraded by GcAVs possessing lysosomal degradation en-
zymes (Nakagawa et al., 2004). This antimicrobial autoph-
agy, which has been likened to “eating the enemy,” was
recently classified as xenophagy selective for degradation
of intracellular bacteria and viruses (Levine, 2005). Al-
though antimicrobial autophagosomes (xenophagosomes)
were morphologically shown to be formed by the fusion of
multiple small precursor structures of xenophagosomes
(Nakagawa et al., 2004, Yamaguchi et al., 2009), their mech-
anism of fusion with lysosomes remains far from clear and
is a major topic of investigation.
Soluble N-ethylmaleimide-sensitive factor attachment pro-
tein receptors (SNAREs) are generally accepted as major
players in the final stage of docking and subsequent fusion
of diverse vesicle-mediated transport events (Hong, 2005).
SNAREs are functionally classified into v-SNAREs, which
are associated with the vesicle/container, and t-SNAREs,
which are associated with the target compartment (Hong,
2005). SNAREs are also structurally divided into Q-SNAREs
(those having a Gln/Q residue) and R-SNAREs (those hav-
ing an Arg/R residue), and Q-SNAREs are further subdi-
vided into Qa-, Qb-, and Qc-SNAREs based on the amino
acid sequence of the SNARE domain. It is unknown whether
SNAREs are involved in xenophagosome-lysosome or ca-
nonical autophagosome-lysosome fusion events.
In the present study, we investigated the involvement of
Vti1b in combinations with the v-SNAREs VAMP7 and
VAMP8 in the fusion of GcAVs (xenophagosomes) with
lysosomes. The results of our experiments using small inter-
fering RNA (siRNA)-knockdown strongly indicate that the
SNARE proteins Vti1b and VAMP8 mediate xenophago-
some-lysosome fusion. Furthermore, those SNARE proteins
were also found to mediate the fusion of canonical autopha-
gosomes with lysosomes.
MATERIALS AND METHODS
Cell Culture and Transfection
All cell lines were cultured in DMEM (Wako Pure Chemicals, Osaka, Japan)
supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen,
Carlsbad, CA). Human breast adenocarcinoma MCF7 cells stably expressing
enhanced green fluorescent protein (EGFP)-LC3 were a kind gift from Dr.
Koichi Matsunaga (Yoshimori laboratory). Human cervical epithelial HeLa
cells stably expressing EGFP-LC3 were constructed as described previ-
ously (Mizushima et al., 2001). Human lung adenocarcinoma epithelial
A549 cells stably expressing EGFP-LC3 were constructed as described pre-
viously (Matsunaga et al., 2009). Transfection was performed using Lipo-
fectamine 2000 (Invitrogen) according to the manufacturer’s protocol. For
amino acid starvation, cells were cultured in Earle’s balanced aalt (EBS)
solution (Sigma-Aldrich, St. Louis, MO) without amino acids and fetal bovine
serum. Stable transformants were selected in complete medium containing
500 ?g/ml G418 (Sigma-Aldrich).
The following antibodies were used: mouse monoclonal anti-LAMP1 (clone
H4A3; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-
VAMP7 (clone 158.2; Covalab, Villeurbanne, France), mouse monoclonal
anti-Vti1b (clone 7; BD Biosciences, Sparks, MD), mouse monoclonal anti-
Syntaxin 8 (clone 48; BD Biosciences), mouse monoclonal anti-?-actin (clone
AC-74; Sigma-Aldrich), rabbit polyclonal anti-LAMP1 (Abcam, Cambridge,
MA), rabbit polyclonal anti-VAMP8 (Covalab), mouse polyclonal anti-Syn-
taxin 7 (Abnova, Taipei, Taiwan), and rabbit polyclonal anti-LC3 (MBL,
Nagoya, Japan). Alexa Fluor-conjugated secondary antibodies (goat anti-
mouse immunoglobulin [Ig]G and goat anti-rabbit IgG) were purchased from
Invitrogen and used for fluorescence microscopy.
Western blotting was done as described previously (Kamimoto et al., 2006).
To determine the intensities of the blotting bands, each was selected using the
selection tool in ImageJ software (http://rsb.info.nih.gov/ij/), and each band
intensity was quantified using the Plot lane command.
siRNAs and Plasmids
Two sets of siRNA duplexes for each SNARE were used to knock down
VAMP7 (siRNA duplex sets: SYBL1-HSS110395 and SYBL1-HSS110396),
VAMP8 (VAMP8-HSS112730 and VAMP8-HSS112731), Vti1b (Vti1b-
HSS145663 and Vti1b-HSS145664), Syntaxin 7 (STX7-HSS112238 and STX7-
HSS112239), Syntaxin 8 (STX8-HSS114122 and STX8-HSS114123), and a
siRNA-negative control (Stealth RNAi; all from Invitrogen). siRNA-SYBL1-
HSS110395 corresponded to nucleotides 240-264 of human vamp7 (GenBank
accession NM_005638) located at the NH2-terminal domain. siRNA-SYBL1-
HSS110396 corresponded to nucleotides 517-541, which are located in the
coiled-coil domain (R-SNARE motif). siRNA-VAMP8-HSS112730 corre-
sponded to nucleotides 194-218 of human vamp8 (GenBank accession
NM_003761), which are located in the coiled-coil domain (R-SNARE motif).
