From the
The voltage-dependent anion channel of the outer mitochondrial
membrane, VDAC (also known as mitochondrial porin), is a small abundant
protein which forms a voltage-gated pore when incorporated into planar
lipid bilayers. This protein forms the primary pathway for movement of
major metabolites through the outer membrane. Recently, it has been
demonstrated that two human VDAC genes, HVDAC1 and HVDAC2, produce
three proteins that differ most significantly at their amino termini.
These results suggest that the distinct amino termini lead to the
targeting of individual VDAC isoforms to different cellular
compartments. Consistent with this hypothesis, recent reports suggest
that HVDAC1 is found in the plasma membrane of mammalian cells. To
define the subcellular location of HVDAC isoforms, HVDAC genes were
modified so that the encoded proteins contain COOH-terminal epitopes
recognized by either of two monoclonal antibodies. Introduction of
these epitope tags had no effect on the function of modified VDAC
proteins. Epitope-tagged proteins were then individually expressed in
COS7 cells or rat astrocytes and the intracellular location of each
isoform subsequently identified by subcellular fractionation, light
level immunofluorescence, and immunoelectron microscopy. Our results
demonstrate that each HVDAC protein is exclusively located in fractions
or subcellular regions containing mitochondrial marker proteins. In
addition, immunofluorescence and immunoelectron microscopy show that an
individual mitochondrion can contain both HVDAC1 and HVDAC2. Our
results call into question previous reports demonstrating VDAC
molecules in the plasma membrane and suggest that functional
differences between individual VDAC isoforms may result in distinct
regulatory processes within a single mitochondrion.
The voltage-dependent anion channel of the mitochondrial outer
membrane (VDAC,
Although single genes in yeast and
Neurospora encode VDAC proteins (12,13), molecular cloning and mapping
studies indicate that multiple VDAC genes are present in mammals which
can potentially encode up to five isoforms of the VDAC protein
(14-16). Genes encoding two of the human isoforms, HVDAC1 and
HVDAC2, have been characterized in the greatest detail (14). When
expressed and purified from yeast lacking endogenous VDAC genes, each
human protein can form channels with the physiological properties
expected for VDAC when incorporated into planar phospholipid bilayers.
In addition, expression of either human protein in such yeast strains
can complement phenotypic defects associated with elimination of the
endogenous yeast VDAC gene. The HVDAC1 and HVDAC2 genes encode proteins
that are 75% identical, the majority of amino acid differences the
result of conservative substitutions. However, HVDAC2 contains an 11
amino acid amino-terminal extension relative to HVDAC1 (Fig. 1)
(14). A putative splice variant of HVDAC2, HVDAC2`, has also been
identified which differs from the HVDAC2 gene by the replacement of the
11 amino acid amino-terminal extension with a different 26 amino acid
amino-terminal extension relative to HVDAC1 (Fig. 1) (15).
Northern blots and polymerase chain reaction amplification of
transcripts suggest that HVDAC1 and HVDAC2 are expressed ubiquitously
while HVDAC2` expression has only been specifically demonstrated in T
and B lymphocytes (14, 15). Functionally, HVDAC1 and HVDAC2 differ in
their ability to bind hexokinase; HVDAC1-containing yeast mitochondria
are able to bind hexokinase while HVDAC2-containing yeast mitochondria
do not. Thus, these two human genes appear to encode at least three
different proteins which differ most significantly in their
amino-terminal regions.
To precisely determine the subcellular
location of HVDAC1 and define the cellular compartments containing
other members of the VDAC family of proteins, genes for HVDAC1, HVDAC2,
and HVDAC2` were modified so that the encoded proteins contain
COOH-terminal epitopes recognized by either of two characterized
monoclonal antibodies. These epitope tags appeared to have no effect on
the function of modified VDAC proteins. Epitope-tagged HVDAC isoforms
were then individually expressed in COS7 cells or rat astrocytes by
transient transfection. Subcellular fractionation, light level
immunocytochemistry, and immunoelectron microscopy demonstrate that
epitope tagged HVDAC1, HVDAC2, and HVDAC2` are exclusively located in
mitochondria. In addition, immunofluorescence and immunoelectron
microscopy have been used to demonstrate that an individual
mitochondrion can contain both HVDAC1 and HVDAC2. Our results call into
question previous reports demonstrating VDAC molecules in the plasma
membrane and suggest that functional differences between individual
VDAC isoforms may result in distinct regulatory processes within a
single mitochondrion.
