Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase.
ABSTRACT Autophagy mediates the cellular response to nutrient deprivation, protein aggregation, and pathogen invasion in human. Dysfunction of autophagy has been implicated in multiple human diseases including cancer. The identification of novel autophagy factors in mammalian cells will provide critical mechanistic insights into how this complicated cellular pathway responds to a broad range of challenges. Here, we report the cloning of an autophagy-specific protein that we called Barkor (Beclin 1-associated autophagy-related key regulator) through direct interaction with Beclin 1 in the human phosphatidylinositol 3-kinase class III complex. Barkor shares 18% sequence identity and 32% sequence similarity with yeast Atg14. Elimination of Barkor expression by RNA interference compromises starvation- and rapamycin-induced LC3 lipidation and autophagosome formation. Overexpression of Barkor leads to autophagy activation and increased number and enlarged volume of autophagosomes. Tellingly, Barkor is also required for suppression of the autophagy-mediated intracellular survival of Salmonella typhimurium in mammalian cells. Mechanistically, Barkor competes with UV radiation resistance associated gene product (UVRAG) for interaction with Beclin 1, and the complex formation of Barkor and Beclin1 is required for their localizations to autophagosomes. Therefore, we define a regulatory signaling pathway mediated by Barkor that positively controls autophagy through Beclin 1 and represents a potential target for drug development in the treatment of human diseases implicated in autophagic dysfunction.
- SourceAvailable from: Ewa Toton[Show abstract] [Hide abstract]
ABSTRACT: The Nomenclature Committee on Cell Death (NCCD, 2009) defines different types of cell death on the basis of morphological, enzymological, immunological and functional criteria. Four basic types of cell death are distinguished from the biochemical point of view: necrosis, apoptosis, autophagy and cornification. Autophagy (macroautophagy) is a highly conserved process by which defective organelles, non-functional proteins and lipids become sequestered within structures called autophagosomes, which fuse with lysosomes, and the engulfed components are then degraded by lysosomal enzymes. The role of autophagy is not only the elimination of components, it also serves as a dynamic recycling system that produces new materials and energy for cellular renovation and homeostasis. Beclin-1 is a protein that plays a central role in autophagy; it interacts with multiple cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, IP3R, PINK and survivin) to promote the formation of the Beclin-1-Vps34-Vps15 complex which triggers the autophagy protein cascade. Beclin-1 dysfunction may lead to immune disorders, liver and neurodegenerative diseases as well as cancer. A positive and negative correlation between the expression pattern and/or activity of Beclin-1 and carcinogenesis has been demonstrated. Here we describe recent advances in understanding the molecular dynamics and regulation of autophagy and we discuss Beclin-1's contribution to anticancer therapy.Journal of physiology and pharmacology: an official journal of the Polish Physiological Society 08/2014; 65(4):459-67. · 2.48 Impact Factor
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ABSTRACT: Autophagy and apoptosis are two important catabolic processes contributing to the maintenance of cellular and tissue homeostasis. Autophagy controls the turnover of protein aggregates and damaged organelles within cells, while apoptosis is the principal mechanism by which unwanted cells are dismantled and eliminated from organisms. Despite marked differences between these two pathways, they are highly interconnected in determining the fate of cells. Intriguingly, caspases, the primary drivers of apoptotic cell death, play a critical role in mediating the complex crosstalk between autophagy and apoptosis. Pro-apoptotic signals can converge to activate caspases to execute apoptotic cell death. In addition, activated caspases can degrade autophagy proteins (i.e., Beclin-1, Atg5, and Atg7) to shut down the autophagic response. Moreover, caspases can convert pro-autophagic proteins into pro-apoptotic proteints to trigger apoptotic cell death instead. It is clear that caspases are important in both apoptosis and autophagy, thus a detailed deciphering of the role of caspases in these two processes is still required to clarify the functional relationship between them. In this article, we provide a current overview of caspases in its interplay between autophagy and apoptosis. We emphasized that defining the role of caspases in autophagy-apoptosis crosstalk will provide a framework for more precise manipulation of these two processes during cell death.International journal of biological sciences 01/2014; 10(9):1072-1083. · 4.37 Impact Factor