siRNA-VAMP8-HSS112731 corresponded to nucleotides 23-47 located in the
NH2-terminal domain. siRNA-Vti1b-HSS145663 and iRNA-Vti1b-HSS145664
corresponded to nucleotides 318-342 and 387-411 of human vti1b (GenBank
accession NM_006370), respectively. These nucleotides are located in the
NH2-terminal domain. siRNA-STX7-HSS112238 corresponded to nucleotides
666-690 of human syntaxin7 (GenBank accession NM_003569), which are
located in the coiled-coil domain (Q-SNARE motif). siRNA-STX7-HSS112239
corresponded to nucleotides 742-766 located in the transmembrane domain.
siRNA-STX8-HSS114122 corresponded to nucleotides 462-486 of human syn-
taxin8 (GenBank accession NM_004853), which are located in the coiled-coil
domain (Q-SNARE motif). siRNA-STX8-HSS114123 corresponded to nucleo-
tides 663-687 located in the transmembrane domain. A plasmid encoding
monomeric Cherry (mCherry) protein was a generous gift from Dr. Roger Y.
Tsien (University of California, San Diego, San Diego, CA). To construct an
mCherry-C1 plasmid, polymerase chain reaction (PCR) was used to generate
mCherry cDNA with the exogenous restriction sites of NheI and BglII at the
5? and 3? ends, respectively, lacking the termination codon. The PCR fragment
obtained after digestion with each restriction enzyme was used to replace
EGFP cDNA of pEGFP-C1. To construct mCherry-Sec20, Slt1, Syntaxin 6,
VAMP7, and Vti1b plasmids, cDNA was cloned from genomic DNA isolated
from HeLa cells and then inserted into pmCherry C1 using engineered SalI
and BamHI sites. To construct an mCherry-VAMP8 plasmid, cDNA was
cloned from genomic DNA isolated from HeLa cells and inserted into pm-
Cherry C1 using engineered SalI and KpnI sites. The expression vectors for
mRFP-GFP-LC3 (tf-LC3) and mRFP-LC3 plasmids have been described pre-
viously (Kimura et al., 2007).
For immunostaining, the cells were washed with phosphate-buffered saline
(PBS), fixed with 3% paraformaldehyde in PBS for 15 min, and permeabilized
with 50 ?g/ml digitonin in blocking solution (0.1% gelatin in PBS) for 10 min.
After being washed twice with PBS, the cells were incubated in blocking
solution for 20 min and subsequently with primary antibodies diluted with
blocking solution at room temperature for 1 h. After being washed twice with
PBS, the cells were probed with secondary antibodies conjugated with Alexa
405, 488, 594, or 633 (Invitrogen). Samples were examined under a fluores-
cence laser scanning confocal microscope (model LSM510; Carl Zeiss, Thorn-
wood, NY) using Zeiss LSM Image Browser software (Carl Zeiss).
Identification of GAS and GcAV was performed as described previously
(Nakagawa et al., 2004; Yamaguchi et al., 2009). In brief, green fluorescent
protein (GFP)-LC3 and DNA of GAS were observed in GFP and 4,6-dia-
midino-2-phenylindole (DAPI) channels, respectively. Obtained images were
merged to compare the two signal patterns. GFP-LC3 puncta closely sur-
rounding and containing a GAS chain were identified as GcAV. To quantify
the colocalization among different compartments, GFP-LC3, SNAREs, and
LAMP1 were observed in the GFP, red fluorescent protein (RFP), and cyanine
(Cy)5 channels, respectively. Obtained images were merged, and then the
overlapped areas of the images were measured using the overlay tool of LSM
Image Browser software (Carl Zeiss). Merged compartments with ?95%
overlap were determined to be colocalized. Colocalization of monomeric
(m)RFP with GFP pixels of tfLC3, LAMP1, or dextran was also determined
using the profile tool of LSM Image Browser software (Carl Zeiss), as de-
scribed previously (Kimura et al., 2007). Obtained images were merged to
compare the two signal patterns, and plot profiles derived from the merged
images were quantified. Colocalization of RFP-LC3, GFP-LC3, and each
SNARE was observed in the GFP, RFP, and Cy5 channels, respectively.
Obtained puncta images were merged to compare the three signal patterns,
and colocalization of these three signals was determined by manual counting.