Rat
astrocytes were a kind gift of Dr. Felix Eckenstein (Oregon Health
Sciences University, Portland, OR). Astrocytes and COS7 cells were
grown in monolayers in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum in an atmosphere of 5% CO
The HVDAC-FLAG and HVDAC-HA constructs were inserted into the
multicloning sites of pCD-PS (27) and pRC/RSV (Invitrogen, San Diego,
CA) mammalian expression vectors. For the expression of HVDAC-FLAG in
astrocytes, a modified pcDNA3 (Invitrogen, San Diego, CA) kindly
provided by Dr. John Adelman (Vollum Institute) was used. For transient
expression of epitope-tagged HVDAC molecules in COS7 cells and
astrocytes, cells were transfected essentially as described in Chen and
Okayama (28).
Cells were also
processed for light level immunocytochemical analysis by the methods
outlined in Dermietzel et al.(19) .
Subcellular fractions of transfected
cells were prepared by first washing cells with ice-cold PBS, 0.1%
glucose and incubation in 10 mM Tris-Cl, pH 7.4, 10
mM NaCl, and 2 mM M
Protein fractions were separated on 10%
polyacrylamide-SDS gels and transferred to nitrocellulose by
electroblotting. Blots were then incubated with appropriate dilutions
of primary polyclonal or monoclonal antibodies as indicated and
antibody binding visualized using enhanced chemiluminescence (ECL,
Amersham).
Previous characterization of the HVDAC1 and HVDAC2 proteins has
demonstrated that each protein is capable of complementing the growth
defects associated with deletion of the endogenous yeast VDAC gene
(14). Deletion of the yeast VDAC gene results in cells that are viable
on glucose-based media at either 30 or 37 °C but fail to grow at 37
°C on non-fermentable carbon sources (i.e. glycerol) (34).
Although the physiological basis of this phenotype is currently
unknown, these growth defects can be corrected by expression of a
plasmid-based yeast VDAC gene or by expression of either HVDAC1 or
HVDAC2 cDNAs (14). These results demonstrate that each human protein
can functionally replace the yeast VDAC gene in yeast cells. To
determine whether introduction of either epitope tag at the COOH
terminus of the human proteins signifi-cantly altered their function,
cDNAs encoding epitope-tagged HVDAC1, HVDAC2, or HVDAC2` proteins were
transformed into yeast cells lacking an endogenous yeast VDAC gene.
Mitochondrial membranes prepared from strains containing each
tagged-HVDAC cDNA were then prepared, separated on SDS-polyacrylamide
gels, and proteins transferred to nitrocellulose. Bound proteins were
then probed with antibodies to the appropriate epitope. As shown in
Fig. 2
for FLAG-HVDAC molecules, tagged proteins are efficiently
targeted to yeast mitochondria and each has the appropriate relative
mobility given the expected differences in NH
Higher eukaryotes, including mammals and plants, express a
family of VDAC proteins each encoded by a distinct gene (14-16,
25, 35, 36). Two human genes encoding VDAC isoforms (HVDAC1 and HVDAC2)
have been characterized in greatest detail (14, 16). These genes
generate three proteins that differ primarily by the addition of
distinct NH
The targeting of
individual tagged isoforms within mammalian cells was assessed
following transient transfection into two different cell types. In COS7
cells, three different approaches all lead to the conclusion that each
HVDAC isoform is located largely, if not exclusively, in mitochondria.
First, cells individually transfected with each HVDAC isoform were
lysed and crude subcellular fractions prepared by differential
centrifugation. In each case, Western blots of ensuing fractions
demonstrated that tagged HVDAC molecules were exclusively located in
fractions containing mitochondria as identified by reprobing blots with
an antibody to subunit IV of mitochondrial protein cytochrome c oxidase (Fig. 5). Second, transfected cells were examined by
indirect immunocytochemical techniques. In each case, tagged HVDAC
molecules are exclusively confined to subcellular structures identified
as mitochondria by the inclusion of a second antibody to the cytochrome
c oxidase holoenzyme during staining (Fig. 6). In these
cases, there is essentially no plasma membrane staining of either
transfected or untransfected cells. Third, immunoelectron microscopic
examination of transfected cells was used to unambiguously localize
HVDAC1 and HVDAC2 to mitochondria (Fig. 9). Again, in each case
epitope-tagged HVDAC molecules are located exclusively in mitochondria
and no staining above background is present in plasma membrane regions
or any other cellular compartment. Thus, these studies in COS7 cells
make it unlikely that any significant fraction of the HVDAC1, HVDAC2,
or HVDAC2` molecules expressed in these cells is present in any
cellular compartment other than mitochondria.
