BIG1, a Brefeldin A–Inhibited Guanine Nucleotide-Exchange
Protein Modulates ATP-Binding Cassette Transporter A-1
Trafficking and Function
Sisi Lin, Chun Zhou, Edward Neufeld, Yu-Hua Wang, Suo-Wen Xu, Liang Lu, Ying Wang,
Zhi-Ping Liu, Dong Li, Cuixian Li, Shaorui Chen, Kang Le, Heqing Huang, Peiqing Liu, Joel Moss,
Martha Vaughan, Xiaoyan Shen
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Objective—Cell-surface localization and intracellular trafficking are essential for the function of ATP-binding cassette
transporter A-1 (ABCA1). However, regulation of these activities is still largely unknown. Brefeldin A, an uncompetitive
inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of
ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1
trafficking and cholesterol efflux, and further to explore the potential mechanism.
Methods and Results—By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles
preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells, and inhibited by 60% cholesterol
release. In contrast, BIG1 overexpression increased surface ABCA1 and cholesterol secretion. With partial restoration of
BIG1 through overexpression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione
cleavage revealed that BIG1 small interfering RNA dramatically decreased the internalization and recycling of ABCA1.
This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation
of ADP-ribosylation factor 1.
Conclusion—BIG1, through its ability to activate ADP-ribosylation factor 1, regulates cell-surface levels and function of
ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action. (Arterioscler Thromb Vasc
Biol. 2013;33:e31-e38.)
Key Words: ABCA1 ◼ BIG1 ◼ cholesterol efflux ◼ trafficking
T
sites is exquisitely controlled, and is essential for regulation
of intracellular cholesterol. Decreasing ABCA1 internalization by deletion of its PEST sequence leads to decreased
cholesterol efflux from late endosomal cholesterol pools.10
Overexpression of the small GTPase Rab8, which is involved
in trafficking from the trans-Golgi network (TGN) to the PM
and from the endosomes to the PM, facilitates ABCA1 surface
expression and stimulates the delivery of cholesterol from late
endosomal compartments to apolipoprotein A-I (apoA-I).11,12
However, the processes regulating specific subcellular localization of ABCA1 are still largely unknown.
Brefeldin A (BFA), an uncompetitive inhibitor of ADPribosylation factor (ARF)–guanine nucleotide-exchange
factor (GEF) activity, has been reported to disturb the
dynamics and intracellular distribution of ABCA1, and thus
inhibit cholesterol efflux.6,13,14 Through binding the ArfGDP-ARF–GEF complex, BFA locks it in a conformation
that prevents nucleotide dissociation, resulting in specific
he ATP-binding cassette transporter A-1 (ABCA1)
belongs to the ATP-binding cassette superfamily of integral membrane proteins that are responsible for the ATPpowered translocation of cholesterol, phospholipids, and other
substrates across membranes.1 The discovery that mutations in
the ABCA1 gene cause Tangier disease and familial hypoalphalipoproteinemia clearly demonstrated ABCA1 as the
key protein responsible for nascent high-density lipoprotein
(HDL) particle, and a critical molecule regulating an initial
step of reverse cholesterol transport.2,3 Thus, ABCA1 has been
considered as a potential target in the treatment of atherosclerotic vascular disease.4
ABCA1 mostly localizes to the plasma membrane (PM)
and endocytic vesicles, and rapidly shuttles between the
cell surface and intracellular compartments.5,6 The importance of its PM localization for proper ABCA1 function
has been confirmed by a number of independent studies.7–9
Trafficking of ABCA1 between specific intracellular and PM
Received on: July 16, 2012; final version accepted on: November 12, 2012.
From the Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China (S.L., C.Z.,
Y-H.W., L.L., Y.W., Z.-P.L., D.L., C.L., S.C., H.H., P.L., X.S.); Cardiovascular and Pulmonary Branch (S.-W.X., K.L., J.M., M.V.), Laboratory of Cardiac
Energetics (E.N.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD; and Pharmacy Department, Zhejiang Provincial
People’s Hospital, Zhejiang, China (S.L.).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.300720/-/DC1.
Correspondence to Xiaoyan Shen, MD, PhD, Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University,
No. 132, East Wai-Huan Rd, College Town, Guangzhou 510006, PR China. Email [email protected]
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
e31
DOI: 10.1161/ATVBAHA.112.300720
e32 Arterioscler Thromb Vasc Biol February 2013
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
blockade of ARF activation by a subset of its GEFs, and thus
interferes reversibly with vesicular transport between the cell
surface and intracellular compartments.15,16 There are 3 BFAsensitive GEFs: brefeldin A-inhibited guanine nucleotideexchange protein (BIG) 1, BIG2, and Golgi-specific brefeldin
A-resistance guanine nucleotide exchange factor 1 (GBF1).
BIG1 and BIG2 are 74% identical in amino acid sequence and
90% identical in Sec7 domains that are responsible for ARF
activation. Both BIG1 and BIG2 are associated mainly with
the regulation of trafficking through TGN and endosomes,17,18
whereas GBF1 functions primarily in transport between ER–
Golgi intermediate and cis-Golgi compartments.19 Therefore,
we hypothesized that BIGs might play a role in regulating
ABCA1 transport and function.
Here, we present evidence that BIG1 is associated with
ABCA1 in vesicles from rat liver. BIG1, but not BIG2, was
required for trafficking and surface localization of ABCA1.
Moreover, we demonstrated that BIG1 activity was crucial
for ABCA1 function in cholesterol efflux. This study evaluated whether BIG1-mediated vesicle trafficking represents
a transcription-independent mechanism for regulation of
ABCA1 function.
Materials and Methods
A detailed Materials and Methods section is presented in the onlineonly Data Supplement.
Vesicle Immunoprecipitation
Membranes from rat liver were subjected to differential centrifugation. Fractions containing vesicles enriched in clathrin and adaptor
protein-1 were used for vesicle immunoprecipitation (IP) by magnetic Dynabeads (Dynal).
Labeling and Tracing of ABCA1
Cell-surface proteins were biotinylated with EZ-Link Sulfo-NHS-SSBiotin (Pierce) and isolated by incubating with streptavidin–agarose
beads (Pierce). Biotinylated proteins remaining on the cell surface
after internalization or recycling were cleaved by reduced glutathione
(pH 8.0).
Results
Association of ABCA1 With BIG1
In Vivo and In Vitro
Because BFA, an inhibitor of BIG1 and BIG2 GEF activity,
was reported to inhibit cholesterol efflux by disturbing intracellular trafficking of ABCA1,6,13,14,20 we hypothesized that BIGs
might play a role in regulating ABCA1 transport and function. BIGs have been found in trans-Golgi compartments and
colocalized with clathrin and adaptor protein-1.21,22 To test the
hypothesis, membrane fractions containing vesicles enriched
in clathrin and adaptor protein-1 from rat liver were immunoprecipitated with anti-BIG1 or BIG2 antibodies. ABCA1 was
detected in the vesicle preparation by anti-BIG1 pull-down
(Figure 1A), indicating colocalization of the 2 proteins in the
same vesicle complex.