N. Furuta et al.
Molecular Biology of the Cell1002
Alexa Fluor 488-conjugated dextran (Mr10 000) was purchased from In-
vitrogen. The reagent was added to the medium of HeLa cells to a final
concentration of 0.5 mg/ml, followed by incubation at 37°C with 5% CO2
overnight. The next day, the cells were transferred to fresh medium, incu-
bated for 4 h to chase, and transfected with siRNA for the control, VAMP7,
VAMP8, and Vti1b. At 24 h after siRNA transfection, the cells were trans-
fected with plasmids expressing mRFP-LC3. At 24 h after plasmid transfec-
tion, the cells were starved in EBS solution for 3 h and then fixed and analyzed
with confocal microscopy. Detection of cathepsin L activity by fluorescent
microscopy was performed using a Magic Red Cathepsin L detection kit
(Immunochemistry Technologies, Bloomington, MN) according to the man-
For Western blotting of LC3, E64d and pepstatin A were purchased from the
Peptide Institute (Osaka, Japan). Cells were transfected with siRNA for the
control, VAMP7, VAMP8, and Vti1b. After 48 h of incubation, the cells were
cultured in EBS solution with E64d (10 ?g/ml) and pepstatin A (30 ?g/ml),
or without proteinase inhibitors (control; dimethyl sulfoxide [DMSO] 1.1
?g/ml) for appropriate times. Lysate samples were examined by Western
blotting using the anti-LC3 and actin antibodies.
Infection with GAS (strain JRS4) was performed as described previously
(Nakagawa et al., 2004). In brief, bacterial cells were added to cell cultures
without antibiotics for 1 h, and then infected cells were washed with PBS and
antibiotics (100 ?g/ml gentamicin and 100 U/ml penicillin G) were added for
an appropriate period to kill extracellular bacteria.
Gas Invasion Assays
GAS cells were incubated separately with 0.1 mCi of [methyl-3H]thymidine
(GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) for 12 h,
after which they were harvested and washed with PBS.3H-Labeled GAS
(multiplicity of infection [MOI]), 100] was added to HeLa cells for 1 h and
then washed three times with PBS to remove external nonadherent bacteria
cells. Next, GAS-infected HeLa cells were incubated for 2 h in medium
containing antibiotics, after which the cells were disrupted by addition of 100
?l of distilled water with incubation at 37°C for 10 min. The numbers of
invading organisms were determined using a liquid scintillation counter
(model LSC-5100; Aloka, Tokyo, Japan) and from the amounts of3H recov-
ered from infected cells, with the results expressed as percentages of the total
numbers of GAS organisms added.
Bacterial Viability Assay
A colony-forming units viability assay was performed as described previ-
ously (Nakagawa et al., 2004). GAS (MOI, 100) was added to HeLa cells for 1 h
and then washed three times with PBS to remove external nonadherent
bacteria cells. Next, GAS infected HeLa cells were incubated for 2 h in
medium containing antibiotics, after which the cells were disrupted by addi-
tion of 100 ?l of distilled water with incubation at 37°C for 10 min. Serial
dilutions of the lysates were plated on THY agar plates, and colony counting
All values shown in figures are presented as the mean ? SD. Statistical
significance (p value) was determined using one-way analysis of variance
(ANOVA) with Scheffe posttest (SPSS, Chicago, IL).
GcAVs. (A) HeLa cells stably expressing GFP-LC3 were
transfected with plasmids to express mCherry-Sec20,
Slt1, Syntaxin 6, Vti1b, VAMP7, and VAMP8. At 24 h
after transfection, the cells were infected with GAS for
180 min at an MOI of 100, as described in Materials and
Methods. Cellular and bacterial DNAs were stained with
DAPI. The boxed regions in the top panels are enlarged
(B) Colocalization frequencies of GcAVs with several
mCherry signals were manually determined as the per-
centage of the total number of GcAVs. Data shown repre-
sent results of ?60 cells and each ratio (percentage) rep-
resents the mean value ? SD from three independent
Colocalization of GFP-LC3 with SNAREs in
VAMP8/Vti1b Mediate Autophagic Fusion
Vol. 21, March 15, 2010 1003
Localization of GFP-LC3 with SNAREs in GcAVs
To determine whether SNARE proteins become localized in
GcAVs of GAS-infected HeLa cells, endosome SNAREs
(VAMP7, VAMP8, and Vti1b), endoplasmic reticulum (ER)-
residing SNAREs (Sec20 and Slt1), and trans-Golgi network
(TGN) residing Syntaxin 6 were examined. As shown in
Figure 1, GFP-LC3 puncta associated with GcAVs were
found to colocalize with those of VAMP7, VAMP8, and
Vti1b, whereas the other SNAREs were not markedly
overlapped with GFP-LC3.
To determine whether these localizations occurred before or
after lysosomal fusion, we next examined the colocalization of
VAMP7, VAMP8, and Vti1b with LAMP1 and GFP-LC3 (Fig-
puncta were negligibly merged with GFP-LC3 and LAMP1,
whereas they gradually became colocalized with them over
time, indicating that VAMP7 and VAMP8 become localized in
GcAVs after fusion with lysosomes. In contrast, GcAVs were
found to possess Vti1b from an early stage, and LAMP1
merged with Vti1b and GFP-LC3 in LAMP1-acquired GcAVs.