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ABSTRACT: Salmonella enterica resides within the cytosol or a membrane-bound vacuole in epithelial cells.•Intestinal and gall bladder epithelial cells are sites of cytosolic colonization in vivo.•Cytosolic Salmonella are transcriptionally distinct from vacuolar bacteria.•Epithelial cells containing cytosolic Salmonella are expelled by pyroptosis.Current Opinion in Microbiology 02/2015; 23. · 7.22 Impact Factor
Identification of Barkor as a mammalian
autophagy-specific factor for Beclin 1 and class III
Qiming Suna, Weiliang Fana, Keling Chena, Xiaojun Dingb, She Chenb, and Qing Zhonga,1
aDivision of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; andbNational
Institute of Biological Sciences, Beijing 102206, China
Communicated by Xiaodong Wang, University of Texas Southwestern Medical Center, Dallas, TX, October 17, 2008 (received for review October 10, 2008)
Autophagy mediates the cellular response to nutrient deprivation,
protein aggregation, and pathogen invasion in human. Dysfunction
of autophagy has been implicated in multiple human diseases includ-
ing cancer. The identification of novel autophagy factors in mamma-
lian cells will provide critical mechanistic insights into how this
complicated cellular pathway responds to a broad range of chal-
lenges. Here, we report the cloning of an autophagy-specific protein
that we called Barkor (Beclin 1-associated autophagy-related key
regulator) through direct interaction with Beclin 1 in the human
phosphatidylinositol 3-kinase class III complex. Barkor shares 18%
sequence identity and 32% sequence similarity with yeast Atg14.
Elimination of Barkor expression by RNA interference compromises
starvation- and rapamycin-induced LC3 lipidation and autophago-
some formation. Overexpression of Barkor leads to autophagy acti-
vation and increased number and enlarged volume of autophago-
somes. Tellingly, Barkor is also required for suppression of the
autophagy-mediated intracellular survival of Salmonella typhi-
murium in mammalian cells. Mechanistically, Barkor competes with
UV radiation resistance associated gene product (UVRAG) for inter-
is required for their localizations to autophagosomes. Therefore, we
define a regulatory signaling pathway mediated by Barkor that
positively controls autophagy through Beclin 1 and represents a
potential target for drug development in the treatment of human
diseases implicated in autophagic dysfunction.
Atg14 ? autophagosome ? LC3 ? Salmonella ? UVRAG
phosphatidylinositol 3-kinase (PI3KC3) complex, which also con-
tains a PI3K catalytic subunit and a regulatory subunit (p150) (4).
Beclin 1 was identified as a haploid insufficient tumor suppressor
gene (3). It is monoallelically deleted in ovarian, breast, and
prostate cancers. Heterozygous Beclin 1?/?mice have reduced
autophagy activity and increased incidence of spontaneous tumors
(5, 6). Allelic loss of Beclin 1 leads to genome instability upon
metabolic stress (7, 8). All of this evidence illustrates a role for
Beclin 1 and autophagy in cancer development.
Notably, Beclin 1 and PI3KC3 have pleiotropic functions in
multiple cellular processes. PI3KC3 is not only required for auto-
Functional equivalents of Beclin 1/PI3KC3/p150 in yeast, Vps30/
Atg6-Vps15-Vps34, are known to play a critical role in autophagy
and in vacuolar protein sorting (VPS) (1, 10). The specificity of
PI3KC3 in yeast is determined by different complex compositions.
Two regulatory proteins, Atg14 and Vps38, direct the core PI3K
complex to either the phagophore assembly site (PAS) for auto-
phagy or the endosome for VPS (10, 11), respectively, to execute
their functions in autophagy or VPS. Atg14 is required for medi-
ating the localization of the core PI3KC3 complex to PAS and is
Atg8, Atg16, and the Atg12-Atg5 conjugate to the PAS for mem-
ne of the central regulators of autophagy in mammalian cells
is Beclin 1 (1–3). Beclin 1 is a component of the class III
is responsible for the endosomal localization of the PI3K complex
(11). Surprisingly, such regulatory mechanisms directing PI3KC3
specificity have not been identified in mammals.