Sustained uptake of cholesterol by human macrophages
upregulates ABCA1 expression.23,24 ABCA1 levels in liver
cells, but not macrophages, modulate susceptibility to atherosclerosis.25 To explore the possible mutual regulation of
ABCA1 and BIG1 in hyperlipidemia, apoE−/− mice with
their genetic abnormalities in lipid metabolism were used.
As reported,26 atorvastatin dose-dependently reduced serum
lipid content (serum total cholesterol, triglycerides, and lowdensity lipoprotein [LDL] cholesterol) in apoE−/− mice
fed with high-fat diet (Figure IA in the online-only Data
Supplement). Hepatic levels of ABCA1 and BIG1 were significantly higher in apoE−/− mice than wild-type (WT) mice, and
were decreased by atorvastatin in a dose-dependent fashion
(Figure IB in the online-only Data Supplement). The concurrent changes in amounts of BIG1 and ABCA1 in liver together
with the co-IP suggests a close relationship between the two
proteins in vivo.
Oxidized low-density lipoprotein (oxLDL) has been implicated in the pathogenesis of atherosclerosis, and reported to
upregulate ABCA1 gene expression through activation of
the nuclear liver X receptors.27 In HepG2 cells, ABCA1 and
BIG1 protein expression increased significantly after addition of oxLDL. Upregulation as a result of oxLDL exposure
was inhibited in a dose-dependent manner by atorvastatin
(Figure 1B), but atorvastatin had no effect on the expression
of BIG1 or ABCA1, without oxLDL treatment (Figure 1C).
In BIG1-depleted cells, oxLDL did not alter BIG1 expression, and the increase in ABCA1 by oxLDL stimulation was
partly inhibited (Figure 1D). ApoA-I has been demonstrated
also to increase ABCA1 levels by binding to and preventing degradation of the ABCA1 transporter.28,29 Treatment
of cells with apoA-I increased ABCA1 without affecting
BIG1 level (Figure 1E). These observations suggest that
the expression of ABCA1 and BIG1 were modulated by
cell lipid content, which differs from the apoA-I–ABCA1
interaction.
Function of ABCA1 in HepG2 Cells
Requires BIG1, but not BIG2
To explore roles of BIG1 and BIG2 in apoA-I–ABCA1-dependent cholesterol efflux, we used small interfering RNA to
deplete BIG1 or BIG2 in HepG2 cells (Figure 2A). As shown
in Figure 2B, only BIG1 small interfering RNA reduced cholesterol efflux to apoA-I (≈60% decrease) by HepG2 cells.
Similarly, decreased release of cellular c­ holesterol into HepG2
cell culture medium was observed only in BIG1-depleted cells
by Amplex Red cholesterol Assay (Figure 2C). Cholesterol
accumulation was evaluated by staining fixed cells with filipin,
a specific marker for unesterified cholesterol. We observed a
significant accumulation of filipin staining in BIG1-depleted
cells compared with Mock, nontargeting, or BIG2-depleted
cells (Figure 2D), corresponding to the decreased efflux
(Figure 2B). The amount of apoA-I present in cells and in
medium was also detected by Western blot. In BIG1-depleted
cells, both cell-associated and medium apoA-I were decreased
(Figure 2E). The mRNA level of apoA-I was also measured by
real-time PCR. Our result revealed that apoA-I mRNA slightly
increased in BIG1-depleted cells, but failed to reach statistical significance (Figure 2E; P>0.05). Together, these findings are consistent with a role for BIG1 in ABCA1-mediated
lipidation of apoA-I.
Lin et al BIG1 Regulates ABCA1 Trafficking and Function e33
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Figure 1. Association of ATP-binding cassette transporter A-1 (ABCA1) with brefeldin A–inhibited guanine nucleotide-exchange protein
(BIG) 1 in vivo and in vitro. A, Membranes from rat liver were subjected to differential centrifugation. Markers of vesicles clathrin and
adaptor protein-1 (AP-1) (adaptin γ) in the precipitates recovered by centrifugation at 19 000g (F1) or 43 000g (F2) were detected by
Western blotting (A1). Samples from F2 were used for immunoprecipitation (IP) by anti-BIG1 antibody, followed by Western blotting with
antibodies against BIG1 or ABCA1 (A2), and electron microscopic image (A3, scale bar: 100 nm). B and C, HepG2 cells stimulated with
(B) or without oxidized low-density lipoprotein (oxLDL; C, 50 μg/mL) were treated with atorvastatin (Ato, L: 2 μmol/L, H: 10 μmol/L) for 24
hours. The expression of ABCA1 and BIG1 were detected. D, After BIG1 depletion by small interfering RNA, HepG2 cells were incubated
with oxLDL (50 μg/mL), and the expression of ABCA1 and BIG1 were detected. E, The expression of ABCA1 and BIG1 in HepG2 cells
stimulated by apoA-1 (10 μg/mL) for 8 hours were detected. Results are representative of 3 independent experiments. Densitometric
quantification was analyzed by ImageQuantTL software. α-tubulin was used as the loading control. Data are means±SEM. #, $ indicates
P<0.05; **, ##, P<0.01; and ***, ###, P<0.001.
BIG1 Is Required for Targeting
of ABCA1 to the PM
Because localization of ABCA1 at the PM is vital for its
lipid transport function, we hypothesized that BIG1 may
play a role in targeting of ABCA1 to the PM. As shown in
Figure 3A, surface ABCA1 of HepG2 or differentiated THP1
cells decreased significantly after BIG1-depletion, but total
ABCA1 content was unchanged. To further verify our hypothesis, we overexpressed BIG1 in nontreated or BIG1-depleted
cells. Expression of green fluorescent protein-BIG1 (WT)
increased cell-surface ABCA1 without affecting the total content of ABCA1. In contrast, overexpression of hemagglutininBIG1 (E793K), a dominant-negative mutant, which does not
activate ARFs, had no effect on total amount or the surface
level of ABCA1 (Figure 3B). In addition, overexpression of
BIG1-WT partially reversed the reduction in surface ABCA1,
in cells treated by BIG1 small interfering RNA (Figure 3C).
Consistent with increased content of cell-surface ABCA1
after BIG1-WT expression, an increase in the amount of cholesterol in the culture medium was observed after BIG1-WT,
but not hemagglutinin-BIG1 (E793K), overexpression
(Figure 3D). A reverse situation was seen in BFA-treated
cells (Figure 3E). The medium cholesterol content correlated with the level of cell-surface ABCA1. Differences
between effects of BIG1-E793K overexpression and BIG1
depletion on surface ABCA1 expression and secreted cholesterol could be caused by low efficiency of plasmid
transfection.