These results suggest that VAMP7 and VAMP8 originate from
lysosomes, whereas Vti1b is derived from xenophagosomes
before fusion with lysosomes.
Knockdown of VAMP8 and Vti1b Disturbs Bactericidal
Effects of GcAVs
Next, we examined the effects of knockdown of VAMP7,
VAMP8, and Vti1b with siRNAs on GcAVs. The expression
of these SNARE proteins was significantly suppressed in
HeLa cells (Figure 4A). After infecting HeLa cells with GAS,
VAMP7, VAMP8, and Vti1b in GcAVs. HeLa
cells stably expressing GFP-LC3 were infected
with GAS for 60 min at an MOI of 100. In-
fected cells were washed and incubated with
antibiotics for the indicated time periods and
then fixed and incubated with anti-LAMP1,
VAMP7, VAMP8, and Vti1b antibodies. Cel-
lular and bacterial DNAs were stained with
DAPI. The boxed regions in the left panels are
enlarged in the right panels. Bars, 10 ?m (left)
and 5 ?m (right).
Colocalization of GFP-LC3 with
colocalization frequencies of GcAVs with VAMP7, VAMP8, and Vti1b
in Figure 2 were determined. SNARE- and LAMP1-negative cells are
shown in green, SNARE-positive and LAMP1-negative cells in white,
SNARE-negative and LAMP1-positive cells in red, and SNARE- and
LAMP1-positive cells in blue. The numbers of those signals were
manually counted and are presented as a percentage of the total
number of GcAVs. Data shown represent results of ?60 cells.
Colocalization of GcAVs with SNAREs and LAMP1. The
N. Furuta et al.
Molecular Biology of the Cell1004
GFP-LC3–positive GcAVs were clearly merged with LAMP1
in the control and VAMP7-depleted cells (Figure 4, B and C),
whereas siRNA knockdown of VAMP8 and Vti1b appar-
ently inhibited the colocalization of GFP-LC3 with LAMP1.
However, GcAV formation efficiency was not altered by
knockdown of these SNAREs (Figure 4D). These results
indicate that VAMP8 and Vti1b are directly involved with
the fusion of xenophagosomes with lysosomes. The effects of
knockdown of these SNAREs were also analyzed in regard
to their cellular bactericidal effects. Although the invasive
efficiency of GAS into cells was not altered by knockdown
(Figure 4E), bactericidal efficiency was significantly dimin-
ished in VAMP8- and Vti1b-depleted cells (Figure 4F).
Knockdown of VAMP8 and Vti1b Disturbs Maturation of
We also analyzed the effects of knockdown of SNARE pro-
teins on canonical autophagy using HeLa cells. It is known
that LC3 contains two forms: LC3-I that resides in cytosol as
a soluble protein and LC3-II that is associated with auto-
phagosomes (Eskelinen, 2005). Under starvation, LC3-I is
converted to LC3-II, and autophagosomes are visualized
as GFP-LC3 puncta. In the control cells, punctate GFP-LC3
structures were clearly observed at 120 min and then
disappeared, with subsequent longer starvation of 240 min
(Figure 5, A and B), due to the degradation of LC3-II in
autolysosomes as well as detachment from autolysosomes
(Eskelinen, 2005). Although knockdown of VAMP8 or Vti1b
negligibly altered the number of GFP-LC3 dots in both
starved and fed conditions at 120 min, it apparently inhib-
ited the disappearance of GFP-LC3 puncta at 240 min.
Knockdown of VAMP7 resulted in little effects in regard to
LC-II protein level. In contrast, considerable amounts of
LC3-II protein remained in the VAMP8- and Vti1b-depleted
cells (Figure 5, C and D). In a previous study, after cells were
treated with lysosomal protease inhibitors such as E64d and
pepstatin A, degradation of LC3-II was inhibited, whereas
that of LC3-I was not affected (Mizushima and Yoshimori,
disturbed the antimicrobial effects of GcAVs.
(A) HeLa cells were transfected with siRNA
for the control, VAMP7, VAMP8, and Vti1b.
At 48 h after transfection, the cells were lysed
and then examined by Western blotting using
anti-Vti1b, -VAMP7, -VAMP8, and actin anti-
bodies. (B) HeLa cells expressing GFP-LC3
were treated with siRNA in the same manner
as described in A. After 48 h, cells were in-
fected with GAS for 60 min at an MOI of 100.
After an additional 120 min of incubation
with antibiotics for bacterial killing, the cells
were fixed and incubated with anti-LAMP1
antibodies and observed with a confocal mi-
croscope. Cellular and bacterial DNAs were
stained with DAPI. The boxed regions in the
left panels are shown enlarged in the right
panels. Bars, 10 ?m (left) and 5 ?m (right). (C)
Colocalization frequencies of GcAVs with
LAMP1 signals were manually determined
and are presented as the percentage of total
number of GcAVs. Data shown represent re-
sults of ?60 cells, and each ratio (percentage)
represents the mean value ? SD from three
independent experiments. *p ? 0.01; **p ?
