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Copyright © 2002, The American Society for Cell Biology ARL2 and BART Enter Mitochondria and Bind the Adenine Nucleotide
Transporter John Pringle, Monitoring Editor ‡Corresponding author. E-mail address:
rkahn/at/emory.edu. Received May 17, 2001; Revised October 18, 2001; Accepted October 24, 2001.  This article has been cited by other articles in PMC. | |||||||||||||||||||||
Abstract The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding
partner binder of ARL2 (BART) are ubiquitously expressed in rodent and
human tissues and are most abundant in brain. Both ARL2 and BART are
predominantly cytosolic, but a pool of each was found associated with
mitochondria in a protease-resistant form. ARL2 was found to lack
covalent N-myristoylation, present on all other members of the ARF
family, thereby preserving the N-terminal amphipathic α-helix as a
potential mitochondrial import sequence. An overlay assay was developed
to identify binding partners for the BART·ARL2·GTP complex and
revealed a specific interaction with a protein in bovine brain
mitochondria. Purification and partial microsequencing identified the
protein as an adenine nucleotide transporter (ANT). The overlay assay
was performed on mitochondria isolated from five different tissues from
either wild-type or transgenic mice deleted for ANT1. Results confirmed
that ANT1 is the predominant binding partner for the BART·ARL2·GTP
complex and that the structurally homologous ANT2 protein does not bind
the complex. Cardiac and skeletal muscle mitochondria from
ant1−/ant1−
mice had increased levels of ARL2, relative to that seen in
mitochondria from wild-type animals. We conclude that the amount of
ARL2 in mitochondria is subject to regulation via an ANT1-sensitive
pathway in muscle tissues. | |||||||||||||||||||||
INTRODUCTION ADP-ribosylation factor-like 2 (ARL2) is a 21-kDa GTPase that
shares 45% primary sequence identity with ADP-ribosylation factor 1
(ARF1; Clark et al., 1993 BART is a soluble 19-kDa protein originally identified and purified
from bovine brain. The binding of ARL2 to BART is of high affinity
(≤20 nM) and dependent on the binding of GTP to ARL2 (Sharer and Kahn,
1999 Primary sites of action for regulatory GTPases in the RAS superfamily include the plasma membrane (e.g., RAS and RHO), the membranes of the secretory pathway (e.g., Golgi complex; ARFs, and RABs), and the nucleus (RAN). However, no GTPases in the RAS superfamily have previously been identified in mitochondria. In the studies described below, we provide evidence that a portion of ARL2 is in mitochondria and that it binds through its effector, BART, to the adenine nucleotide transporter (ANT). ANT proteins are abundant, integral components of the mitochondrial
inner membrane, where they homodimerize and form channels used to
exchange ATP and ADP, thereby controlling the levels of ATP in the
cytoplasm (Fiore et al., 1998 Previous reports have described the presence of multiple species of
GTP-binding proteins in mitochondria from rat liver (Lithgow et
al., 1991 | |||||||||||||||||||||
MATERIALS AND METHODS Antibodies Polyclonal antisera were raised in rabbits against recombinant
ARL2 (R-86336) or a BART-His6 fusion protein
(R-46712) as previously described (Sharer and Kahn, 1999  ). Each of
these antibodies is sufficiently sensitive to detect ≤1 ng of
purified, recombinant protein by immunoblotting. The
specificity of the ARL2 antibody was tested against human ARF1–6,
ARL1, and ARL3 with no cross-reactivity evident in
immunoblots. Each was affinity purified in two steps.
Immunoglobulins were first enriched on a 1-ml HiTrap Protein G column
(Amersham Biosciences, Piscataway, NJ). The specific antibodies
were then affinity purified by passing over antigen-specific resins,
composed of ARL2 or BART-His6 covalently bound to
Affigel 15 beads (Bio-Rad, Hercules, CA) according to manufacturer's
instructions. Polyclonal antibodies specific for rodent ANT1 and ANT2
have been described previously (Graham et al., 1997 ).