BIG1 Depletion Inhibited ABCA1
Internalization and Recycling
ABCA1/apoA-I retroendocytosis was reported to contribute
significantly to HDL formation, when cells had accumulated excess cholesterol.30 To further elucidate the intracellular pathways of ABCA1 trafficking after BIG1-depletion,
the internalization and recycling of surface ABCA1 were
traced. Cell-surface proteins were biotinylated and allowed
to internalize for indicated time. Proteins remaining on the
cell surface were cleaved by reduced glutathione. Internalized
biotinylated proteins were isolated by streptavidin–agarose
beads. As shown in Figure 4A, the internalized ABCA1 in
BIG1-depleted cells was much less than in nontargeting
control cells, indicating a dramatically decreased capability
of internalization in BIG1-depleted cells. Furthermore, the
e34 Arterioscler Thromb Vasc Biol February 2013
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Figure 2. Brefeldin A–inhibited guanine nucleotide-exchange protein (BIG) 1, but not BIG2, depletion impaired the function of ATPbinding cassette transporter A-1 (ABCA1). A, HepG2 cells were incubated with vehicle (Mock) or with non-targeting control, BIG1, or
BIG2 small interfering RNA for 72 hours, followed by immunoblotting to detect RNA interference efficiency. B, HepG2 cells treated with
vehicle, or nontargeting, BIG1, or BIG2 small interfering RNA were incubated with [H3]-labeled cholesterol, followed by efflux to apoA-I (10
μg/mL). Mock was used as a control (set at 100%). n=3; ***P<0.001. C, After nontargeting, BIG1, or BIG2 small interfering RNA treatment,
cholesterol content in condition medium was measured by the Amplex Red Cholesterol Assay Kit (n=6, ***P<0.001). D, Cholesterol accumulation in HepG2 cells after small interfering RNA was displayed by filipin-labeling. The images were taken using a confocal microscope
(Zeiss 710). E, HepG2 cells were incubated with vehicle (Mock) or with nontargeting or BIG1 small interfering RNA for 72 hours. The levels
of apoA I in total cell lysates (T) or culture medium (S) after trichloroacetic acid (TCA) precipitation were detected by Western blotting. The
levels of BIG1 and apoA-I mRNA were detected by real-time PCR. ***P<0.001 vs non-targeting.
amount of ABCA1 recycling back to the cell surface was also
reduced by BIG1-depletion. Although both internalization and
recycling were inhibited by BIG1 depletion, some ABCA1
was still transported between the PM and intracellular compartments (Figure 4B). As ABCA1 was reported to cycle
intracellularly via a Rab4- or Rab8-mediated pathway,12,31 it
is conceivable that BIG1 participates in only one part of the
whole recycling process.
In HeLa cells without endogenous ABCA1, stably expressed
ABCA1-green fluorescent protein resides on the surface and
also in intracellular endosomes (Figure III in the online-only
Data Supplement). Depletion of endogenous BIG1 resulted in a
loss of filipin-labeling in endosome-like clusters and exhibited
much greater accumulation of intracellular cholesterol (Figure
4C), suggesting that mobilization of cellular ABCA1-green
fluorescent protein through BIG1 contributed significantly to
cholesterol release. Sucrose gradient fractionation also revealed
that BIG1 depletion significantly affected the distribution of
ABCA1 and late endosome marker Rab7, but had no effect
on early endosome marker EEA1, recycling endosome marker
transferrin receptor, or lysosome marker lamp2. ABCA1 in
fractions 2, 3, and 6 in which enriched EEA1 and Rab7 were
absent in BIG1-depleted cells indicated that the recycling of
ABCA1 was partly eliminated (Figure IV in the online-only
Data Supplement).
Regulation of BIG1 in Surface Localization
and Function of ABCA1 Was Achieved
Through Activation of ARF1
BIG1 functions as an activator of ARFs, which are major
regulators of membrane remodeling and protein trafficking in
eukaryotic cells. Our results from BIG1-E793K overexpression
and BFA inhibition indicated that effects of BIG1 on the surface localization and function of ABCA1 were dependent on
its GEF activity. To determine whether the activation of ARFs
is truly indispensable for ABCA1 function, we compared the
distribution of ARF (1, 3, 4, 5, and 6) between cytosol and
membrane of HepG2 cells treated with nontargeting, or specific BIG1 or BIG2 small interfering RNA, or vehicle alone
(Mock). Only Arf1, which localizes primarily to the Golgi
apparatus, known to regulate both anterograde and retrograde
vesicular traffic,32 was found to be decreased in membrane
fractions after BIG1 depletion (Figure 5A). To assess the
potential involvement of ARF1 in the BIG1–ABCA1 interaction, constructs of WT and 2 mutants of ARF1 with myc tags
were prepared for transient overexpression. Cells overexpressing ARF1-Q71L, which is assumed to be constitutively active
with GTP-bound, or WT-ARF1, showed increased surface
ABCA1. In addition, ARF-Q71L stimulated a much greater
surface distribution of ABCA1 than ARF1-WT. However, in
cells expressing inactive ARF1-T31N, surface ABCA1 was
not affected. No change was seen in total ABCA1 after plasmid transfection. Cholesterol secreted in the culture medium
was also measured using the Amplex Red Cholesterol Assay
Kit. An increased cholesterol content was found in cells overexpressing Arf1-WT and Arf1-Q71L (Figure 5C). There were
no significant differences in the levels of total cell cholesterol.
Discussion
ABCA1 is widely expressed in nearly all tissues of the
body, and is particularly abundant in the liver. Findings
from studies of hepatic overexpression of ABCA1 suggested
that the liver itself is a major source of cholesterol for both
Lin et al BIG1 Regulates ABCA1 Trafficking and Function e35
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Figure 3. Brefeldin A–inhibited guanine nucleotide-exchange protein (BIG) 1 regulated level of cell surface ATP-binding cassette
transporter A-1 (ABCA1). A, After BIG1 or BIG2 depletion, surface
proteins of HepG2 cells (left) or differentiated THP-1 cells (right)
were biotinylated and separated by streptavidin beads. The levels
of ABCA1 and BIG1 were detected by Western blot (S: cell surface
proteins; T: total cell proteins). Densitometric quantification was
analyzed by ImageQuantTL software. Data are means±SEM (n=3).
***P<0.001 vs non-targeting. B, HepG2 cells were overexpressed
with empty vector of EGFP-C2 (empty vector) or BIG1 plasmids
(wild-type (WT): EGFP-WT BIG1; E793K: HA dominant-negative
BIG1). The surface proteins were isolated and analyzed as in Figure 3A. Data are means±SEM (n=3). ** indicates P<0.01 vs empty
vector. C, HepG2 cells treated with BIG1 small interfering RNA
were transiently overexpressed with hemagglutinin (HA)-BIG1WT, followed by ABCA1 and BIG1 detection by Western blotting
and densitometric quantification. Data are means±SEM (n=3).