0.05 by one-way ANOVA and Scheffe ´’s post-
test. (D) Numbers of cells containing GcAVs
were counted and are presented as the per-
centage of the total number of GAS infected
cells. HeLa cells stably expressing GFP-LC3
were transfected with siRNA and infected
with GAS in the same manner as described in
B. Data shown represent results of ?30 cells
and each ratio (percentage) represents the
mean value ? SD from three independent
experiments. (E) Efficiency of GAS invasion
was measured as described in Materials and
Methods. Data are shown as the mean ? SD
from three independent experiments. (F) Vi-
ability of invaded GAS in HeLa cells was
evaluated as described in Materials and
Methods. Data are shown as the mean ? SD
from three independent experiments. *p ?
0.01; **p ? 0.05 by one-way ANOVA and
Scheffe ´’s posttest.
Knockdown of VAMP8 and Vti1b
VAMP8/Vti1b Mediate Autophagic Fusion
Vol. 21, March 15, 2010 1005
2007). That report also demonstrated that the amount of
LC3-II at a certain time point does not indicate the total
amount of autophagic flux, rather that flux is more accu-
rately represented by differences in the amounts of LC3-II
between samples in the presence and absence of lysosomal
protease inhibitors. We examined autophagic flux using
E64d and pepstatin A, and we observed vastly different
densities between LC3-II bands in the presence and absence
of protease inhibitors in both control and VAMP7-depleted
cells, indicating the induction of normal autophagic flux
(Figure 6, A and B). In contrast, there were only slight
differences between band densities with and without the
inhibitors in the VAMP8- and Vti1b-depleted cells. Further-
more, knockdown of VAMP8 and Vti1b clearly disturbed
the maturation of canonical autophagosomes in other cell
types, including MCF7 and A549 cells (Supplemental Fig-
ures 1–4). These results indicate that knockdown of VAMP8
and Vti1b prevents LC3-II degradation in autolysosomes
and suggest that VAMP8 and Vti1b are also involved in
maturation of canonical autophagosomes.
Impaired Degradation of LC3 Proteins in
Autophagosomes of VAMP8- and Vti1b-depleted Cells
Fusion of autophagosomes with lysosomes provides an acidic
environment to digest the interior. A recent study showed that
GFP loses its fluorescence in acidic conditions and is subse-
quently degraded by lysosomal hydrolases, whereas mRFP is
uration of canonical autophagosomes. (A) HeLa cells stably ex-
pressing GFP-LC3 were treated with siRNA in the same manner
as described in Figure 4A. The cells were further cultured in
growth medium (Fed) or EBS solution (Starved) for the indicated
periods and then fixed and observed with a confocal microscope.
Cellular DNA was stained with DAPI (blue). Bars, 10 ?m. (B)
Quantitative analysis of the number of GFP-LC3 puncta per cell
shown in A was performed using ImageJ software. More than 100
cells were examined. Data are shown as the mean ? SD (C)
siRNA-treated HeLa cells were cultured in Fed and Starved
conditions for 240 min. The cellular lysates were subjected to
western blotting using anti-LC3 and actin antibodies. (D) Quan-
titative analysis of Western blot bands shown in C was per-
formed using ImageJ software. Data show the ratios of LC3-II
band intensities to the actin bands. Values are shown as the
mean ? SD from three independent experiments.
Knockdown of VAMP8 and Vti1b disturbed the mat-
VAMP8- and Vti1b-depleted HeLa cells. (A) HeLa cells were treated
with siRNA in the same manner as described in Figure 4A. At 48 h
after transfection, the cells were cultured in starved solution (EBS)
with or without proteinase inhibitors for the indicated times. The
cellular lysates were examined to measure the amounts of LC3-II
proteins with Western blotting using anti-LC3 and actin antibodies.
DMSO was used as the control. (B) Quantitative analysis of the relative
intensities of the LC3-II bands (inhibitor treated/control) after 240 min
in A was performed using ImageJ software. The mean values ? SD are
shown from three independent experiments. *p ? 0.01 by one-way
ANOVA and Scheffe ´’s posttest.