Monoclonal antibodies specific to matrix heat-shock protein 70
(mtHSP70) or cytochrome c were obtained from Affinity
Bioreagents (Golden, CO) and Trevigen (Gaithersburg, MD), respectively.Gel Electrophoresis and Immunoblotting Protein samples (20–40 μg of total protein/lane) were boiled
in Laemmli's sample buffer for 5 min before electrophoresis on 12.5 or
15% discontinuous polyacrylamide gels (Laemmli, 1970 ). Resolved
proteins were electrophoretically transferred to nitrocellulose and
immunoblotted as described (Towbin et al., 1979 ;
Zhang et al., 1994 ) by using affinity-purified R-86336 or
R-46712 at 180 or 150 ng/ml, respectively. Immunoreactive proteins were
visualized using enhanced chemiluminescence detection (PerkinElmer Life
Sciences, Boston, MA).Note that the recombinant BART used as a positive control in immunoblots contains a poly-histidine tag that makes it migrate slightly slower in SDS gels than that from cells or tissues (Figure 1). It was also noted that ARL2, either recombinant or in cell or tissue lysates, can migrate as a doublet (Figures 1 or 3). No covalent modifications or proteolytic processing is known to occur on ARL2, so no explanation for the doublet is currently available. However, there was no consistent difference in behavior of the two ARL2 immunoreactive bands with regard to any of the results described herein.
Cell Culture Sf295 glioblastoma cells (obtained from the National Cancer
Institute tumor cell line repository) and normal rat kidney (NRK) cells
were grown in RPMI containing 10% fetal bovine serum (Invitrogen,
Carlsbad, CA) at 37°C in 10% CO2. For
immunocytochemistry, cells were seeded at a density of ~1 ×
105 cells/ml on glass coverslips and grown to
50–80% confluence before collection and processing. For subcellular
fractionation studies, ~5 × 105 cells
were seeded into ten 150-mm2 dishes and allowed
to reach 80% confluence. Cells were then harvested by scraping with a
Teflon cell scraper in phosphate-buffered saline (PBS) and pelleted
before further treatment. For transient transfection experiments, NRK
cells were seeded at a density of 1 × 105
cells/ml on glass coverslips and incubated for 24 h before
transfection with pcARL2myc (pcDNA3 containing the ARL2 coding region
fused to a C-terminal myc epitope) by using FuGENE6 (Roche Molecular
Biochemicals, Indianapolis, IN), as per manufacturer's instructions.Fluorescence Microscopy Cells were prepared for immunocytochemistry as previously
described (Zhang et al., 1994 ). All cells were viewed and
images captured with an Olympus BX60 epifluorescence microscope fitted
with a Dage-MTI model charge-coupled device-300-RC camera and Image Pro
capturing and imaging software. Labeling of mitochondria with
Mitotracker (Molecular Probes, Eugene, OR) was achieved by incubating
cells in culture with 0.2 μM Mitotracker for 30 min before fixation.Subcellular Fractionation/Isolation of Mitochondria All subcellular fractionation and organelle isolation techniques
were adapted from previously reported, well established methods
(Greenawalt, 1974 ; Graham et al., 1994 ; Spector et
al., 1998 ). All steps were carried out at 4°C. Sf295 cells were
collected by centrifugation and washed once with PBS and twice with
mitochondrial isolation buffer (10 mM HEPES pH 7.4, 70 mM sucrose, 200
mM mannitol, 1 mM EDTA, protease inhibitor cocktail [Sigma, St. Louis,
MO]; Greenawalt, 1974 ). Cells were then resuspended in mitochondria
isolation buffer, passed five times through a 23-gauge needle, and
disrupted by N2 cavitation (25 min at 500 psi).