How the function of Beclin 1 is specifically directed toward
autophagosomes in mammalian cells has remained elusive. We
speculate that there are autophagy-specific factors mediating
Beclin 1 activity in autophagy. We used a biochemical approach
to purify and proteomic methods to characterize the Beclin 1
complex. Here, we report the identification of a Beclin 1-
associated protein that promotes autophagy specifically through
the interaction with Beclin 1.
Identification of Barkor as a Beclin 1-Interacting Protein. To search
for Beclin 1 regulatory proteins, we generated a cell line from
human osteosarcoma U2OS cells that is stably transfected with
ZZ-Beclin 1-FLAG under the control of doxycycline [supporting
by the titration of doxycycline, and a dose (20 ng/mL) that induces
expression of tagged Beclin 1 close to the endogenous level was
selected (Fig. S1B). The tagged Beclin 1 was purified from cell
extracts by sequential affinity chromatography steps, and the final
excised and analyzed by mass spectrometry. In addition to the
known components of the Beclin 1 complex, namely the PI3K
catalytic subunit, p150 regulatory subunit, and UVRAG, we also
identified a 68-kDa protein by mass spectrometry, KIAA0831 (Fig.
key regulator). We were able to purify the same complex from
human embryonic kidney 293T cells expressing tagged Beclin 1,
(Fig. 1B). Bioinformatic analysis revealed that Barkor contains an
N-terminal zinc finger motif and a central coiled-coil domain
(CCD) (Fig. S2) and a domain organization similar to Atg14 in
similarity with yeast Atg14 (Fig. S3). The identities of these
interacting proteins were further confirmed by immunoblotting
analysis (Fig. S4). Although another Beclin 1-interacting protein,
Bcl-2 (14), could not be visualized by silver staining, its presence in
the final eluate was validated by immunoblotting (Fig. S4). The
interaction of Barkor and Beclin 1 was further confirmed by the
and S.C. contributed new reagents/analytic tools; Q.S., W.F., and Q.Z. analyzed data; and
Q.S. and Q.Z. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
University of California, 316 Barker Hall, Berkeley, CA 94720. E-mail: qingzhong@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 9, 2008 ?
vol. 105 ?
no. 49 ?
reciprocal endogenous coimmunoprecipitation of Barkor and Be-
clin 1 with each other’s antibodies (Fig. 1C).
Barkor Is Important for Efficient Production of PI3P in Vivo. Because
Beclin 1 is a major component of the PI3KC3 complex, we checked
whether Barkor is also a component of this complex. Indeed,
Barkor and Beclin 1 were coimmunoprecipitated with PI3KC3
antibody (Fig. S5), indicating that Barkor is part of the PI3KC3
The interaction between Beclin 1 and PI3KC3 was not affected
by either Barkor-knockdown (Fig. S6 A and B) or overexpression
(Fig. S6C). Because Barkor interacts directly with Beclin 1, we
asked whether Beclin 1 is required for the association between
PI3KC3 and Barkor. Indeed, in Beclin 1-knockdown (Fig. S6D)
1D, lane 7) was dramatically reduced compared with that in Beclin
1-proficient cells (Fig. 1D, lane 3). The amount of PI3KC3 in
Barkor immunoprecipitate in Beclin 1-knockdown cells (Fig. 1D,
lane 8) was also greatly compromised compared with that in Beclin
1-proficient cells (Fig. 1D, lane 4). In summary, Beclin 1 is required
for the interaction between PI3KC3 and Barkor.