To address potential
cell-specific targeting of HVDAC isoforms to other cellular
compartments, the cellular distribution of HVDAC isoforms was examined
in rat astrocytes transfected with constructs encoding epitope-tagged
HVDAC1, HVDAC2, and HVDAC2` molecules. These cells have recently been
reported to contain rat homologs of HVDAC1 in their plasma membranes
(19). However, immunocytochemical analysis of transfected astrocytes
has demonstrated that each epitope-tagged HVDAC molecule is located
exclusively in mitochondria as again identified by inclusion of
cytochrome c oxidase antibodies (Fig. 7). In the case of
astrocytes, some plasma membrane staining with anti-FLAG antibodies is
evident in transfected cells. However, this staining is clearly not
specific, since untransfected cells show identical levels of plasma
membrane staining. In addition, differences between the results
presented here for astrocytes and COS7 cells and those reported by
others cannot be attributed to differences in fixation technique since
identical mitochondrial staining patterns were observed regardless of
cell fixation with paraformaldehyde or ethanol, as used in the study of
Dermietzel et al. (19).
The location and nature of protein
sequence motifs responsible for directing mammalian components of the
mitochondria to this organelle remain to be defined. However, the fact
that each of the HVDAC molecules examined in this study can be
efficiently, and, it appears, exclusively targeted to mitochondria in
both yeast and mammalian cells suggests that these targeting motifs are
conserved and effectively recognized by the targeting machinery in both
organisms. In lower eukaryotes like yeast and Neurospora, proteins of
the outer mitochondrial membrane lack cleavable NH
Cloning of cDNAs for HVDAC2 and HVDAC2` indicate that each is
expressed with a different NH
It
is possible that the expression systems used in this study result in
overexpression of epitope-tagged forms of HVDACs relative to the
endogenous, unmodified proteins. In this event, one might have expected
a portion of the tagged forms of these molecules to be mislocalized to
a variety of cellular locations due to saturation of the normal
mitochondrial targeting machinery. However, in each case,
plasmid-encoded molecules are exclusively located in mitochondria and
not other cellular compartments. This suggests either that these
molecules are not overexpressed in transfected cells or that if
overexpressed, the machinery responsible for targeting VDAC to
appropriate cellular compartments has not been saturated in these
cells. It is also formally possible that the addition of sequences
encoding COOH-terminal epitopes interferes in some way with normal
targeting of one or more of the HVDAC molecules to the plasma membrane.
In this event, targeting to the mitochondria might represent a
``default'' pathway once plasma membrane targeting has been
eliminated by inclusion of epitope sequences. If COOH-terminal epitopes
interfere with plasma membrane targeting, this interference must be
independent of amino acid sequence since molecules containing either of
two very different amino acid sequences, the FLAG or HA epitopes, are
targeted only to mitochondria.
The results presented here call into
question previous reports of the presence of HVDAC1 in the plasma
membrane of mammalian cells. In general, three lines of experimentation
have been used to demonstrate the presence of these molecules in this
cellular compartment. HVDAC1 has been observed to
``co-purify'' with proteins normally found in the plasma
membrane, like the GABA
Immunoelectron microsopic examination of the distribution of HVDAC
isoforms indicates that these proteins are not uniformly distributed
over the surface of the mitochondria but are located within discrete
patches. This would be consistent with the proposal (7, 9), and recent
experimental evidence (41), that VDAC is concentrated at contact sites
between the inner and outer mitochondrial membranes. Unfortunately, the
resolution of the images produced in frozen thin sections was not
sufficient to directly identify contact sites as the points of antibody
binding in this study.
Previous studies have indicated that
essentially all cell types and tissues express both HVDAC1 and HVDAC2
(14). We demonstrate here that both isoforms are directed to
mitochondria. In addition, an individual mitochondrion can contain two
isoforms of VDAC since in cells expressing both HVDAC1 and HVDAC2, and
all mitochondria apparently contain both isoforms. At the electron
microscopic level, these two different isoforms appear to be present
within the same restricted patch. Although the co-existence of two
isoforms in the same mitochondria may be an artifact of the expression
systems used in this study, these results raise the possibility that in
normal mammalian cells individual mitochondria contain a mixture of
VDAC isoforms. Direct demonstration of this possibility awaits the
generation of antibodies which will specifically distinguish each
wild-type protein. The only known functional difference between HVDAC1
and HVDAC2 is that only HVDAC1 can bind hexokinase, although sequence
differences in specific domains suggest that other biochemical
differences may exist (14). Since the association of hexokinase
with VDAC is dymanic and regulated, co-localization of these two
isoforms within the same mitochondrial domain suggests that
mitochondria can draw on pools of each molecule to establish
functionally distinct complexes, perhaps at contact sites and perhaps
composed of different spectra of proteins, depending on the
requirements of the cell. The nature of these complexes and their
functional differences await further experimentation.
We thank Dr. Elizabeth Blachly-Dyson for help in the
construction of cDNAs encoding epitope-tagged HVDAC molecules and Dr.