The cell lysate was then subjected to centrifugation at 1000 ×
g for 10 min, yielding a nuclear pellet and postnuclear
supernatant. The pellet was carefully resuspended in isolation buffer,
by using either a large-bore pipette or a few strokes with a
loose-fitting Dounce homogenizer, and collected again by centrifugation
at 1000 × g, yielding the final nuclear pellet. The
heavy mitochondrial fraction was obtained from the postnuclear
supernatant after centrifugation at 3000 × g for 10
min. This pellet was resuspended and the 3000 × g spin
was repeated to obtain the final heavy mitochondrial pellet. The
supernatant from the 3000 × g spins was then subjected
to 15,000 × g for 10 min. The resulting light
mitochondrial pellet was resuspended and sequential 3000 and
15,000 × g spins yielded the final light mitochondrial
pellet. Finally, the 15,000 × g supernatant was
further fractionated at 100,000 × g for 60 min,
yielding the 100,000 × g supernatant (S100) and pellet
(P100).Three different methods were used to further enrich for mitochondria
after preparation of the crude mitochondrial fraction by differential
centrifugation. For additional purification of mitochondria, velocity
or equilibrium density gradient centrifugation was used. For rapid
isolation of purified mitochondria, heavy or combined heavy and light
mitochondria were applied to 30% Percoll in mitochondria isolation
buffer and resolved at 95,000 × g for 30 min. The
lower portion of the dense mitochondrial band was removed with a
Pasteur pipette and washed twice with mitochondria isolation buffer
(Hovius et al., 1990 For fractionation of mammalian tissues, adult cow brains (Pel-Freeze) or freshly excised organs from fasted mice or rats were washed in ice cold mitochondria isolation buffer, minced with surgical scissors, and homogenized with a chilled Teflon-glass homogenizer. The crude lysate was filtered through five layers of cheesecloth and processed for subcellular fractions as described above for cultured cells. Test for N-myristoylation Target proteins were coexpressed in Escherichia coli
with N-myristoyltransferase (NMT) as described previously (Duronio
et al., 1990 ; Randazzo and Kahn, 1995 ; Van Valkenburgh
et al., 2001 ). Briefly, cultures were grown to an
A600 of ~0.6 in Luria broth, at which time 1 mM
isopropyl β-d-thiogalactoside and 40 μCi/ml
[3H]myristic acid were added and cultures were
allowed to grow for an additional 90 min. Cells were collected by
centrifugation and resuspended in SDS sample buffer. Proteins were
resolved on a 15% SDS-polyacrylamide gel (50 μg of protein/lane).
The gel was then treated for fluorography by using Enhance (Amersham
Biosciences), dried, and exposed to film. Plasmids used for protein
expression include the following: pBB131, yeast NMT (Duronio et
al., 1990 ); pNMT1 (Randazzo and Kahn, 1995 ), N-terminal truncation
of human NMT1; pHV641 (Van Valkenburgh and Kahn, 2001 ), full-length
human NMT1; pHV644 (Van Valkenburgh and Kahn, 2001 ), full-length human
NMT2; pJCY1–74 (Kahn et al., 1995 ), yeast ARF1; pJCY1–75
(Kahn et al., 1995 ), yeast [G2A]ARF1; pOW12 (Weiss
et al., 1989 ), human ARF1; and pJCH14-4 (Clark et
al., 1993 ), human ARL2.For expression and analysis in mammalian cells (Jones et
al., 1990 Treatments of Mitochondria Protease sensitivity was determined using freshly prepared
mitochondria and S100 from rat brain. Thermolysin (Sigma) was added to
40–60 μg of brain proteins at a ratio of 1:5 and incubated at 30°C
for 10 min. Reactions were terminated by the addition of protease
inhibitors and controls included the addition of the inhibitors before
protease.Fractionation of mitochondria was achieved by sequential treatment of
freshly isolated rat brain mitochondria with low (0.1) and high (0.25)
digitonin (Calbiochem, San Diego, CA):protein ratios for 15 min at
0°C. The higher concentration of digitonin has been previously shown
(Hovius et al., 1990 Overlay Assays Binding of the ARL2(GTP)·BART complex or in vitro translated
ARL2 and/or BART to other proteins was assayed using a modified version
of the gel overlay assay described in Sharer and Kahn (1999) . For
assays using radioactive GTP, recombinant ARL2 (12 μM) was loaded
with 8 μCi of [α-32P]GTP in 10 mM
3-(N-morpholino)propanesulfonic acid, pH 7.1, 1 mM EDTA, 1.5
mg/ml bovine serum albumin, 0.5 mM magnesium acetate, 0.1% Triton
X-100 for 10 min at 30°C. The buffer was then increased to 2 mM
magnesium acetate, and BART-His6 (19 μM) was
added and incubated for 10 min. Potential binding proteins were
resolved by SDS-PAGE and electrophoretically transferred to
nitrocellulose. Proteins were renatured by incubating the filter in
renaturation buffer [10 mM 3-(N-morpholino)propanesulfonic
acid, pH 7.1, 100 mM potassium acetate, 5 mM magnesium acetate, 0.25%
Tween 20, 5 mM dithiothreitol, 0.1% Triton X-100, and 0.5% bovine
serum albumin] for at least 1 h before the
ARL-[α-32P]GTP-BART mixture was added. After
15 min at room temperature, the filter was washed three times with
~30 ml of binding buffer before exposure to PhosphorImager screens
(Molecular Dynamics, Sunnyvale, CA).For analysis with in vitro translated ARL2 or BART, the coding regions
in pcDNA3 (5 μg of DNA/112-μl reaction; Sharer and Kahn, 1999 Protein Purification and Microsequencing Bovine brain mitochondrial proteins (~340 μg) were incubated
in 0.1 M Na2CO3 pH 11.5.