To test whether Barkor might regulate PI3KC3 activity, we
knockdown cells. PI3KC3 phosphorylates the 3?-hydroxyl position
of the phosphatidylinositol (PtdIns) ring to produce PtdIns3P
(PI3P) (9). The production of PI3P by PI3KC3 could be visualized
and quantified by fluorescence of the GFP-tagged double FYVE
binds to PI3P, the only end product of PI3KC3, we could measure
PI3KC3 activity by detecting FYVE fluorescence. PI3P production
was diminished in Barkor knockdown cells compared to that in
wild-type cells, and could be further depleted by treatment of the
PI3K inhibitor 3-methyladeline (3-MA) (Fig. 1 E and F).
Barkor Is Required for LC3 Conjugation and Autophagosome Assem-
bly. To demonstrate the role of Barkor in autophagy directly, we
generated doxycycline-inducible RNAi-knockdown cell lines for
marker of autophagy activity is LC3 conjugation to phosphati-
dylethanolamine (PE), which is strongly induced by stimuli such as
starvation or rapamycin treatment (16). The LC3-conjugated form
(also called LC3II) migrates slightly faster than the cytosolic free
form (LC3I). In wild-type cells, the LC3II form was dramatically
increased upon starvation (Fig. 2A, lanes 3 and 7) compared with
that in untreated cells (Fig. 2A, lanes 1 and 5). However, in
Barkor-inducible knockdown cells, the LC3II form was de-
1-knockdown cells (Fig. 2A, lane 4). Similarly, LC3II was
strongly induced in rapamycin-treated wild-type cells (Fig. 2B,
lane 3), but not in the Barkor-knockdown cells (Fig. 2B, lane 7).
Pretreatment with the protease inhibitors pepstatin and E-64D
accumulated the LC3II form in rapamycin-treated (Fig. 2B, lane
4) and untreated (Fig. 2B, lane 2) Barkor-proficient cells, but
had no effect on LC3 conjugation in Barkor-deficient cells (Fig.
2B, lanes 6 and 8). All of these data indicate that Barkor is
essential for LC3 conjugation to PE and for autophagy activa-
tion. Consistently, LC3 puncta were also dramatically compro-
mised in Barkor knockdown cells (Fig. S8).
To visualize autophagosome formation directly, we performed
an electron microscopic analysis. During autophagy, cytoplasmic
components, including proteins and organelles, are engulfed by
double-membrane autophagosomes, which fuse to lysosomal vesi-
cles to form autolysosomes where the contents are degraded into
their components (17). Autophagic vacuoles (AVs) that include
mission electron microscope and are shown as double-membrane
vesicles (autophagosomes) or single-membrane vesicles (autolyso-
somes) that contain intracellular contents including cytosol and
organelles (mitochondria and/or endoplasmic reticulum) (Fig. 2E,
marked by arrows) (17). In Barkor wild-type cells, we observed
abundant AVs in response to nutrient deprivation (Fig. 2 C, E, and
F). AVs were rarely observed in Barkor-knockdown cells (Fig. 2 D
We then asked whether forced expression of Barkor would
stimulate autophagosome formation. For this purpose, we set up
a Barkor stable overexpression (OE) cell line in U2OS, and
All the marked bands were identified by mass spectrometry. (B) A similar Beclin 1 complex was purified from human kidney embryonic HEK293T cells. (C) Reciprocal
1 bridges the interaction between PI3KC3 and Barkor. Beclin 1-knockdown 293T cells or control cells were transfected with FLAG-PI3KC3 and Myc-Barkor. Whole-cell
lysates were immunoprecipitated with anti-FLAG or Myc antibodies and analyzed. (E) Barkor-knockdown decreases the activity of PI3KC3 in vivo. Barkor-knockdown
U2OS cells were transfected with FYVE2-EGFP expression vector. Thirty hours after transfection, cells were treated with 5 mM 3-MA for another 4 h. FYVE2-EGFP was
quantified in F.
www.pnas.org?cgi?doi?10.1073?pnas.0810452105 Sun et al.
autophagic vacuole formation was observed in these cells. The
number of AVs was dramatically increased in Barkor OE cells
(Fig. 2 H–J) compared with that in parental cells (Fig. 2 G and
J). Also, AVs in Barkor OE cells were more heterogeneous, and
we observed a significant amount of large AVs (Fig. 2 H and I).