Blachly-Dyson and Dr. Gary Thomas for critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
also known as mitochondrial
porin) is a small (30-35 kDa), abundant pore-forming protein
found in the outer membranes of mitochondria from cells of all
eukaryotic kingdoms (for reviews see Refs. 1-3). The purified
protein, when incorporated into planar phospholipid bilayers, forms
large (
3 nm) voltage-gated pores that have been examined in great
detail by biophysical methods and are a model for voltage regulation of
membrane protein structure (for review see Ref. 4). Physiologically,
VDAC is thought to function as the primary pathway for the movement of
adenine nucleotides and other metabolites through the mitochondrial
outer membrane, thus controlling the traffic of these essential
compounds to and from this organelle as well as the entry of other
substrates into a variety of metabolic pathways. VDAC has also been
shown to be the site for binding of hexokinase and glycerol kinase to
the mitochondrial outer membrane. The binding of these enzymes to the
mitochondrion is dynamic, varying between different tissues, during
development, and depending on the metabolic state of the cell (for
reviews see Refs. 5, 6). It has been proposed that binding to the outer
membrane allows these enzymes preferential access to mitochondrial ATP,
thus regulating metabolism (7), although this view has been questioned
(8). Consistent with the notion that binding of these enzymes is an
important metabolic regulatory event, binding may occur specifically at
contact sites between the inner and outer mitochondrial membranes
potentially linking cytoplasmic metabolism and ATP production, as
regulated by hexokinase and glycerol kinase, to mitochondrial
respiration and oxidative phosphorylation (7, 9). Furthermore,
malignant cells found in tumors have an increased percentage of
mitochondrially bound hexokinase compared to normal cells (10) and it
appears that VDAC is part of a complex forming the mitochondrial
benzodiazepine receptor (11), a distinct receptor that is similar to
the central receptor in its affinity for diazepam but differs in its
affinity for other drugs.
Figure 1:
Comparison of amino-terminal sequences
of HVDAC isoforms as encoded by each cDNA. Residues 1-8 of HVDAC1
are aligned with residues 1-19 of HVDAC2 (14) and residues
1-35 of HVDAC2` (15).
These results have led to the hypothesis
that the distinct amino termini present in HVDAC1, HVDAC2, and HVDAC2`
target these molecules to different subcellular compartments.
Consistent with this hypothesis, a variety of studies suggest that
HVDAC1 is found in the plasma membrane of a number of different
mammalian cell types. Several reports using patch clamping techniques
have documented the presence of plasma membrane channels with
physiological properties similar to mitochondrial VDAC channels
incorporated into planar phospholipid bilayers (17, 18). More recently,
this channel activity has been correlated with the presence of homologs
of HVDAC1 in the plasma membrane of bovine astrocytes
(19) . In
addition, both polyclonal and monoclonal antibodies generated to
NH-terminal epitopes present in HVDAC1 purified from human
B lymphocytes appear to specifically label the plasma membrane by
immunocytochemical techniques (20-23). These antibodies also
label the plasma membrane of bovine astrocytes and block a high
conductance anion channel found in the plasma membrane of these cells
(19). The mouse homolog of HVDAC1 has also been found in plasma
membrane fractions enriched for caveolae, a plasma membrane microdomain
(24). Finally, the rat homolog of HVDAC1 has been found to co-purify
with the rat GABA
receptor complex found in the plasma
membrane of neurons (25).
Antibodies and Cell Lines
Mouse monoclonal
antibodies (M2) recognizing the FLAG epitope were purchased from
International Biotechnologies (New Haven, CT) and rabbit polyclonal
antibodies recognizing this epitope were a kind gift of Dr. Gary
Ciment, Oregon Health Sciences University. Mouse monoclonal antibodies
recognizing the HA (12CA5) epitope were purchased from Boehringer
Mannheim, and rabbit polyclonal antibodies (PRB-101C) recognizing this
epitope were purchased from BabCo (Richmond, CA). Fluorescein- and
rhodamine-conjugated secondary antibodies and avidin were purchased
from Vector Laboratories (Burlingame, CA). Gold-conjugated secondary
antibodies were purchased Amersham Corp. Polyclonal antibodies to
mammalian cytochrome c oxidase holoenzyme and the monoclonal
antibody against cytochrome c oxidase subunit IV
(10G8-D12-C12) were kind gifts from Dr. Jan-Willem Taanman (University
of Oregon, Eugene, OR). Polyclonal antibody against Signal Sequence
Receptor was provided by Dr. Rapopport (Berlin, Germany) (26).
.