***P<0.001; #P<0.05. D and E, HepG2 cells overexpressing WTBIG1 or BIG1-E793K (D), or treated with brefeldin A (BFA; 5 μg/
mL, 8 hours). The secreted cholesterol in condition medium were
detected by Amplex Red analysis; n=3, *P<0.05.
plasma HDL acceptors and nascent HDL particles, which
mediate cholesterol efflux from peripheral cells.33,34 More
important, results from liver-specific ABCA1-knockout mice
demonstrated that hepatic ABCA1 is the single most important
source of nascent apoA-I, and maintains the majority of
the plasma HDL pool.35 Studies using ABCA1-knockout
bone marrow transplanted into WT mice demonstrated that
ABCA1-mediated cholesterol efflux from macrophages did
not significantly contribute to plasma HDL cholesterol level.36
These combined results indicate that appropriate regulation
of hepatic ABCA1 is critical for a selective increase in HDL
cholesterol levels. It is thus important to understand how the
expression and function of ABCA1 is regulated in hepatocytes.
In our study, we used the human hepatoma cell line HepG2
as a model system, combined with rat liver and apoE–/– mice,
to identify BIG1 as the novel regulator of ABCA1, and to
demonstrate the underlying mechanism.
BFA, often used as an inhibitor of vesicle trafficking
between the TGN and PM, inhibits the activity of ARF-GEFs
and arrests ARFs in the GDP-bound form.37 It was reported
that BFA disturbed the dynamics and intracellular distribution
of ABCA1,6 and inhibited apoA-I–mediated lipid efflux from
macrophages and fibroblasts.20 Therefore, we hypothesized
that BIGs, BFA-sensitive ARF-GEFs, might play a role in
regulating ABCA1 transport and function. To identify BIGs
involvement in the regulation of hepatic ABCA1 function,
vesicles isolated from rat liver were immunoprecipitated
with anti-BIG1 or BIG2 antibodies. Interestingly, ABCA1
was present in vesicles precipitated with the anti-BIG1, but
not anti-BIG2, antibodies, indicating a potential interaction
between the 2 proteins. It has been clearly established that
regulation of ABCA1 expression is primarily via cellular
cholesterol level, through the nuclear receptor liver X
receptor.38 To explore the possible regulation of BIG1 in
hepatic ABCA1 function, the relationship between varying
BIG1 and ABCA1 expression in liver was observed. In apoE−/−
mice fed with high-fat diet, increased hepatic ABCA1 and
BIG1 were found along with high serum lipid content (serum
total cholesterol, triglycerides, and LDL cholesterol). When
serum lipids were decreased by atorvastatin treatment, both
ABCA1 and BIG1 were downregulated. Consistent with in
vivo findings, high concentrations of oxLDL increased both
ABCA1 and BIG1 proteins in HepG2 cells. Increases in
ABCA1 and BIG1 were counteracted by atorvastatin treatment
in a dose-dependent manner. BIG1 depletion, however, had no
effect on total amount of ABCA1, but reduced the elevation
of ABCA1 induced by oxLDL. Concurrently increased or
decreased levels of BIG1 and ABCA1, together with the
co-IP of ABCA1-BIG1 in hepatic vesicles, identified a novel
interaction between the 2 proteins. Regulation of ABCA1 at
the PM and endocytic compartments is important for lipid
processing because the functionality of ABCA1 at these
sites is considered essential for the initiation of cholesterol
removal from cells.5,6 We showed that BIG1 overexpression
increased levels of surface ABCA1, and facilitated HepG2
cells to discard excess cholesterol when exposed to oxLDL.
In BIG1-depleted cells with partial restoration of BIG1
through overexpression, ABCA1 localization at the PM was
also restored, consistent with a role for BIG1 in the process.
Recycling of ABCA1 from endocytic compartments to the
PM was markedly reduced in BIG1-depleted cells. Because
of these findings and the discovery of the interaction between
BIG1 and ABCA1, it seems likely that BIG1 plays a significant
role in the pathophysiology of atherosclerosis.
BIG1 is often found in trans-Golgi compartments and colocalizes with clathrin and adaptor protein-1.21,22 Silencing of
BIG1 expression can halt protein trafficking at sites where it
is required to activate ARFs, and thereby initiate vesicle generation for the next stage of transport to the cell surface. Our
previous studies revealed that BIG1 is required for integrity
of the Golgi structure.39 Kinesin family member 21A, a plusend–directed motor protein that moves cargo on microtubules
away from TGN, was identified as a novel binding partner of
e36 Arterioscler Thromb Vasc Biol February 2013
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Figure 4. Effect of brefeldin A–inhibited guanine nucleotide-exchange protein (BIG) 1 depletion on internalization and recycling of surface ATPbinding cassette transporter A-1 (ABCA1). A, Internalized, biotinylated ABCA1 (ABCA1-In) in the control of BIG1-depleted cells at indicated
time points was separated, followed by Western blot detection. B, After internalization at 37°C for 1 hour, biotinylated proteins in the control or
BIG1-depleted cells were recycled at 37°C for the indicated time. The intracellular remaining biotinylated ABCA1 (IC) or the total biotinylated
ABCA1 (IC+S, including that on the surface) after recycling was detected. Transferrin receptor was used as a control. Densitometric quantification normalized to transferrin receptor was analyzed by ImageQuantTL software. Data are means±SEM (n=3). C, HeLa cells stably expressing
wild-type (WT)-ABCA1-green fluorescent protein (GFP) were transfected with BIG1 small interfering RNA, followed by filipin staining.
BIG1. The newly recognized interaction integrates functions
of BIG1 in local vesicle formation with longer range transport processes toward the cell surface.40 Thus, it appears that
BIG1 regulates ABCA1 cell-surface expression via governing
its internalization and trafficking. It has been clearly established that the PM is an important site at which ABCA1 plays
an antiatherogenic role.7–9 Internalization and trafficking of
ABCA1 is functionally important in mediating ABCA1 cellsurface expression, as well as cholesterol efflux from intracellular cholesterol pools.41 Like the other cell-surface proteins,
once ABCA1 arrives at the TGN, it is sorted for delivery over
long distances to the PM by large membrane carriers, in which
BIG1 has been found to be involved.40 Consistent with the
role of BIG1 in that process, our results from BIG1 depletion or overexpression revealed that BIG1 is required to maintain surface ABCA1 levels and cholesterol efflux to apoA-I.