The average size of AVs in Barkor OE cells was nearly doubled
compared with that in control cells (Fig. 2K). Consistently,
overexpression of Barkor in HEK293T cells led to autophagy
activation, illustrated by increasing amounts of the LC3II form
(Fig. 2L). All of these data demonstrate that Barkor is important
in autophagosome formation and expansion.
Barkor Is Critical for Autophagy-Mediated Bacterial Clearance. Au-
to suppress bacterial infection (18). It has been reported that
infection by Salmonella typhimurium, a causative agent for food
poisoning and typhoid fever, is controlled by autophagy (19–21).
We first asked whether autophagy is required for controlling
bacterial infection in nonphagocytic mammalian cells. Mouse em-
bryonic fibroblasts (MEFs) knocked out of Atg7 (22), an essential
gene for autophagy, were infected with Salmonella marked with
GFP, and uptake of Salmonella was monitored microscopically by
green fluorescence. As expected, Atg7?/?MEFs were more per-
missive for intracellular replication by Salmonella than wild-type
cells, allowing remarkably increased GFP fluorescence in the
cytosol (Fig. 3A). We further performed a quantitative assay to
Atg7-knockout cells compared with wild-type cells (Fig. 3B), con-
firming that autophagy is required for Salmonella amplification in
nonphagocytic mammalian cells.
A similar phenomenon was observed in Barkor-knockdown
cells, namely that there was more bacterial growth when
Barkor protein was eliminated (Fig. 3C). The same quantita-
tive assay for bacterial growth indicated that a 2- to 3-fold
increase in bacterial replication could be detected in Barkor-
deficient over Barkor-proficient cells (Fig. 3D). This result
demonstrates that Barkor is crucial for autophagy-mediated
bacterial elimination in mammalian cells.
Barkor Interacts with Beclin 1 Through CCDs. We performed an
in-depth analysis of the interaction between Barkor and Beclin
1. We constructed a series of vectors that express various
putative structures. Barkor contains an N-terminal zinc finger
motif and a central CCD (Fig. 4A), and Beclin 1 consists of 3
domains: an N-terminal BH3 domain, a central CCD, and an
evolutionarily conserved domain at the C terminus (Fig. 4B)
(23). IP assays showed that all of the Barkor fragments contain-
ing CCD, including CCD alone (Fig. 4A, lanes 2, 4, 5, and 6),
immunoprecipitated Beclin 1, whereas Barkor fragments lacking
CCD failed to bind (Fig. 4A, lanes 3 and 7), demonstrating that
Barkor specifically binds to Beclin 1 through its CCD (Fig. 4A).
Additionally, Beclin 1 specifically interacts with Barkor through
its CCD as well (Fig. 4B).
Barkor and UVRAG Form Mutually Exclusive Complexes with Beclin 1.
UVRAG is a recently identified positive regulator of Beclin 1
(24) and interacts with Beclin 1 through a CCD interaction.
Because the same binding surface of Beclin 1 is used to bind
to both Barkor and UVRAG, we speculated that Barkor and
UVRAG might form mutually exclusive complexes with Beclin
1 through competition. To test this hypothesis, we examined
the direct interaction among Barkor, UVRAG, and Beclin 1
in an in vitro binding assay. In this assay, we purified different
recombinant CCDs of Beclin 1, Barkor, and UVRAG from
Escherichia coli (Fig. S9) and performed in vitro binding
reactions. As shown in Fig. 4C (Bottom), both Barkor CCD
(lane 4) and UVRAG CCD (lane 6) bound to Beclin 1 CCD
directly. Similar experiments were performed by using Barkor
CCD (Fig. S10A) or UVRAG CCD (Fig. S10B) as baits; both
CCDs bind to Beclin 1 but not to each other.