Construction of Epitope-tagged HVDAC Molecules and Cell
Transfection
For HVDAC1, fragments were generated by polymerase
chain reaction using oligonucleotides spanning an internal
EcoRI site and inserting a novel ClaI site at the
stop codon. For HVDAC2 and HVDAC2`, fragments were generated by
polymerase chain reaction using oligonucleotides spanning an internal
BglII site and inserting a novel ClaI site at the
stop codon. These fragments were then used to replace the corresponding
fragments in each cDNA. Double-stranded DNA cassettes encoding the FLAG
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) and HA
(Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) epitopes containing 5`
ClaI sites and 3` XhoI sites followed by a stop codon
were inserted between the newly created ClaI sites at the COOH
terminus of each gene and a vector XhoI site to create cDNAs
encoding HVDAC1, HVDAC2, and HVDAC2` epitope-containing molecules.
Light-level Immunofluorescence
COS7 cells and rat
astrocytes were seeded onto glass coverslips and transfected as
described above. Transfected cells were fixed in phosphate-buffered
saline (PBS) containing 4% paraformaldehyde for 20 min at room
temperature. Fixed cells were then washed with PBS containing 0.2%
bovine serum albumin and 0.1% Triton X-100 (washing buffer) three times
and incubated in PBS containing 10% horse serum and 0.1% Triton X-100
for 1 h at room temperature. Primary antibodies (monoclonal or
polyclonal) were diluted in washing buffer, applied to coverslips, and
coverslips placed at 4 °C. Following overnight incubation,
coverslips were washed five times with washing buffer and then
incubated with fluorescein isothiocyante-conjugated anti-mouse or
anti-rabbit antibodies depending on the source of primary antibody. For
rhodamine detection, coverslips were incubated with biotin-conjugated
anti-mouse or anti-rabbit antibodies depending on the source of primary
antibody, washed five times with washing buffer, incubated with
avidin-rhodamine for 30 min followed by five washes.
Immunoelectron Microscopy
Transfected cells were
washed once with Versene (0.53 mM EDTA in PBS) and incubated
in 1 ml of Versene at room temperature for 3-5 min. Cell were
lifted from the dish by tapping the culture dish and fixed with 2%
paraformaldehyde, 0.1% glutaraldehyde in 50 mM PIPES, pH 7.2,
for 1 h on ice. Fixed cells were rinsed three times with 0.1 M
PIPES, embedded in agar, infiltrated with polyvinylpyrolidone and
sucrose (29), and prepared for frozen thin sectioning (30, 31). After
incubating with primary monoclonal or polyclonal antibodies, anti-mouse
or anti-rabbit secondary antibodies conjugated to gold particles
(anti-mouse, 5 or 10 nm: anti-rabbit, 15 nm) were applied to sections.
Immunolabeled sections were embedded in methylcellulose (31) and viewed
with a JOEL JEM-100CXII transmission electron microscope.
Biochemical Methods and
Immunoblotting
Unfractionated protein samples from transfected
and control cells were prepared by washing cells with PBS three times
followed by incubation in RIPA buffer (0.01 M Tris-HCl, pH
7.6, 0.15 M NaCl, 1% sodium deoxycholate, 1% Triton X-100,
0.1% SDS, 1% aprotinin, and 2 mM sodium vanadate) on ice.
Cellular material was then harvested by scraping and further incubation
on ice for 10 min. Samples were then centrifuged at 12,000
g for 10 min at 4 °C in a microcentrifuge and supernatants
mixed with SDS sample buffer.
Cl
for 10
min on ice. Cells were then harvested by scraping, homogenized with a
Dounce tissue grinder, and spun at 600
g for 5 min at
4 °C. Phenylmethylsulfonyl fluoride (2 mM) was added prior
to centrifugation. The resulting supernatant was centrifuged at 20,000
g for 10 min to produce a low speed pellet and
supernatant. The low speed supernatant was subjected to a subsequent
centrifugation at 235,000
g for 1 h. The low speed
(20,000
g) and the high speed (235,000
g) pellets were resuspended in the buffer used for washing
cells containing phenylmethylsulfonyl fluoride. The protein
concentration of various fractions was determined by the method of
Bradford (32, 33).
Expression of Epitope-tagged HVDAC Molecules in
Yeast
HVDAC1, HVDAC2, and HVDAC2` cDNAs containing epitope tags
were inserted into a single copy yeast plasmid (pSEYC58) so that
expression is driven by the endogenous yeast VDAC promoter. Resulting
plasmids were then introduced into a yeast strain lacking the
endogenous yeast VDAC gene (34) by transformation. Mitochondrial
protein of yeast transformants was prepared according to Blachly-Dyson
et al. (34).