Further, results from tracing the internalization and recycling
of surface ABCA1 after BIG1-depletion revealed a dramatic
inhibition of the capability of internalization and recycling. In
HeLa cells stably expressing ABCA1-green fluorescent protein, depletion of endogenous BIG1 resulted in loss of endosome-like filipin-labeling clusters and exhibited much more
intracellular cholesterol accumulation, consistent with the
findings from internalization and recycling. However, some
ABCA1 was still transported between the PM and intracellular compartments after BIG1 depletion, indicating that other
pathways also participate in the recycling of ABCA1, such as
the Rab4- or Rab8-mediated pathway.12,31
In the present study, the effects of BIG1 on cell-surface
ABCA1 expression and cholesterol secretion is dependent on
its GEF activity, as dominant-negative BIG1 overexpression
did not increase surface ABCA1 levels and promote
cholesterol secretion, as did WT BIG1. Previous studies
reported that BIG1 preferentially functions as a GEF of class
I ARFs (ARF1 and ARF3), by catalyzing replacement of
ARF-bound GDP with GTP.42 We found that BIG1 depletion
decreased membrane binding by ARF1 in HepG2 cells,
consistent with decreased ARF1 activity. Overexpression of
WT ARF1 resulted in increased cell-surface ABCA1, which
was even more enhanced by the constitutively active ARF1
(Q71L) mutant, suggesting that ARF1 activation is important
Figure 5. The activation of ADP-ribosylation factor 1 (ARF1) by
brefeldin A–inhibited guanine nucleotide-exchange protein (BIG)
1 is required for the surface expression and function of ATPbinding cassette transporter A-1 (ABCA1). A, Proteins in cytosol
and membrane fractions were isolated from HepG2 cells incubated with nontargeting control, BIG1, or BIG2 small interfering
RNA, followed by immunoblotting with indicated antibodies.
B, Surface ABCA1 from HepG2 cells transfected with plasmids
of empty vector, myc-ARF1-wild-type (WT), or with Q71L or
T31N ARF1 mutants were detected. C, Cellular cholesterol
content and secreted cholesterol in medium were detected,
respectively, after ARF1 WT or mutant overexpression. Data are
means±SEM; n=3; *P<0.05; **P<0.01.
Lin et al BIG1 Regulates ABCA1 Trafficking and Function e37
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
for ABCA1 surface localization regulated by BIG1. Actually,
ARF-like 7 has been found to be involved in transport between
the perinuclear compartment and the PM, apparently linked
to the ABCA1-mediated cholesterol secretion pathway.43
Nevertheless, the dominant-negative ARF1 (T31N) exhibited
no obvious suppression of surface ABCA1 or cholesterol
secretion, perhaps because of low levels of the mutant
proteins. Both BIG1 and ARF1 have been reported to function
in the Golgi complex and clathrin-coated vesicles.21,22,44 The
ABCA1/apoA-I complex is also endocytosed via the clathrin
pathway.30 It appears that the newly synthesized ABCA1 is
transported to the surface in the same manner. Clearly, further
studies are required to evaluate these possibilities.
In summary, the present work identifies BIG1 as an important intracellular regulator that mediates cell-surface ABCA1
expression, and facilitates cholesterol efflux to apoA-I by
governing its intracellular trafficking. This novel function
of BIG1 requires GEF activity to accelerate activation of
ARF1. Interestingly, a xLxxKN motif serves as a Golgi exit
signal and directs ABCA transporters to a post-Golgi vesicular sorting station, where additional signals may be required
for selective delivery of individual transporters to final subcellular destinations.45 Thus, BIG1 regulation may be related
to xLxxKN motif, which requires further research. Our study
represents a transcription-independent mechanism for regulating ABCA1 function.
Sources of Funding
This study was supported by National Natural Science Foundation
of China (No. 31070924 and 81173056), Projects of International
Cooperation and Exchanges, Science and Technology Planning Project
of Guangdong Province, China (No. 1011420600004), and Research
Fund for the Doctoral Program of Higher Education of China (No.
20100171110052). Edward Neufeld, Joel Moss, and Martha Vaughan
were supported by the Intramural Research Program, National
Institutes of Health, National Heart, Lung, and Blood Institute.
Disclosures
None.
References
1. Oram JF, Vaughan AM. ATP-Binding cassette cholesterol transporters
and cardiovascular disease. Circ Res. 2006;99:1031–1043.
2. Bodzioch M, Orsó E, Klucken J, et al. The gene encoding ATPbinding cassette transporter 1 is mutated in Tangier disease. Nat Genet.
1999;22:347–351.
3. Yang J, Chang E, Cherry AM, Bangs CD, Oei Y, Bodnar A, Bronstein A,
Chiu CP, Herron GS. Human endothelial cell life extension by telomerase expression. J Biol Chem. 1999;274:26141–26148.
4. Attie AD. ABCA1: at the nexus of cholesterol, HDL and atherosclerosis.
Trends Biochem Sci. 2007;32:172–179.
5. Neufeld EB, Stonik JA, Demosky SJ Jr, Knapper CL, Combs CA,
Cooney A, Comly M, Dwyer N, Blanchette-Mackie J, Remaley AT,
Santamarina-Fojo S, Brewer HB Jr. The ABCA1 transporter modulates
late endocytic trafficking: insights from the correction of the genetic
defect in Tangier disease. J Biol Chem. 2004;279:15571–15578.
6. Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, Comly
M, Dwyer NK, Zhang M, Blanchette-Mackie J, Santamarina-Fojo S,
Brewer HB Jr. Cellular localization and trafficking of the human ABCA1
transporter. J Biol Chem. 2001;276:27584–27590.
7. Singaraja RR, Visscher H, James ER, Chroni A, Coutinho JM, Brunham
LR, Kang MH, Zannis VI, Chimini G, Hayden MR. Specific mutations
in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro. Circ Res. 2006;99:389–397.
8. Witting SR, Maiorano JN, Davidson WS. Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1. J Biol Chem. 2003;278:
40121–40127.
9. Singaraja RR, Kang MH, Vaid K, Sanders SS, Vilas GL, Arstikaitis P,
Coutinho J, Drisdel RC, El-Husseini AD, Green WN, Berthiaume L,
Hayden MR. Palmitoylation of ATP-binding cassette transporter A1 is
essential for its trafficking and function. Circ Res. 2009;105:138–147.
10. Chen W, Wang N, Tall AR. A PEST deletion mutant of ABCA1 shows
impaired internalization and defective cholesterol efflux from late endosomes. J Biol Chem. 2005;280:29277–29281.
11. Linder MD, Uronen RL, Hölttä-Vuori M, van der Sluijs P, Peränen J, Ikonen
E. Rab8-dependent recycling promotes endosomal cholesterol removal in
normal and sphingolipidosis cells. Mol Biol Cell. 2007;18:47–56.
12. Linder MD, Mäyränpää MI, Peränen J, Pietilä TE, Pietiäinen VM, Uronen
RL, Olkkonen VM, Kovanen PT, Ikonen E. Rab8 regulates ABCA1 cell
surface expression and facilitates cholesterol efflux in primary human
macrophages. Arterioscler Thromb Vasc Biol. 2009;29:883–888.