We further investigated whether Barkor and UVRAG form
LC3 conjugation was examined in Beclin 1 and Barkor-knockdown U2OS cells in
complete medium (DMEM ? 10% FBS) or starvation medium (Earle’s balanced
treated with 500 nM rapamycin overnight. Proteases inhibitors (2 ?g/mL E64D
and 2 ?g/mL pepstatin for 4 h) were used to block lysosomal degradation. (C–E).
Electron microscopic (EM) analysis of Barkor-knockdown cells. Both control cells
transmission electron microscopy. (E) High-magnification picture of the framed
area in C showing AVs (marked by arrows) that contain intracellular contents.
[Scale bars: 2 ?M (C), 2 ?M (D), and 1 ?M (E).] (F) AVs per cross-sectioned cell
(mean ? SD; n ? 21) under EM were calculated and summarized. CM, complete
medium. Arrows indicate autophagic vacuole. (G–I) Barkor-overexpression (OE)
U2OS cells (H and I) and U2OS parental cells (G) were observed under EM. (I)
High-magnification picture of the framed area in H shows AVs (marked by
arrows) that contain intracellular contents. [Scale bars: 1 ?M (G–I).] (J) AVs per
cross-sectioned cell under EM were calculated. (K) The average size of AVs in
LC3 conjugation was examined in these cells.
Sun et al. PNAS ?
December 9, 2008 ?
vol. 105 ?
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mutually exclusive subcomplexes with Beclin 1 in vivo. We
performed coimmunoprecipitation experiments to detect Be-
clin 1, Barkor, and UVRAG interactions in vivo. Beclin 1
antibody (Fig. 4D, lane 3) but not control antibody (Fig. 4D,
lane 2) immunoprecipitated with both Barkor and UVRAG.
However, Beclin 1 interacted with both Barkor and UVRAG,
mediated suppression of bacterial replication in
vivo. (A) Atg7?/?and Atg7?/?MEFs cells were
infected with wild-type GFP-marked S. typhi-
anti-tubulin antibody (red). (B) Atg7?/?or
(SL1344) for indicated times. The infected cells
were treated with gentamicin sulfate to block
extracellular bacterial amplification and then
lysed, and internalized bacteria were plated on
plates. (C) Barkor-knockdown U2OS cells were
induced by doxycycline for 2 days and infected
with S. typhimurium as described in A. (D) The
bacterial growth in Barkor-knockdown U2OS
cells was measured as described in B.
Barkor is indispensable for autophagy-
subcomplexes with Beclin 1 through their
CCDs. (A) Barkor binds to Beclin 1 through
its CCD. 293T cells were transfected with
FLAG-Beclin 1, Myc-Barkor, or its Myc-
tagged mutants. Whole-cell lysates (WCLs)
were immunoprecipitated (IP) with anti-
anti-FLAG. § indicates a nonspecific band.
293T cells were transfected with Myc-
Barkor, FLAG-Beclin 1, or its FLAG-tagged
mutants. WCLs were immunoprecipitated
ti-Myc. (C) Direct interaction of Beclin 1
with Barkor or UVRAG. A Ni-column was
incubated first with His-Beclin 1-CC and
then with FLAG-tagged Beclin 1-CC, Bar-
kor-CC, and UVRAG-CC. Proteins bound to
and UVRAG form distinct subcomplexes
with Beclin 1. 293T cells were transfected
with FLAG-UVRAG, Myc-Barkor, and HA-
Beclin 1. WCLs were immunoprecipitated
with anti-FLAG, anti-HA, or Myc, and the
immunoprecipitates were analyzed. (E)
Barkor competes with UVRAG for binding
Beclin 1 in vitro; increasing doses of the
Barkor CCD were then added to the reac-
bound to beads were analyzed by SDS/
PAGE stained with Coomassie blue. (F)
UVRAG competes with Barkor–Beclin 1 in-
teraction in vivo. HA-Beclin 1 was cotrans-
fected into HEK293T cells with Myc-Barkor
noprecipitated with anti-HA followed by
the IB with antibodies against Myc, FLAG,
Barkor and UVRAG form distinct
www.pnas.org?cgi?doi?10.1073?pnas.0810452105 Sun et al.