HVDAC Constructs and Expression in Yeast
Human
isoforms of the outer mitochondrial membrane protein VDAC (HVDAC) share
extensive amino acid homology (roughly 75%) and do not differ
sufficiently in any one domain to allow the generation of antibodies
that will distinguish each isoform. To facilitate analysis of the
subcellular localization of individual HVDAC isoforms, we took
advantage of the fact that both monoclonal and polyclonal antibodies
are available which specifically recognize epitopes of defined amino
acid sequence. In this study, two such epitopes were employed; the
synthetic FLAG epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) and an epitope
derived from the human influenza hemagglutinin protein
(Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, HA). Cassettes encoding these
epitope were inserted into HVDAC1, HVDAC2, and HVDAC2` cDNAs so that
resulting proteins (HVDAC-FLAG or HVDAC-HA) contain either epitope at
their extreme carboxyl termini (``Materials and Methods'').
termini;
HVDAC1 migrates more rapidly than HVDAC2, consistent with the
additional NH
-terminal 11 amino acids present in HVDAC2,
and HVDAC2` migrates more slowly than HVDAC2, consistent with the
additional 15 NH
-terminal residues present in HVDAC2`
relative to HVDAC2. Similar results were obtained for HA-tagged
molecules (not shown).
Figure 2:
Immunoblot of yeast mitochondrial
membranes prepared from cells lacking VDAC (lane 1), or
expressing HVDAC1 (lane 2), HVDAC2 (lane 3), and
HVDAC2` (lane 4) each containing the FLAG epitope. The blot
was probed with mouse anti-FLAG monoclonal antibodies (3 µg/ml).
Mitochondrial membranes and immunoblots were prepared as described
under ``Materials and Methods.'' Each lane contains 10 µg
of protein.
As shown in Fig. 3, yeast strains
lacking the endogenous VDAC gene () are unable to grow on
non-fermentable carbon sources at 37 °C. Reintroduction of plasmids
containing the wild-type yeast VDAC gene (YVDAC) permits the growth of
these cells at the restrictive temperature. Introduction of plasmids
containing epitope-tagged forms of HVDAC1, HVDAC2, or HVDAC2` also
permits growth of these cells at the restrictive temperature. Since
wild-type forms of these human proteins complement this growth defect
(14), these results indicate that the function of the tagged molecules
is not significantly altered by the presence of either epitope at the
COOH terminus. In addition, epitope-tagged HVDAC molecules purified
from yeast are able to form channels when incorporated into planar
phospholipid bilayers (not shown).
Figure 3:
Differential growth of stains lacking VDAC
and strains expressing epitope-tagged HVDAC molecules on media
containing glyerol. A yeast strain in which the chromosomal VDAC gene
was deleted ()
was transformed with plasmids
mediating the expression of the wild-type yeast VDAC (YVDAC)
and individual FLAG-tagged or HA-tagged HVDAC proteins. The resulting
strains were then streaked on media containing 2% glycerol as the sole
carbon source and incubated at the indicated
temperatures.
Expression of HVDACs in Mammalian Cells
Expression
of tagged HVDAC constructs in mammalian cells was achieved by
subcloning cDNAs encoding each tagged construct into either pCD/PS or
pRC/RSV followed by transient transfection into COS7 cells. Forty-eight
h following transfection, immunocytochemical analysis (see below)
indicated that roughly 20-40% of transfected cells expressed
either epitope-tagged form of HVDAC1 and HVDAC2 while roughly 10%
express epitope-tagged forms of HVDAC2`. Western blots of total
cellular protein prepared from transfected cells and probed with
monoclonal antibodies to either the FLAG or HA epitope demonstrated
that single proteins of appropriate size were present in cells
containing either tagged form of HVDAC1 or HVDAC2
(Fig. 4A). In addition, Western blots of total cellular
protein prepared from cells co-transfected with both HA-tagged HVDAC1
and FLAG-tagged HVDAC2 demonstrate that roughly equal levels of both
tagged proteins are expressed in these cells (Fig. 4B).
However, two bands were detected in cells transfected with
epitope-tagged forms of HVDAC2`. Bands of similar relative molecular
weight arise by use of alternate 5` translational initiation points
following in vitro translation of HVDAC2` transcripts (15). No
Western positive bands were observed in untransfected COS7 cells or
cells transfected with vector alone (Fig. 4A).
Figure 4:
Immunoblots of total protein prepared from
transfected COS7 cells. A, samples prepared from untransfected
cells (lane 1), cells transfected with vector alone (lane
2), and vector containing HVDAC1-FLAG (lane 3),
HVDAC2-FLAG (lane 4), and HVDAC2`-FLAG (lane 5)
constructs. Also shown for comparison are samples prepared from yeast
mitochondria containing HVADC1-FLAG (lane 6), HVDAC2-FLAG
(lane 7), and HVDAC2`-FLAG (lane 8). B,
samples prepared from cells co-transfected with vectors containing
HVDAC1-HA and HVDAC2-FLAG constructs. Blots were probed with either
mouse anti-FLAG (1.7 µg/ml) or mouse ant-HA (0.5 µg/ml)
monoclonal antibodies. Each lane contains protein from an equal number
of transfected cells.