13. Mendez AJ. Monensin and brefeldin A inhibit high density lipoproteinmediated cholesterol efflux from cholesterol-enriched cells. Implications
for intracellular cholesterol transport. J Biol Chem. 1995;270:5891–5900.
14. Remaley AT, Schumacher UK, Stonik JA, Farsi BD, Nazih H, Brewer
HB Jr. Decreased reverse cholesterol transport from Tangier disease
fibroblasts. Acceptor specificity and effect of brefeldin on lipid efflux.
Arterioscler Thromb Vasc Biol. 1997;17:1813–1821.
15.Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A:
insights into the control of membrane traffic and organelle structure.
J Cell Biol. 1992;116:1071–1080.
16. Mossessova E, Corpina RA, Goldberg J. Crystal structure of ARF1*Sec7
complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism. Mol Cell. 2003;12:1403–1411.
17. Ishizaki R, Shin HW, Mitsuhashi H, Nakayama K. Redundant roles
of BIG2 and BIG1, guanine-nucleotide exchange factors for ADPribosylation factors in membrane traffic between the trans-Golgi network and endosomes. Mol Biol Cell. 2008;19:2650–2660.
18. Shen X, Xu KF, Fan Q, Pacheco-Rodriguez G, Moss J, Vaughan M.
Association of brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling endosomes during transferrin uptake. Proc
Natl Acad Sci USA. 2006;103:2635–2640.
19. Zhao X, Claude A, Chun J, Shields DJ, Presley JF, Melançon P. GBF1,
a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi
protein traffic. J Cell Sci. 2006;119:3743–3753.
20. Zha X, Gauthier A, Genest J, McPherson R. Secretory vesicular transport
from the Golgi is altered during ATP-binding cassette protein A1 (ABCA1)mediated cholesterol efflux. J Biol Chem. 2003;278:10002–10005.
21. Yamaji R, Adamik R, Takeda K, Togawa A, Pacheco-Rodriguez G,
Ferrans VJ, Moss J, Vaughan M. Identification and localization of two
brefeldin A-inhibited guanine nucleotide-exchange proteins for ADPribosylation factors in a macromolecular complex. Proc Natl Acad Sci
USA. 2000;97:2567–2572.
22. Zhao X, Lasell TK, Melançon P. Localization of large ADP-ribosylation
factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic. Mol Biol Cell.
2002;13:119–133.
23. Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G,
Kaminski WE, Schmitz G. Molecular cloning of the human ATP-binding
cassette transporter 1 (hABC1): evidence for sterol-dependent regulation
in macrophages. Biochem Biophys Res Commun. 1999;257:29–33.
24. Orsó E, Broccardo C, Kaminski WE, et al. Transport of lipids from golgi
to plasma membrane is defective in tangier disease patients and Abc1deficient mice. Nat Genet. 2000;24:192–196.
25. Brunham LR, Singaraja RR, Duong M, Timmins JM, Fievet C, Bissada
N, Kang MH, Samra A, Fruchart JC, McManus B, Staels B, Parks JS,
Hayden MR. Tissue-specific roles of ABCA1 influence susceptibility to
atherosclerosis. Arterioscler Thromb Vasc Biol. 2009;29:548–554.
26.Nachtigal P, Pospisilova N, Vecerova L, Micuda S, Brcakova E,
Pospechova K, Semecky V. Atorvastatin increases endoglin, SMAD2,
phosphorylated SMAD2/3 and eNOS expression in apoE/LDLR double
knockout mice. J Atheroscler Thromb. 2009;16:265–274.
27. Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao
D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P. A PPAR
gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell. 2001;7:161–171.
e38 Arterioscler Thromb Vasc Biol February 2013
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
28. Arakawa R, Yokoyama S. Helical apolipoproteins stabilize ATP-binding
cassette transporter A1 by protecting it from thiol protease-mediated
degradation. J Biol Chem. 2002;277:22426–22429.
29. Lu R, Arakawa R, Ito-Osumi C, Iwamoto N, Yokoyama S. ApoA-I facilitates ABCA1 recycle/accumulation to cell surface by inhibiting its intracellular degradation and increases HDL generation. Arterioscler Thromb
Vasc Biol. 2008;28:1820–1824.
30. Azuma Y, Takada M, Shin HW, Kioka N, Nakayama K, Ueda K.
Retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation. Genes Cells. 2009;14:191–204.
31. Tanaka AR, Kano F, Yamamoto A, Ueda K, Murata M. Formation of
cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1. Genes Cells. 2008;13:
889–904.
32. Casanova JE. Regulation of Arf activation: the Sec7 family of guanine
nucleotide exchange factors. Traffic. 2007;8:1476–1485.
33. Wellington CL, Brunham LR, Zhou S, Singaraja RR, Visscher H, Gelfer
A, Ross C, James E, Liu G, Huber MT, Yang YZ, Parks RJ, Groen A,
Fruchart-Najib J, Hayden MR. Alterations of plasma lipids in mice via
adenoviral-mediated hepatic overexpression of human ABCA1. J Lipid
Res. 2003;44:1470–1480.
34. Basso F, Freeman L, Knapper CL, Remaley A, Stonik J, Neufeld EB,
Tansey T, Amar MJ, Fruchart-Najib J, Duverger N, Santamarina-Fojo
S, Brewer HB Jr. Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations.
J Lipid Res. 2003;44:296–302.
35. Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR,
Mulya A, Gebre AK, Coutinho JM, Colvin PL, Smith TL, Hayden MR,
Maeda N, Parks JS. Targeted inactivation of hepatic ABCA1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.
J Clin Invest. 2005;115:1333–1342.
36. Haghpassand M, Bourassa PA, Francone OL, Aiello RJ. Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL
levels. J Clin Invest. 2001;108:1315–1320.
37. Helms JB, Rothman JE. Inhibition by brefeldin A of a Golgi membrane
enzyme that catalyses exchange of guanine nucleotide bound to ARF.
Nature. 1992;360:352–354.
38.Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC,
Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by
the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci USA.
2000;97:12097–12102.
39. Shen X, Hong MS, Moss J, Vaughan M. BIG1, a brefeldin A-inhibited
guanine nucleotide-exchange protein, is required for correct glycosylation and function of integrin beta1. Proc Natl Acad Sci USA.
2007;104:1230–1235.
40. Shen X, Meza-Carmen V, Puxeddu E, Wang G, Moss J, Vaughan M.
Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein
(BIG) 1 and kinesin motor protein KIF21A. Proc Natl Acad Sci USA.
2008;105:18788–18793.
41. Kang MH, Singaraja R, Hayden MR. Adenosine-triphosphate-binding
cassette transporter-1 trafficking and function. Trends Cardiovasc Med.
2010;20:41–49.
42.Moss J, Vaughan M. Molecules in the ARF orbit. J Biol Chem.
1998;273:21431–21434.