After 60 min at 4°C, the mitochondria were collected and boiled in
SDS sample buffer, and proteins were resolved by SDS-PAGE. Two
identical gels were used to determine ARL2·BART binding in the gel
overlay assay and to identify the corresponding band after staining
with colloidal brilliant blue (Sigma). The latter gel was extensively
washed in 25% methanol to remove SDS, and ~40 pmol of the 32-kDa
protein was then excised and submitted to the Yale Cancer Center Mass
Spectrometry Resource and W.M. Keck Foundation Resource Laboratory for
analysis. Tryptic peptides were subjected to Q-TOF MS/MS
analysis. | |||||||||||||||||||||
RESULTS ARL2 and BART Are Most Abundant in Brain and Derived Cell Lines Human and rat ARL2 share 95% amino acid sequence identity, and
human BART and a translated mouse expressed sequence tag clone encoding
a predicted BART protein are also 95% identical.1
Polyclonal antibodies raised against recombinant human ARL2 or BART
were used to determine the levels of protein expression. Each antiserum
binds rat or human ARL2 or BART with essentially the same sensitivity
and specificity.Expression patterns for ARL2 and BART were very similar in a number of
rat (Figure 1) and mouse tissues and in several human, mouse, or rat
cell lines. Thus, the levels of expression of the two proteins in
different tissues have also been conserved in mammals. Although ARL2
and BART were detected in all tissues examined, each was particularly
abundant in brain, especially the hippocampus and cortex, with somewhat
less in the cerebellum. We estimate that each protein was present in
these tissues at ~0.01% of total cell protein. Considerably less
total protein was detected in heart. The latter result differs somewhat
from Northern blot analyses, in which levels of BART mRNA in heart were
nearly as high as in brain (Sharer and Kahn, 1999 ARL2 and BART Localize to Mitochondria The subcellular localization of endogenous ARL2 and BART was
initially investigated in sf295 cells by using indirect
immunofluorescence. ARL2 or BART antisera produced distinctive
mitochondrial labeling (Figure 2), as
indicated by colocalization with the vital mitochondrial stain
Mitotracker, or with antibodies against cytochrome c or
matrix heat-shock protein 70 (our unpublished data). Staining of
mitochondria by each of the four antisera was evident in sf295 cells
permeabilized with Triton X-100 (0.1%) but not in those permeabilized
with saponin (0.2%). This is consistent with the conclusion that each
antigen is inside mitochondria, because mitochondrial membranes are
known to be low in cholesterol and are thus poorly permeabilized by
saponin. In contrast to sf295 cells, little or no specific staining of
endogenous ARL2 or BART was evident in NRK, CHO, or COS-7 cells,
consistent with the relatively low levels of the two proteins in these
cells, as detected by immunoblot analyses.
Subcellular Fractionation Confirms the Association of ARL2 and BART
with Mitochondria To confirm and extend the location of ARL2 and BART in cultured
cells, sf295 cells were analyzed by subcellular fractionation. Cells
were harvested, lysed, and subjected to differential centrifugation to
enrich for specific cellular components. The resulting fractions were
then assayed by immunoblot analyses for the presence of
ARL2, BART, ARL3 (53% identical to ARL2), or cytochrome c
(a marker for mitochondria). All four proteins were readily detected in
total cell lysates (25 μg of protein/lane) and were excluded from the
nuclear fraction (Figure 3). ARL2, BART,
and cytochrome c were each enriched in the mitochondria
fraction, but ARL3 was not. In contrast, 80–90% of ARL2, ARL3, and
BART, but not cytochrome c, were present in the 100,000
× g supernatant (S100). Little or none of the four proteins
appeared in the high-speed pellet, which consisted primarily of
membrane vesicles.