LC3-II proteins accumulated without degradation in
N. Furuta et al.
Molecular Biology of the Cell1006
stable under degradation conditions (Kimura et al., 2007). In
contrast, the life span of xenophagosomes (GcAVs) is signif-
icantly longer than that of canonical autophagosomes, and
GFP-LC3 notably remains on GcAVs for up to several hours
or even days (Nakagawa et al., 2004). Therefore, mRFP-LC3
as well as mRFP-GFP in tandem with fluorescently tagged
LC3 (tfLC3) have been devised for dissecting the maturation
process of autophagosomes to autolysosomes (Kimura et al.,
2007). Using tfLC3, we analyzed the distributions of mRFP
and GFP signals in LC3 proteins of canonical autophago-
somes in VAMP8- and Vti1b-depleted cells. mRFP puncta
were inconsistently merged with those of GFP in the control
as well as VAMP7-depleted cells at 180 min after starvation,
indicating that GFP signals were attenuated by the acidic
condition in the autolysosomes, whereas mRFP remained
(Figure 7A). In contrast, the puncta of these markers were
clearly colocalized in VAMP8- and Vti1b-depleted cells, in-
dicating negligible attenuation of GFP, because few LC3
proteins were sorted to autolysosomes. Quantitative analy-
sis also indicated that a majority of GFP signals remained
with mRFP in autophagosomes in VAMP8- and Vbti1b-
depleted cells (Figure 7B), indicating prevention of fusion
between autophagosomes and lysosomes in those cells.
To determine whether lysosomal enzyme failure in
VAMP8- and Vti1b-depleted cells is the cause of this phe-
notype, the distribution of mRFP-LC3 in lytic compartments
was further examined. RFP signals were clearly merged
with LAMP1 in the control and VAMP7-depleted cells after
180 min of starvation (Figure 8, A and C), whereas their
coexistence was merely observed in VAMP8- and Vti1b-
depleted cells. Next, we used another lysosome marker,
dextran (Bright et al., 2005), and preloaded HeLa cells with
Alexa 488 dextran for marking lysosomes via endocytosis
and then starved them for 180 min, after which the colocal-
ization of dextran with mRFP-LC3 was examined as de-
scribed previously (Kimura et al., 2007). This assay also
indicated that colocalization of the markers for autophago-
somes and lysosomes was significantly inhibited by siRNA
for VAMP8- and Vti1b, respectively (Figure 8, B and D).
We also examined whether knockdown of Vti1b or
VAMP8 had a negative influence on lysosomal maturation
and proteolytic activation. The lysosomal cysteine pepti-
dase cathepsin L significantly contributes to terminal deg-
radation of proteins in lysosomes (Mohamed and Sloane,
2006). To validate the effects on lysosomal maturation and
proteolytic activation, we examined cathepsin L activities in
HeLa and MCF7 cells after SNARE knockdown by using a
Magic Red Cathepsin L detection kit, which enables deter-
mination of cathepsin L activity in whole cells with fluores-
cent microscopy (Droga Mazovec et al., 2008; Razi et al.,
2009). As shown in Supplemental Figure 5, fluorescent
signals were clearly observed around the nuclei in control
cells under both fed and starved conditions, whereas they
were apparently attenuated by the addition of lysosomal
protease inhibitors (E64d and pepstatin A). In contrast, flu-
orescence was clearly observed in VAMP7-, VAMP8-, and
Vti1b-depleted cells in a manner similar to the control cells.
These results indicate that lysosomal maturation and pro-
teolytic activation were not diminished by knockdown of
VAMP8 and Vti1b.
Vti1b interacts with Syntaxin 7 and Syntaxin 8 to form the
t-SNARE complex, which mediates the formation of both
late endosomes and lysosomes (Antonin et al., 2000). Thus,
we examined whether Syntaxin 7 and Syntaxin 8 are also
involved in autophagic fusion with lysosomes. It was ob-
served that GFP-LC3-positive GcAVs were clearly merged
with LAMP1 in Syntaxin 7- and Syntaxin 8-depleted cells in
a manner similar to the control cells (Figure 9). In addition,
knockdown of Syntaxin 7 and Syntaxin 8 did not interfere
maturation of canonical autophagosome (Figure 10). These
results indicate that Syntaxin 7 and Syntaxin 8 are not in-
volved in the formation of GcAVs or canonical autophagy.
Collectively, these findings strongly suggest that the com-
binational SNARE proteins VAMP8 and Vti1b also mediate
the fusion of autophagosomes with lysosomes.
Localization of VAMP8 and Vti1b
We also examined the localization of VAMP8 and Vti1b in
terms of canonical autophagy by using tfLC3. At 60 min
after beginning starvation, mRFP-LC3 (autolysosome
marker) accounted for ?15% of the LC3 puncta, whereas
the residual portion was positive for mRFP-GFP-LC3 (auto-
phagosome marker; Supplemental Figure 6A). mRFP-GFP-
LC3 was negligibly colocalized with VAMP8 at 60 min after
beginning starvation, and mRFP-LC3 puncta were clearly
merged with those of VAMP8, indicating that VAMP8 re-
sides in autolysosomes (Supplemental Figure 6, B and C). In
contrast, mRFP-GFP-LC3 was apparently merged with
Vti1b, whereas a few mRFP-LC3 puncta were colocalized
with Vti1b (Supplemental Figure 6, B and C), showing that
Vti1b resides in autophagosomes. These results also indicate
that autophagosome-lysosome fusion is likely mediated by a
somes from VAMP8- and Vti1b-depleted cells. (A) HeLa cells were
transfected with siRNA for the control, VAMP7, VAMP8, and Vti1b.