The
subcellular location of each epitope-tagged HVDAC molecule was
initially determined by differential centrifugation of extracts
prepared from COS7 cells transfected with HVDAC constructs expressing
FLAG-tagged molecules. Aliquots of fractions enriched for soluble
components of the cytoplasm (high speed supernatant), mitochondria (low
speed pellet), and other membrane components (high speed pellet) were
then separated on SDS-polyacrylamide gels and Western blots probed with
anti-FLAG monoclonal antibodies. To identify mitochondrial fractions,
blots were subsequently reprobed with a monoclonal antibody to subunit
IV of mitochondrial cytochrome c oxidase. As shown in
Fig. 5
, HVDAC-FLAG proteins and subunit IV were both exclusively
detected in low speed pellets expected to be enriched in mitochondria
and not in soluble cytoplasmic fractions or in high speed pellets
containing other cellular membranes including the plasma membrane. This
analysis strongly suggests that epitope-tagged HVDAC1, HVDAC2, and
HVDAC2` molecules are located in cellular fractions enriched in
mitochondria.
Figure 5:
Immunoblots of subcellular fractions
prepared from transfected COS7 cells. Fractions were prepared as
described under ``Materials and Methods.'' In each case,
lanes represent samples of high speed supernatant (lane 1),
high speed pellet (lane 2), low speed pellet (lane
3), and samples prepared from yeast mitochondria containing the
appropriate FLAG-tagged HVDAC protein (lane 4). The amount of
protein loaded in each case was varied due to differences in
transfection efficiency in specific populations of cells. a,
fractions prepared from cells transfected with HVDAC1-FLAG constructs.
Each lane contains 30 µg of protein. b, fractions prepared
from cells transfected with HVDAC2-FLAG constructs. Each lane contains
50 µg of protein. c, fractions prepared from cells
transfected with HVDAC2`-FLAG constructs. Each lane contains 100 µg
of protein. Blots were probed with mouse anti-FLAG monoclonal
antibodies (3 µg/ml) or monoclonal antibodies to subunit IV of
mitochondrial cytochrome c oxidase. Arrow indicates
the position of subunit IV (17.2 kDa).
Light Microscopic Localization of HVDAC
Isoforms
Mitochondrial localization of epitope-tagged HVDAC
molecules was confirmed by indirect immunofluorescence microscopy. COS7
cells were seeded on glass coverslips and transfected with cDNAs
encoding either HA or FLAG-tagged forms of HVDAC1, HVDAC2, and HVDAC2`.
After fixation, cells were incubated with mouse monoclonal antibodies
to the appropriate epitope and a rabbit polyclonal antibody raised
against the mitochondrial cytochrome c oxidase complex to
identify the location of mitochondria. Antibodies to the signal
sequence receptor were used to identify the position of the rough
endoplasmic reticulum. As shown in Fig. 6for cells expressing
FLAG-tagged HVDAC proteins, antibodies to the FLAG epitope specifically
label the same subcellular structures as antibodies to mitochondrial
cytochrome c oxidase. In both cases, staining appears
exclusively in a punctate or vermiform organelles distributed
throughout the cytoplasm with higher concentrations in perinuclear
areas, as expected for mitochondria. The appearance of the stained
organelles appears to alternate between punctate and vermiform in a
cell-specific fashion independent of the antibody used for staining
(FLAG, HA or cytochrome c oxidase). In addition, close
examination of stained organelles at this level suggests that HVDAC
isoforms are unevenly distributed within an individual mitochondrion
compared to cytochrome c oxidase staining (for example,
compare Fig. 6, E and F). Signal sequence
receptor staining, on the other hand, is clearly different and is
present in a diffuse, reticulate pattern characteristic of the ER. In
no case is there significant plasma membrane-associated staining in
cells transfected with any FLAG-tagged HVDAC constructs nor is plasma
membrane staining more intense in transfected cells when compared to
untransfected cells (Fig. 6). Identical results were obtained
with cells transfected with HA-tagged HVDAC constructs (not shown).
Figure 6:
Colocalization of epitope-tagged HVDAC
molecules and cytochrome c oxidase in transfected COS7 cells.
Cells expressing HVDAC1-FLAG (a and b), HVDAC2-FLAG
(c and d), and HVDAC2`-FLAG (e and
f) stained with mouse anti-FLAG monoclonal antibodies
(a, c, and e) and the same field of cells
stained rabbit polyclonal antibodies to the mitochondrial cytochrome
c oxidase holoenzyme (b, d, and f).