but no interaction was detected between Barkor and UVRAG
We then asked whether Barkor competes with UVRAG for
1–UVRAG (Fig. 4E) complex formation. Excess amounts of
Barkor CCD were added to the reaction mixture at different
concentrations to compete with UVRAG–Beclin 1 binding. As
expected, UVRAG CCD was displaced from the Beclin 1 complex
in a dose-dependent manner (Fig. 4E). A similar competition assay
was performed in vivo by coimmunoprecipitation. Barkor could be
efficiently coimmunoprecipitated with antibodies against HA-
diminished when UVRAG was overexpressed (Fig. 4F, lane 6).
Therefore, an excess amount of UVRAG could compete with the
Beclin 1–Barkor interaction in vivo. These results indicate that
Barkor and UVRAG interact with Beclin 1 in a mutually exclusive
manner through direct competition.
Subcellular Localization of Barkor Is Regulated by Autophagy Stress.
We first investigated Barkor subcellular localization in human
osteosarcoma U2OS cells transfected with GFP-Barkor. Approxi-
mately 20% of GFP-positive cells displayed a scarce punctate
staining, and the rest showed a diffuse cytoplasmic staining (Fig.
5AI). The percentage of cells containing abundant Barkor foci was
dramatically augmented (?80%) by treatment with the autophagy
inducer rapamycin (Fig. 5AII) or nutrient withdrawal (Fig. 5AIII).
Treatment with the autophagy inhibitor 3-MA converted the
punctate pattern of Barkor to a diffuse cytoplasmic staining (Fig.
5AIV). A statistical analysis of foci per cell or number of cells with
foci was also consistent with the observations (Fig. 5 B and C). The
Barkor punctate staining colocalized nearly perfectly with LC3 in
the unstressed condition (Fig. 5D I–III) or upon rapamycin treat-
predominantly on autophagosomes, which is regulated by
autophagy stimuli. As a control, there was no apparent overlap
between Barkor and the early endosome marker EEA1 before or
after rapamycin treatment (Fig. 5D VII–IX and data not shown).
Barkor Promotes Beclin 1 Translocation to Autophagosomes. Wenext
asked whether Barkor would affect Beclin 1 distribution through
direct interaction. In yeast, Atg6 localizes to the PAS, and this
localization is required for the recruitment of downstream auto-
phagy proteins (11, 12). However, in mammalian cells, Beclin 1
normally localizes to the trans-Golgi network (4) (Fig. 5E I–III). It
Given the location of Barkor on autophagosomes (Fig. 5D), we
speculate that Barkor might promote the translocation of Beclin 1
from the trans-Golgi network to autophagosomes.
We examined the localization of Beclin 1 in the presence of
Barkor expression. When Barkor (GFP-tagged) and Beclin 1
(RFP-tagged) were coexpressed, nearly all Barkor and Beclin 1
proteins were colocalized in cytoplasmic foci (Fig. 5E IV–VI).
LC3 staining (Fig. 5E VII–IX), indicating that Beclin 1 is localized
to the autophagosome. The distribution of Barkor and Beclin 1 on
autophagosomes is mediated by their interaction because a Barkor
detected in transfected U2OS cells upon mock treatment (I), 500 nM rapamycin
Barkor-EGFP punctate staining-positive cells. (D) Colocalization of Barkor and
and then mock treated (I–III) or treated with 500 nM rapamycin (IV–IX) for 12 h.
(I–VI) GFP-Barkor (green) was costained with Myc-LC3 (red). (VII–IX) GFP-Barkor
(green) was costained with endogenous EEA1 (red) (an endosome marker).
Barkor promotes Beclin 1 translocation to autophagosomes through
(E) U2OS cells were transfected with RFP-Beclin 1. (I–III) RFP-Beclin 1 was
costained with endogenous TGN38 (green) (a trans-Golgi network marker).