The mitochondria examined herein consisted of combined heavy (3000 × g pellet) and light (15,000 × g pellet) mitochondria fractions. When examined separately, ARL2 and BART were each detected in both pellets (our unpublished data). The heavy mitochondria fraction is generally of higher purity, whereas the light mitochondria fraction is known to be contaminated with components of the Golgi apparatus, endoplasmic reticulum, lysosomes, and peroxisomes. One or both of these fractions are routinely used as a starting point in the isolation of highly enriched mitochondria preparations. The heavy mitochondria fraction from sf295 cells was applied to linear
10–30% gradients of Optiprep (Graham et al., 1994
ARL2 and BART Localize within Mitochondria Given the extensive literature on the binding of ARFs to Golgi and
other membranes, and their role in coat protein recruitment, we
anticipated finding the ARL2 and BART on the cytoplasmic surface of the
outer mitochondrial membrane. However, treatment of purified, intact
sf295 mitochondria with high salt (0.5 M NaCl) or alkaline carbonate
(0.1 M Na2CO3, pH 11.5),
which efficiently removes peripherally bound proteins from biological
membranes (Cavenagh et al., 1996 ), failed to extract the
ARL2 or BART (our unpublished data). Protease sensitivities of
the ARL2, BART, and mtHSP70 associated with purified mitochondria were
also examined and compared with that of ARL2 and BART in the S100
fraction (Figure 5). Treatment of the
S100 fraction with thermolysin (5:1 for 10 min at 30°C) resulted in
the complete degradation of the proteins, whereas the ARL2, BART, and
mtHSP70 in the mitochondrial fraction were all resistant to the
protease (Figure 5). Partial or complete solubilization of the
organelles with detergent rendered the otherwise resistant ARL2 and
BART susceptible to protease digestion (our unpublished data).
The same results were obtained when purified rat brain mitochondria
were used as the source. These data are consistent with the conclusion
that this fraction of ARL2 and BART is not extrinsic, cytosolic-facing
proteins but resides inside mitochondria.
To identify where ARL2 and BART localize within mitochondria we
performed differential detergent extractions studies. Progressive
disruption of the outer mitochondrial membrane has been observed when
mitochondria are exposed to digitonin, at detergent:protein ratios from
0.1 to 0.25 (Hovius et al., 1990
Predicted Amphipathic N Terminus of ARL2 Is not Myristoylated The discovery that ARL2 and BART can enter mitochondria prompted
analysis of both proteins for potential import sequences. ARL2, like
all members of the ARF family, contains an N-terminal domain that is
predicted to form an amphipathic α-helix. A common feature of
proteins imported into the mitochondrial matrix is an N terminus that
contains several positively charged residues and is predicted to form
an amphipathic α-helix (von Heijne, 1986 ). Every member of the ARF
family, including ARL2, has a glycine at position 2 and each ARF tested
to date, including ARL1, has been found to be cotranslationally,
covalently modified by the addition of myristic acid (Kahn et
al., 1988 ; Randazzo and Kahn, 1995 ; Lee et al., 1997 ).
The presence of this moiety at the N terminus would probably present
problems in import, so we tested the possibility that cytosolic and
mitochondrial pools of ARL2 may contain different modifications to
their N termini.ARL2 was examined in two different systems for N-myristoylation
competence: in bacteria expressing yeast or human NMT and in cultured
mammalian cells. In the former system, used previously to demonstrate
acylation of other ARF family proteins (Randazzo and Kahn, 1995
Human ARFs are inefficiently modified in the bacterial system, so we also monitored the incorporation of [3H]myristate into ARL2 in mammalian cells expressing endogenous NMTs. CHO cells were transiently transfected with plasmids directing the expression of C-terminal myc-tagged human ARF5 or ARL2, and N-myristoylation was monitored after metabolic labeling with [3H]myristate, as described under MATERIALS AND METHODS. A labeled band of the expected Mr was detected in cells expressing ARF5 (Figure 7B). In spite of the higher levels of ARL2 expression achieved in these cells, there was no detectible labeling of the immunoprecipitated protein (Figure 7B). We conclude that, in contrast to every other member of the ARF family tested previously, human ARL2 is not N-myristoylated. In related studies, we found that human ARL3 is also not N-myristoylated (our unpublished data). The lack of myristoylation of ARL2 eliminates this potential barrier to import, and thus no differences in covalent modifications between cytosolic and mitochondrial ARL2 could be discerned. ARL2·GTP·BART Complex Binds Bovine ANT The high affinity of BART for ARL2·GTP (≤20 nM) made it
possible to screen for proteins that bound the complex, but not
ARL2·GTP alone, by using a modification of the gel overlay assay.
Little or no binding of the
ARL2·[α-32P]GTP·BART complex could be
detected in the gel overlay assay when whole cell lysates were probed.
With knowledge of the presence of ARL2 and BART in mitochondria, bovine
brain fractions enriched for mitochondria were assayed and a protein of
32 kDa was found to bind the complex specifically (Figure
8). Binding activity was also evident in
mitochondria isolated from rat brain tissue and sf295 cells. No
activity was detected when the probe was
[α-32P]GTP alone or
ARL2·[α-32P]GTP (Figure 8). Thus, using the
overlay assay, we were able to detect the presence of a specific
binding partner in mitochondria for the ARL2·GTP·BART complex.