At 24 h after transfection, the cells were further transfected with
plasmids expressing tf-LC3. After 24 h of incubation, the cells were
subjected to a starved condition for 180 min and then fixed and
observed with a confocal microscope. The boxed regions in the left
panels are enlarged in the right panels. Bars, 10 ?m (left) and 5 ?m
(right). (B) The colocalization frequencies of mRFP with GFP signals
shown as tf-LC3 pixels in A were determined using LSM Image
Browser software (Carl Zeiss) and are presented as the percentage
of total number of mRFP pixels. Values are shown as the mean ? SD
of ?60 cell images. *p ? 0.01 by one-way ANOVA and Scheffe ´’s
Impaired degradation of LC3 proteins in autophago-
VAMP8/Vti1b Mediate Autophagic Fusion
Vol. 21, March 15, 20101007
combination of autophagosome-derived Vti1b and lysoso-
Our results showed that the fusion events of antimicrobial
and canonical autophagosomes with lysosomes are medi-
ated by the combinational SNARE proteins VAMP8 and
Vti1b. In contrast, Syntaxin 7 and Syntaxin 8, which also
function as t-SNAREs, were not shown to be involved in that
fusion. This is the first report of the involvement of SNAREs
in the autophagic process in mammals and several of our
findings are worthy of special mention.
First, it is of interest that the late stages of autophagic
maturation interconnect with the endocytic pathway by
sharing the SNARE machinery, which is known to be in-
volved in homotypic fusion within late endosomes. In this
study, a unique combination of endocytic SNAREs was
shown to function in autophagy, whereas previous studies
have suggested that autophagosomal maturation has several
other features similar to the progression of endosomes to
lysosomes. For example, it was revealed that autophago-
some–endosome fusion depends on Vps4/SKD1 (Nara et al.,
2002), Rab11 (Fader et al., 2008), homotypic fusion and pro-
tein sorting (HOPS) complex (Liang et al., 2008), Hrs (Tamai
et al., 2007), and the ESCRT III complex (Rusten et al., 2007;
Lee et al., 2007), whereas autophagosome-lysosome fusion
was shown to be mediated by Rab7 (Gutierrez et al., 2004;
Jager et al., 2004; Yamaguchi et al., 2009), UV radiation resis-
tance-associated gene (Liang et al., 2008), the HOPS complex
(Lindmo et al., 2006), presenilin (Esselens et al., 2004), and
LAMP2 (Eskelinen et al., 2004). It is also interesting that
Vti1b was found to be recruited to autophagosomes before
lysosomal fusion. Although the mechanism of Vti1b deliv-
ery to autophagosomes remains to be determined, it is an
important issue, as it is possible that it is acquired by fusion
with endosomes. We also found that xenophagosomes and
autophagosomes share the same SNARE machinery to fuse
with lysosomes, even though antimicrobial autophagy against
GAS differs from canonical autophagy in several other aspects,
such as size, morphology, and the conditions required for
initiation (Yoshimori and Amano, 2009). We recently re-
ported that GcAVs are formed through the fusion of multi-
ple isolation membrane like-structures (Yamaguchi et al.,
2009). In spite of these discrepancies, our results indicate
that GcAVs and autophagosomes share the same set of
SNARE machinery to fuse with lysosomes.
The present study also revealed the involvement of
SNAREs in autophagosome–lysosome fusion in mammalian
cells. Phenotypical alteration by deletion of Vti1b was exam-
ined previously using knockout (KO) mice (Atlashkin et al.,
2003). Contrary to expectation, most of the vti1b-KO mice in
that study behaved normally and were indistinguishable
from wild-type mice, with no defects in transport to the
lysosomes. Furthermore, only a limited percentage (20%) of
the mice was physically smaller, whereas multivesicular
bodies and autophagic vacuoles were accumulated in hepa-
tocytes. Compensatory activation/hyperfunction of other
related genes/molecules might have prevented the pheno-
typical alteration in those KO mice. In contrast, the present
knockdown assay clearly showed that an alteration was
caused by the deficiency. In addition, recent studies with
yeast have implicated the SNAREs Vam3 (possible mamma-
lian orthologue for syntaxin7) and Vti1 (mammalian homo-
logue for Vti1a) in the fusion of autophagosomes with vacu-
oles of Saccharomyces cerevisiae (Darsow et al., 1997; Fischer
von Mollard and Stevens, 1999; Ishihara et al., 2001). Mam-
malian Vti1a and Vti1b share 30% of their amino acid resi-
LAMP1 in VAMP8- and Vti1b-depleted cells.
(A) HeLa cells were transfected with siRNA
for the control, VAMP7, VAMP8, and Vti1b.