43. Engel T, Lueken A, Bode G, Hobohm U, Lorkowski S, Schlueter B, Rust S,
Cullen P, Pech M, Assmann G, Seedorf U. ADP-ribosylation factor (ARF)like 7 (ARL7) is induced by cholesterol loading and participates in apolipoprotein AI-dependent cholesterol export. FEBS Lett. 2004;566:241–246.
44. Yu X, Breitman M, Goldberg J. A structure-based mechanism for Arf1dependent recruitment of coatomer to membranes. Cell. 2012;148:530–542.
45. Beers MF, Hawkins A, Shuman H, Zhao M, Newitt JL, Maguire JA, Ding
W, Mulugeta S. A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments. J Lipid
Res. 2011;52:1471–1482.
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
BIG1, a Brefeldin A−Inhibited Guanine Nucleotide-Exchange Protein Modulates
ATP-Binding Cassette Transporter A-1 Trafficking and Function
Sisi Lin, Chun Zhou, Edward Neufeld, Yu-Hua Wang, Suo-Wen Xu, Liang Lu, Ying Wang,
Zhi-Ping Liu, Dong Li, Cuixian Li, Shaorui Chen, Kang Le, Heqing Huang, Peiqing Liu, Joel
Moss, Martha Vaughan and Xiaoyan Shen
Arterioscler Thromb Vasc Biol. 2013;33:e31-e38; originally published online December 6,
2012;
doi: 10.1161/ATVBAHA.112.300720
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2012 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/33/2/e31
Data Supplement (unedited) at:
http://atvb.ahajournals.org/content/suppl/2012/12/06/ATVBAHA.112.300720.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
which permission is being requested is located, click Request Permissions in the middle column of the Web
page under Services. Further information about this process is available in the Permissions and Rights
Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/
Supplement Material
Materials and Methods
Antibodies and other materials.
Human apoA-I was purchased from Calbiochem; Preparation of LDL and oxidative
modification were as described previously.1 Anti-BIG1 and BIG2 antibodies were those used
in prior studies.2,3,4,5 mouse monoclonal anti-TGN230, EEA1, and ARF3 antibodies were
purchased from BD Biosciences; anti-ABCA1 from Abcam; anti-HDL from American
Research Products, Inc (Catalog No. :12-7000); anti-transferrin receptor from Invitrogen;
anti-ARF1 antibodies were purchased from Enzo Life Sciences; anti-ARF4 and ARF6
antibodies were purchased from ProteinTech Group; horseradish peroxidase-conjugated
sheep anti-rabbit and anti-mouse immunoglobulins were purchased from Promega; additional
reagents from Sigma.
Cell culture, sample preparation and Western blotting
HepG2 and HeLa cells were cultured in DMEM with 10% fetal bovine serum (FBS) and 1%
penicillin–streptomycin (Invitrogen). When necessary, plates were coated with collagen I
before plating HepG2 cells. THP-1 cells were maintained in RPMI + 10% FBS medium, and
pretreated with 100 nM PMA (Sigma) for 48 hours. Cells were incubated at 37°C in a
humidified incubator (5% CO2/95% O2).
Cytosol and membrane fractions of HepG2 cells were separated using kits from
Beyotime Institute of Biotechnology (P0033). Proteins in conditioned medium
(DMEM+0.5% BSA) were precipitated in 10% trichloroacetic acid.
For Western blotting, sample of proteins were separated by SDS-PAGE in 8-12% gel,
transferred to nitrocellulose membranes, and incubated with the indicated antibodies,
followed by appropriate HRP-conjugated secondary antibodies. Blots were developed using
enhanced chemiluminescence (Pierce) and visualized with Las4000 (GE Healthcare). The
intensities of the blots were quantified by ImageQuantTL (GE Healthcare) according to
manufacturer's instructions.
Vesicle immunoprecipitation
Coated vesicles were isolated from the livers of male Sprague-Dawley (SD) rats as described
previously.6 The vesicles (0.3 ml, ~1 mg protein) were incubated overnight with 5 μg
anti-BIG1 antibody or normal rabbit IgG at 4°C and then with secondary antibody-coated
magnetic Dynabeads (Dynal) for 12 h at 4°C. Electron microscopy (EM) analysis of
immunoisolated vesicles was performed as described previously.7 Beads were washed with
homogenization buffer three times and eluted with 100 µl loading buffer. All animal
experimental procedures were approved by the Institutional Ethical Committee for Animal
Research at the Sun Yat-sen University and were performed in accordance with national law.
siRNA and plasmid over-expression
Specific siRNA against human BIG1 and BIG2 were designed and synthesized by Invitrogen.
Stealth RNAi™ siRNA negative control (Invitrogen) was used as the non-targeting control
(NT) in our siRNA experiments. Plasmids encoding human BIG1 (WT or E793K mutation)
or ARF1 (WT or Q71L or T31L mutations with myc tags) were those used in earlier studies. 4,
5
. Lipofectamine RNAiMAX (Invitrogen) was used for transfection with siRNA and
Genejuice (Novagen) according to the manufacturer’s instructions for protein
over-expression.
Labeling and tracing of ABCA1
Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin (Pierce) for 30
min at 4°C. After quenching, cells were washed and lysed, and membrane proteins were
isolated by incubating with streptavidin-agarose beads (Pierce) at RT for 1 hour. After
recovering the beads, bound proteins were eluted with loading buffer, and analyzed by
Western blotting with anti-ABCA1 antibody as described. To trace internalized surface
ABCA1, biotinylated proteins remaining on the cell surface after internalization were cleaved
by incubating the cells with 50 mmol/L reduced glutathione (pH 8.0) twice for 20 minutes,
and the internalized biotinylated ABCA1 was analyzed by Western blotting.8
To examine ABCA1 recycling, cell surface proteins were pulse-labeled with
sulfo-SS-biotin. After internalization at 37°C for 1 hour, biotinylated proteins remaining on
the cell surface were cleaved with reduced glutathione, followed by incubation for the
indicated time (0.5, 1, and 3 h) to allow recycling of internalized ABCA1. Cell surface
proteins were again removed The biotinylated ABCA1 after second cleavage was analyzed
and compared with that without the second cleavage to estimate amounts of resurfaced
ABCA1.9
Cholesterol quantification and filipin staining
Briefly, HepG2 cells after treatment for 72h with BIG1 and BIG2 siRNA were labeled with 1
μCi/ml [3H]-labeled cholesterol for 24 h. The cells were washed with 0.2% BSA, serum-free
medium, and cholesterol efflux was determined in the presence of 10 μg/ml human apoA-I
(Roche). After incubation for 12 h, radioactivity was quantified in both the medium and the
cells by scintillation counting. Efflux is reported as radioactivity in medium divided by the
sum of the radioactivity in medium and cells. Total cholesterol in cell lysates and conditioned
medium was also measured using the Amplex Red Cholesterol Assay Kit (Molecular
Probes)according to the manufacturer's instructions.10 To assess the distribution of free
cholesterol, cells were fixed and incubated in 0.05 mg/ml filipin (Sigma) for 1 h before
washing with PBS and inspection using a confocal laser scanning microscope (Zeiss 710).