Initial characterization of the 32-kDa protein revealed a tight association with the membrane fraction after chemical or physical disruption of mitochondria. Sequential extractions with detergents and/or alkaline carbonate allowed the partial purification of this activity. It quickly became apparent that the binding activity copurified with a relatively abundant 32-kDa protein band, and extended carbonate extraction alone was sufficient for isolation of the binding protein as a single band in that region of the polyacrylamide gel. Bovine brain mitochondria were treated with alkaline carbonate and the resulting insoluble material was collected by centrifugation and resolved by gel electrophoresis. Approximately 40 pmol of the 32-kDa protein was excised from the gel, digested with trypsin, and subjected to Q-TOF MS/MS analysis to determine sequences of the derived peptides. Five such peptide sequences were obtained in this manner and each was present within one polypeptide in the protein database, bovine adenine nucleotide transporter 3 (ANT3; Figure 9).
ARL2·GTP·BART Binds Mouse ANT1 but not ANT2 Nucleotide transporters are abundant proteins in mitochondria, so
it was especially important to confirm the identity of the ARL2·BART
binding protein. The availability of transgenic mice deleted for ANT1
(Graham et al., 1997 ) allowed for definitive analysis of
binding to different rodent ANT species. Although humans and cows
express three ANT isoforms, rodents contain only two. Thus, the
ant1−/ant1−
mice also afforded an opportunity to assess specificity among isoforms.
The two murine ANT proteins differ in tissue expression and abundance
(Graham et al., 1997 ), with ANT1 expressed in heart,
skeletal muscle, and brain (Graham et al., 1997 ; Figure
10, top), and ANT2 found in all tissues
(Graham et al., 1997 ; Figure 10, bottom). As expected, no
ANT1 protein was detected in mitochondria isolated from tissues
obtained from the transgenic animals (Figure 10, top, right). When the
same mitochondrial preparations were assayed for the binding of the
ARL2·BART complex (Figure 10, middle), activity was present in
tissues from the wild-type animal, and the amount of activity
paralleled the immunoreactive material seen in the ANT1 blot. In
contrast, no activity was detected in mitochondria isolated from the
same tissues of
ant1−/ant1−
animals (Figure 10, middle). These results confirmed the identification
of ANT1 as a binder of the ARL2·BART complex in mouse mitochondria
and support the identification of bovine ANT3 as the protein with
the same activity in bovine brain mitochondria. Interestingly, ANT2 is
the most abundant ANT isoform in several other tissues, with levels
comparable to ANT1 in heart and brain, and yet no binding activity was
ever seen in the absence of ANT1. Thus, the ARL2·BART complex
exhibits specificity for the binding of the ANT1 isoform in mice.
BART Can Bind to ANT Independently of ARL2 In the overlay assay, the radioactive label required for signal
detection is bound to ARL2. Therefore, this method could not be used to
assay for direct binding of BART to ANT. We modified the technique to
allow direct tests of the ability of BART alone to bind ANTs by
generating radiolabeled BART, by using in vitro translation in the
presence of [35S]methionine and cysteine. In
this way we were able to demonstrate that BART was capable of
interacting with ANT1 in the absence of ARL2 (Figure
11). The interaction of in
vitro-translated BART with murine ANT1 was confirmed by comparing
activities in mitochondria isolated from wild-type and transgenic
animals (Figure 11, lanes 5 and 6). The 32-kDa band was detected when
radiolabeled BART was present in the assay (Figure 11, lanes 2, 3, and
5) but not with comparable levels of radiolabeled ARL2 alone (Figure
11, lane 1). Excess unlabeled BART effectively competed the binding of
[35S]BART to ANT (Figure 11, lane 4). The
presence of an ~20-kDa protein was often, although not always (Figure
11, compare lanes 2 and 3 with lanes 5 and 6), seen in these assays,
particularly when radioactive BART was used. Originally thought to be
endogenous ARL2, the interaction is not dependent on added GTP and is
under investigation.