(IV–VI) U2OS cells were transfected with Barkor-EGFP (green) and RFP-Beclin
1 (red), and fluorescence of Barkor-EGFP (green) and RFP-Beclin 1 (red) was
observed. (VII–IX) U2OS cells were transfected with RFP-Beclin 1, Myc-Barkor,
and GFP-LC3, and fluorescence of GFP-LC3 (green) and RFP-Beclin 1 (red) was
observed. (X–XII), U2OS cells were transfected with RFP-Beclin 1 and Barkor
CCD-deletion mutant-fused EGFP, and the fluorescence of GFP-Barkor CCD
deletion (green) and RFP-Beclin 1 (red) was observed.
Sun et al.PNAS ?
December 9, 2008 ?
vol. 105 ?
no. 49 ?
mutant lacking its CCD failed to localize to autophagosomes and
failed to direct Beclin 1 to autophagosomes (Fig. 5E X–XII).
Therefore, complex formation of Barkor and Beclin 1 is required
for their localization to autophagosomes.
Barkor Promotes Autophagy Through Interaction with Beclin 1.Inthis
work, we reported the purification of the Beclin 1 complex from
and p150, and a known autophagy regulatory protein UVRAG, a
unique protein Barkor has also been identified in this complex.
Barkor interacts with Beclin 1 directly through its central CCD in
a way similar to the Beclin 1–UVRAG interaction. Consequently,
with Beclin 1 and actually form distinct complexes in mammalian
knockdown of this protein from mammalian cells compromises
their ability to activate autophagy in response to nutrient
deprivation and bacterial infection. Overexpression of Barkor
leads to autophagy activation and augmentation of autopha-
gosome formation. Finally, the Barkor–Beclin 1 interaction is
required for their localization to autophagosomes.
Barkor Could Be the Mammalian Functional Ortholog of Atg14 in
Yeast. Based on the sequence alignment and functional similarity,
Barkor is a good candidate to be the mammalian functional
ortholog of Atg14, the autophagy-specific regulatory factor for
Atg6/Beclin 1 in yeast (10, 11). Both Barkor and Atg14 possess a
zinc finger motif at the N terminus and a central CCD. Barkor also
shares 18% sequence identity and 32% sequence similarity with
yeast Atg14 (Fig. S3). Critically, both Barkor and Atg14 direct
Beclin 1/Atg6 to the autophagosome.
It is interesting to note that Barkor competes with UVRAG for
its binding to Beclin 1, similar to the interplay between Atg14 and
Vps38 in yeast. Coincidentally, a recent study suggests that
UVRAG is involved in late endosome fusion with the lysosome, a
phenomenon equivalent to vacuolar protein sorting in yeast,
through its interaction with the HOPS/Vps C complex (25). It is
possible that Barkor and UVRAG mediate the activity of Beclin 1
in autophagy and vacuole protein sorting, respectively. However,
evidence for the UVRAG role in autophagy (24) also demands an
autophagosome formation and late autophagosome/lysosome fu-
field. The identification of Barkor and 2 other factors in the Beclin
1 complex will provide an opportunity perhaps to allow in vitro
reconstitution of PI3K function and autophagosome formation.
Materials and Methods
The full-length cDNAs of human Barkor (KIAA0831), Beclin 1, UVRAG, and
Barkor knockdown is GATCCCCGAAGGAAAGGTTAAGCCGATTCAAGA-
GATCGGCTTAACCTTTCCTTCTTTTTA. The rest of the information about re-
agents, cell lines, cell lysates preparation, tandem affinity purification, coim-
munoprecipitation, immunostaining, electronic microscopy, autophagy
analysis, and bacterial infection is listed in SI Experimental Procedures.
ACKNOWLEDGMENTS. We thank all of the Zhong laboratory members for
(Stanford University School of Medicine) for reagents; Xiaodong Wang, Robert
critical reading of the manuscript; Nick V. Grishin at the University of Texas
Southwestern Medical Center for a bioinformatic analysis of Barkor; Dr. Yumay
at Shanghai for Barkor and Beclin 1 antibody production. The work was sup-
ported in part by a New Investigator Award for Aging from the Ellison Medical
Foundation (to Q.Z.).
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