Subcellular fractionation
HepG2 cells, harvested from 100 mm dishes, were resuspended in 1.5 ml of buffer A (25 mM
Tris/HCl, pH 7.4, 100 mM KCl, 1 mM EDTA and 5 mM EGTA) containing a protease
inhibitor cocktail (Roche).11 Cells were broken with glass homogenizer and after
centrifugation at 800 g for 10 minutes, supernatant adjusted to 0.25 M sucrose in a final
volume of 1.5 ml, was applied to the top of a discontinuous sucrose gradient comprising 1ml
of 2 M; 4ml of 1.3 M; 3.5ml of 1.16 M; 2ml of 0.8 M sucrose prepared in buffer A in a 12 ml
ultracentrifugation tube (Beckman).12 Tubes were centrifuged for 2.5 h (180,000 g, 4°C) in a
Beckman SW41 rotor and 1 ml fractions were recovered from the top (12 fractions) for
testing.
Statistics
Data are expressed as mean ± standard error of the mean (SEM). All raw data were processed
by authorized software SPSS 13.0. One-way ANOVA was used to compare the mean values
of the groups. Statistical significance was accepted at P< 0.05.
References
1. Arakawa R, Yokoyama S. Helical apolipoproteins stabilize atp-binding cassette transporter
A1 by protecting it from thiol protease-mediated degradation. J Biol Chem.
2002;277:22426-22429.
2. Shen X, Xu KF, Fan Q, Pacheco-Rodriguez G, Moss J, Vaughan M. Association of
brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2) with recycling
endosomes during transferrin uptake. Proc Natl Acad Sci U S A. 2006;103:2635-2640
3. Yamaji R, Adamik R, Takeda K, Togawa A, Pacheco-Rodriguez G, Ferrans VJ, Moss J,
Vaughan M. Identification and localization of two brefeldin A-inhibited guanine
nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex.
Proc Natl Acad Sci U S A. 2000;97:2567-2572
4. Shen X, Hong MS, Moss J, Vaughan M. Big1, a brefeldin A-inhibited guanine
nucleotide-exchange protein, is required for correct glycosylation and function of integrin
beta1. Proc Natl Acad Sci U S A. 2007;104:1230-1235
5. Shen X, Meza-Carmen V, Puxeddu E, Wang G, Moss J, Vaughan M. Interaction of
brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor
protein KIF21a. Proc Natl Acad Sci U S A. 2008;105:18788-18793
6. Campbell C, Squicciarini J, Shia M, Pilch PF, Fine RE. Identification of a protein kinase as
an intrinsic component of rat liver coated vesicles. Biochemistry. 1984;23:4420-4426
7. Ferguson SM, Savchenko V, Apparsundaram S, Zwick M, Wright J, Heilman CJ, YiH,
Levey AI, Blakely RD. Vesicular localization and activity-dependent trafficking of
presynaptic choline transporters. J Neurosci. 2003;23(30):9697-709.
8. Abdel Shakor AB, Taniguchi M, Kitatani K, Hashimoto M, Asano S, Hayashi A, Nomura
K, Bielawski J, Bielawska A, Watanabe K, Kobayashi T, Igarashi Y, Umehara H, Takeya H,
Okazaki T. Sms1-generated sphingomyelin plays an important role in transferrin
trafficking and cell proliferation. J Biol Chem. 2011;286:36053-36062.
9. Lu R, Arakawa R, Ito-Osumi C, Iwamoto N, Yokoyama S. ApoA-I facilitates ABCA1
recycle/accumulation to cell surface by inhibiting its intracellular degradation and
increases HDL generation. Arterioscler Thromb Vasc Biol. 2008;28:1820-1824.
10. Amundson DM, Zhou M. Fluorometric method for the enzymatic determination of
cholesterol. J Biochem Biophys Methods. 1999;38:43-52.
11. Langmann T, Klucken J, Reil M, Liebisch G, Luciani MF, Chimini G, Kaminski
WE,Schmitz G. Molecular cloning of the human ATP-binding cassette transporter 1
(hABC1): evidence for sterol-dependent regulation in macrophages. Biochem Biophys Res
Commun. 1999;257:29-33.
12. Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transactivation of the ABC1
promoter by the liver X receptor/retinoid X receptor. J Biol Chem.
2000;275:28240-28245.
Figure I
Figure I. Concurrent increased/decreased expression of BIG1 and ABCA1 in ApoE−/−
mice. Lipid profile of the ApoE−/− mice and the effect of atorvastatin treatment. Male
ApoE−/− mice (Mod) and wild-type C57BL/6J mice (NC) fed on high fat diet were dosed
daily via intragastric gavage with 3 (AtoL) or 10 (AtoH) mg/kg atorvastatin calcium salt
trihydrate for 16 weeks, 0.5% CMC-Na as vehicle control. A, Serum total cholesterol (TC),
triglycerides (TG), High-density lipoprotein (HDL) and LDL cholesterol (LDL-C) were
determined by commercially available kits. Data are represented as means ± SEM; n=6 in
each group, *p<0.05, **p<0.01. B, Total lysates from liver of each mouse in the same group
were pooled, and proteins (20 µg) from each group were subjected to test the expression of
ABCA1 and BIG1 by Western blotting.
Figure II
Figure II. Effect of BIG1 siRNA on the level of ABCA1 mRNA and the influence of
oxLDL. A, HepG2 cells were incubated with non-targeting control (NT) or BIG1 siRNA for
72 h, followed by quantitative RT-PCR to detect the levels of BIG1 and ABCA1 mRNA.
*p<0.05, ***p<0.001 vs NT. B, After BIG1 depletion by siRNA, HepG2 cells were incubated
with oxidized LDL (50 μg/ml) and the levels of ABCA1 and BIG1 mRNA were detected by
quantitative RT-PCR. Results are representative of three independent experiments. β-actin
was used as an internal control. Data are means ±S.E.M. n=5. # p<0.05, $$ p<0.01; ###, ***
p<0.001.
Figure III
Figure III. Distribution of ABCA1-GFP in Hela Cell. Hela cells were stably
over-expressing ABCA1-GFP, and were inspected by confocal microscopy. GFP
fluorescence indicates the expression and distribution of exogenous ABCA1.
Figure IV
Figure IV. Equilibrium sucrose density centrifugation. Homogenates were prepared from
HepG2 cells transfected with non-targeting (NT, A) or BIG1 siRNA (B). Fractions were
analyzed for distribution of the following organelle markers: EEA1 (early endosomes), Rab7
(late endosomes), transferrin receptor (TFR; recycling endosome), lamp2 (lysosomes), and
TGN230 (trans-Golgi network).