Intramitochondrial Levels of ARL2 Are Increased in Mice Deleted of
ANT1 The expression of a number of proteins is known to be increased in
mitochondria isolated from
ant1−/ant1−
mice (Esposito et al., 1999 ; Murdock et al.,
1999 ), presumably as part of a compensatory response to the loss of the
translocase. Although some of these are directly involved in oxidative
phosphorylation (e.g., components of complex I and IV, malate
dehydrogenase, and glycogen phosphorylase; Esposito et al.,
1999 ; Murdock et al., 1999 ), others had not previously been
linked with mitochondrial metabolism (e.g., Mcl-I, WS-3, and Skd3;
Esposito et al., 1999 ). To determine the impact of the loss
of ANT1 on total or intramitochondrial levels of ARL2 and/or BART, both
total soluble protein and purified mitochondria were isolated from
ant1−/ant1−
and control mouse tissues and analyzed by immunoblot along
with mtHSP70, which served as a control for gel loading and protein
preparations. Total and mitochondrial levels of BART (our unpublished
data) or mtHSP70 (Figure 12)
were virtually unchanged in all five tissues examined. Of the five
mouse tissues analyzed, only the heart showed increased levels of
soluble ARL2 in knockout versus control animals (Figure 12, middle). A
much more dramatic increase was evident in the levels of heart
mitochondrial ARL2 and was also seen in skeletal muscle mitochondria
(Figure 12, top). Indeed, ARL2 was virtually undetectable in wild-type
skeletal muscle mitochondria but approached levels seen in brain or
heart in the knockout animals. Identical results were obtained with
four separate pairs of parental and transgenic animals. The constancy
of soluble ARL2 levels in skeletal muscle supports the conclusion that
import into mitochondria is under some form of regulation that is
specifically sensitive to the absence of ANT1, at least in this tissue,
but probably also heart.
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DISCUSSION Our results document the presence within mitochondria of a subpopulation of cellular ARL2 and its binding partner, BART, and that ARL2·GTP·BART, or BART alone, binds directly and specifically to certain isoforms of the adenine nucleotide transporter in in vitro assays. Mitochondria from the two muscle tissues were found to have substantially increased levels of ARL2, but not BART, in response to the deletion of ANT1. These observations provide initial support for the hypothesis that ARL2 and BART are imported into mitochondria in a regulated manner and there may bind to ANT1 (rodent) or ANT3 (bovine) to regulate adenylate translocase and mitochondrial function(s). Our conclusion that subpopulations of ARL2 and BART reside within mitochondria is based on the following observations. 1) Indirect immunofluorescence, using antibodies specific to each (endogenous) protein or epitope-tagged, overexpressed protein revealed clear evidence of colocalization with mitochondrial markers in cultured mammalian cells. 2) Immunostaining of mitochondria with ARL2 or BART antisera requires permeabilization of mitochondrial membranes with Triton X-100 and is not seen when only the plasma membrane is permeabilized, with saponin. 3) Both ARL2 and BART are reproducibly detected in fractions defined as being enriched for mitochondria by using previously established cellular fractionation and organelle isolation methods. In particular, the data shown in Figure 4 demonstrate colocalization of ARL2 and BART with mtHSP70 after isolation of heavy mitochondria and iso-osmotic density gradient centrifugation. 4) ARL2, BART, and markers for intramitochondrial proteins in fractions enriched for mitochondria are each protected from protease digestion, whereas the ARL2 and BART in cytosol are completely degraded under identical conditions. In contrast, all other members of the ARF family are absent in enriched mitochondria preparations and bind to membranes in a rapidly reversible manner, making them sensitive to proteases. 5) Solubilization of the outer mitochondrial membrane with digitonin releases the bulk of the mitochondria-associated ARL2 and BART. Unfortunately, our antisera were not useful for immunolocalization at the level of electron microscopy. Most proteins that are targeted to the mitochondrial matrix contain an
amino-terminal presequence, generally 20–60 residues in length, that
is predicted to form an amphipathic α-helix (von Heijne, 1986 The majority of the mitochondria-associated ARL2 and BART is
solubilized when the outer membrane is progressively extracted with
increasing concentrations of digitonin. In contrast, both cytochrome
c and mtHSP70 remained associated with the mitoplasts after
digitonin treatments. The simplest interpretation of these results is
that ARL2 and BART are soluble proteins in the intermembrane space,
although reversible, functional association(s) with either the inner or
outer membrane is likely. Another possibility for the incomplete
solubilization of ARL2 and BART is that they could bind to contact
points, like the (undefined) GTP-binding proteins detected within
mitochondria (Thomson, 1998 Data from studies in the mouse revealed that ARL2·BART bound ANT1 in
vitro but not ANT2, whereas bovine ANT3 was clearly present in the
purified binding protein preparation. Because ANT1 and ANT3 are each
expressed in cow brain and have very similar molecular weights it is
likely that each is present in the protein preparation that was
sequenced. Indeed, three of the five peptides obtained corresponded to
regions of the proteins that were identical. The presence of three
amino acid differences between ANT1 and ANT3 in peptide 1 and one
difference in peptide 3 definitively identified ANT3 as being present
in this preparation. Thus, in contrast to murine ANT1 we lack
confirmatory evidence for the binding of ARL2·BART to bovine ANT3.