At 24 h after transfection, the cells were fur-
ther transfected with plasmids expressing
mRFP-LC3. After 24 h of incubation, the cells
were subjected to a starved condition for 180
min, followed by fixation and incubation with
anti-LAMP1 antibodies and then observed
with a confocal microscope. Cellular DNA
was stained with DAPI. Bars, 10 ?m. (B) HeLa
cells were preloaded with Alexa 488 dextran for
marking lysosomes, as described in Materials
and Methods. Cellular DNA was stained with
DAPI. Bars, 10 ?m. (C) The colocalization fre-
quencies of mRFP shown as LAMP1 pixels
were determined using LSM Image Browser
software (Carl Zeiss) and are presented as the
ues are shown as the mean ? SD of ?30 im-
ages. *p ? 0.01 by one-way ANOVA and
Scheffe ´’s posttest. (D) The colocalization fre-
quency of mRFP-LC3 shown as Alexa 488
dextran pixels was determined using LSM
Image Browser (Carl Zeiss) and presented as
the percentage of total number of mRFP pix-
els. The mean value ? SD of ?30 cell images
is shown. *p ? 0.01 by one-way ANOVA and
Scheffe ´’s posttest.
Colocalization of mRFP-LC3 with
N. Furuta et al.
Molecular Biology of the Cell1008
dues with each other (Advani et al., 1998). Therefore, we
examined the effect of Vti1b depletion on expression of Vti1a
protein and confirmed that there were no differences in
regard to Vti1a protein level between the control and Vti1b
knockdown cells (Supplemental Figure 7). Similarly to Syn-
taxin 7 and Syntaxin 8, Vti1a is not likely to be involved in
autophagic fusion with lysosomes.
The involvement of VAMP8 and Vti1b in bacterial in-
vasion of host cells remains unclear. In this study, knock-
down of VAMP8 and Vti1b showed negligible effects on
the invasive efficiency of GAS, suggesting the nonpartici-
pation of these SNAREs in the bacterial invasive event.
However, it was reported previously that VAMP8 is re-
cruited to bacteria-induced membrane ruffles, which facili-
tates invasion of HeLa cells by Salmonella enterica serovar
Typhimurium (Dai et al., 2007). Furthermore, knockdown of
VAMP8 by siRNA reduced the invasion level of Salmonella,
indicating that Salmonella exploits host SNARE proteins and
vesicle trafficking to promote bacterial entry. GAS uses a
different strategy to enter cells as compared with Salmonella
(Cossart and Sansonetti, 2004) and their diverse underlying
mechanisms might be the cause of these discrepant findings.
The involvement of VAMP8 in bacterial invasion requires
In summary, our results present several new aspects to
help unravel the mechanism underlying regulation of auto-
phagic maturation events for the degradation of cargos.
Autophagy is a versatile cellular machinery that has various
physiological roles and its striking evolution has been
shown to exploit, at least in part, interconnections with
household pathway(s) by sharing ordinary machinery such
as SNAREs. Additional investigations will help to elucidate
the mechanism of not only canonical autophagy but also
This research was supported by grants-in-aid for scientific research (B) from
the Ministry of Education, Culture, Sports, Science and Technology, Japan.
taxin 8-depleted cells. (A) HeLa cells were transfected with siRNA
for the control, Syntaxin 7, and Syntaxin 8. At 48 h after transfection,
the cells were lysed and examined by Western blotting using anti-
Syntaxin 7, -Syntaxin 8, and actin antibodies. (B) HeLa cells stably
expressing GFP-LC3 were transfected with siRNA for the control,
Syntaxin 7, and Syntaxin 8. After 48 h, the cells were infected with
GAS for 180 min at an MOI of 100 as described in Materials and
Methods. After fixation, the cells were incubated with anti-LAMP1
antibodies and observed with a confocal microscope. Cellular and
bacterial DNA were stained with DAPI. The boxed regions in the
top panels are enlarged in the bottom panels. Bars, 10 ?m (top) and
5 ?m (bottom). (C) Colocalization frequencies of GcAVs with
LAMP1 signals were manually determined and are presented as the
percentage of total number of GcAVs. Data shown represent results
of ?30 cells.
Antimicrobial effects of GcAVs on Syntaxin 7 and Syn-
and Syntaxin 8-depleted cells. (A) MCF7 cells were transfected with
siRNA for the control, Syntaxin 7, and Syntaxin 8. At 48 h after
transfection, the cells were lysed and then examined by Western
blotting using anti-Syntaxin 7, -Syntaxin 8, and actin antibodies. (B)
MCF7 cells stably expressing GFP-LC3 were treated with siRNA in
the same manner as described in A. The cells were further cultured
in growth medium (Fed) or EBS solution (Starved) for the indicated
times, then fixed and observed with a confocal microscope. Cellular
DNA was stained with DAPI (blue). Bars, 10 ?m. (C) siRNA-treated
MCF7 cells were cultured in Fed and Starved conditions for 240
min, and then the cellular lysates were subjected to Western blotting
using anti-LC3 and actin antibodies.
Maturation of canonical autophagosomes in Syntaxin 7
VAMP8/Vti1b Mediate Autophagic Fusion
Vol. 21, March 15, 2010 1009
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