The specificity of ARL2·BART or BART alone for rodent ANT1, but not
rodent ANT2, has potential implications regarding the functional
specificities of the different ANT isoforms. Specific binding of
ARL2·BART to ANT1 was suggested by the fact that ANT2
levels2 in mitochondria from brain, liver, and lung are
comparable to levels of ANT1 in heart, skeletal muscle, and brain
(Stepien et al., 1992 BART alone can interact with ANT, independently of ARL2, and a complex
consisting of ARL2, BART and ANT can also form, at least in vitro. This
suggests that BART can bind both ARL2 and ANT simultaneously, although
we cannot yet determine whether binding to one protein alters affinity
for the other. We have been unable to document the binding of ARL2/BART
to ANT1 by using independent techniques (including native blue gels and
coimmunoprecipitation after overexpression of myc-ANT1; our unpublished
data) or to document functional effects of the binding to ANT1
on its activity. Thus, although ARL2 and BART clearly associate with
mitochondria in a specific manner, the hypothesis that the interaction
with ANT1 is a functional one requires further experimental
confirmation. We speculate that ARL2 and BART are part of a mechanism
sensitive to changes in cytoplasmic conditions associated with ANT
function (e.g., levels of ATP/ADP, reactive oxygen species, or pH) and
may comprise part of a signal transduction pathway for modulating one
or more ANT activities. Lack of ANT1 results in metabolic acidosis and
a severe defect in coupled respiration associated with lack of
mitochondrial ATP (Graham et al., 1997 Intramitochondrial levels of ARL2, but not BART, are increased markedly in heart and skeletal muscle mitochondria obtained from animals deleted for ANT1. This finding could in theory reflect increased import of ARL2, decreased degradation in mitochondria, or a decrease in a potential protein export process. It is curious that BART levels do not change in the absence of ANT1, because this is one of the few instances in which ARL2 and BART clearly differ. The change in intramitochondrial ARL2 levels implicates a regulated process tied to ANT1 function in mice. The presence of only a fraction of total cellular ARL2 or BART in
mitochondria leaves open the possibility for other actions by each of
these proteins in cytosol or other organelles. It is unlikely that the
soluble ARL2 and BART are present only as depots for recruitment into
mitochondria. A role for ARL2 in microtubule folding and dynamics has
been proposed recently, based upon its binding to cofactor D
(Bhamidipati et al., 2000 | |||||||||||||||||||||
ACKNOWLEDGMENTS We thank the following people for help during the completion of this work in the form of discussions or training: Jason Kokoszka, Tim Greenamyre, Juan Carlos Amor, Dean Jones, Gerry Shadel, Yunfei Wang, Tracy Sittler-Obertone, David Wolff, and Suzanne Hollinger. This work was supported by National Institutes of Health grants GM-55148 (to R.A.K.), CA-73202 (to J.D.S.), NS-21328 (to D.C.W.), NS-41850 and HL-64017 (to D.C.W.). | |||||||||||||||||||||
Abbreviations used: | |||||||||||||||||||||
Footnotes Article published online ahead of print. Mol. Biol. Cell
10.1091/mbc.01–05-0245. Article and publication date are at
www.molbiolcell.org/cgi/doi/10.1091/mbc.01–05-0245. 1Accession numbers: human ARL2, AAC37606; rat
ARL2, O08697; human BART, AF126062; and mouse EST, BF780835. 2The levels of ANT1 and ANT2 seen in purified
mitochondria from mouse tissues (Figure 10) agreed very well with those
reported Levy et al. (2000) . Expression and sequence
analysis of the mouse adenine nucleotide translocase 1 and 2 genes
([In Process Citation]. Gene 254, 57–66) for these
proteins when total tissue lysates were probed. The one exception was
skeletal muscle, in which our data suggest a higher level of ANT2 was
present. Because the same antibodies are used in each study one
potential explanation for this difference lies in the protein
preparations being probed; purified mitochondria versus tissue lysates. | |||||||||||||||||||||
REFERENCES
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