Also shown are COS7 cells expressing HVDAC1-FLAG stained with mouse
anti-FLAG monoclonal antibodies (g) and a rabbit polyclonal
antibody to the signal sequence receptor of the rough endoplasmic
reticulum (h).
A recent report of plasma membrane-localized VDAC molecules has
suggested the presence of homologs of HVDAC1 in the plasma membrane of
rat astrocytes (19). To examine the subcellular location of
epitope-tagged HVDAC molecules in these cells and to investigate the
possibility that cell type specific differences may exist in the
targeting of these molecules, rat astrocytes were transiently
transfected with constructs encoding FLAG-tagged HVDAC1, HVDAC2, and
HVDAC2` molecules. Cells were then processed for indirect
immunofluorescence as described above for COS7 cells (paraformaldehyde
fixation) or as described in Dermietzel et al. (19) (ethanol
fixation). Following fixation, cells were incubated with mouse
monoclonal antibodies to the FLAG epitope and the rabbit polyclonal
antibody raised against the cytochrome c oxidase complex to
identify the location of mitochondria. As shown in Fig. 7for
astrocytes expressing FLAG-tagged HVDAC2 molecules, the FLAG antibody
identified the same set of punctate or vermiform organelles as was
identified by the cytochrome c oxidase antibody. Similar
results were obtained in cell expressing FLAG-tagged HVDAC1 and HVDAC2`
molecules (not shown). In these cases, plasma membrane staining appears
to be present in transfected cells. However, this staining can be
attributed entirely to a nonspecific interaction of the FLAG antibodies
with astrocyte plasma membrane components since identical levels of
staining are present in non-transfected cells. In addition, plasma
membrane staining in these cells cannot be due to differences in
fixation technique since identical results were obtained regardless of
whether cells were fixed with paraformaldehyde, as was used for COS7
cells, or ethanol, as was used in the studies of Dermietzel et al. (19) (Fig. 7). Taken together, these indirect
immunocytochemical studies confirm initial conclusions reached on the
basis of cell fractionation studies that epitope-tagged HVDAC1, HVDAC2,
and HVDAC2` are located exclusively in mitochondria in at least two
distinct cell types.
Figure 7:
Colocalization of epitope-tagged HVDAC2
molecules and cytochrome c oxidase in transfected rat
astrocytes. Rat astrocytes were transfected with vectors containing
HVDAC2-FLAG constructs and then fixed with ethanol at -20 °C
(a) or paraformaldehyde (b and c) as
described under ``Materials and Methods.'' Fixed cells were
then incubated with mouse anti-FLAG monoclonal antibodies (a and b). The cell shown in b was also stained
with a rabbit polyclonal antibody to the mitochondrial cytochrome c oxidase holoenzyme (c).
The results outlined above indicate that
multiple isoforms of mammalian VDAC proteins are directed to the
mitochondrion. To determine whether an individual mitochondrion
contains only one VDAC isoform or multiple isoforms, COS7 cells were
co-transfected either with constructs encoding HA-tagged HVDAC1 and
FLAG-tagged HVDAC2 or FLAG-tagged HVDAC1 and HA-tagged HVDAC2.
Transfected cells were then processed either for immunofluorescence
microscopy as described above using mouse monoclonal antibodies to one
epitope and rabbit polyclonal antibodies to the other epitope to define
the subcellular location HVDAC1 and HVDAC2 respectively. As shown in
Fig. 8
for cells expressing HA-tagged HVDAC1 and FLAG-tagged
HVDAC2, both antibodies identify an identical set of subcellular
structures distributed in the same perinuclear punctate pattern as was
observed for mitochondria (see Fig. 5and Fig. 6),
indicating that an individual mitochondria can contain at least two
different VDAC isoforms. Similar results were obtained in cells
expressing FLAG-tagged HVDAC1 and HA-tagged HVDAC2 (not shown).
Figure 8:
Colocalization of epitope-tagged HVDAC1
and HVDAC2 in co-transfected COS7 cells. Cells were stained with mouse
anti-HA monoclonal antibodies (a) and a rabbit anti-FLAG
polyclonal antibody (b).
Electron Microscopic Localization of HVDAC
Isoforms
To precisely determine the subcellular location of
epitope-tagged HVDAC isoforms, COS7 cells were transfected with
constructs encoding either HA- or FLAG-tagged HVDAC1 and HVDAC2 and
frozen thin sections prepared for immunoelectron microscopy. Due to low
transfection efficiency, cells transfected with constructs encoding
epitope-tagged forms of HVDAC2` could not be analyzed by this
technique. Frozen thin sections were subsequently incubated with the
appropriate monoclonal antibody and antibody binding detected with a
secondary antibody conjugated to colloidal gold particles. As shown in
Fig. 9
for FLAG-tagged HVDAC1, gold particles were associated with
mitochondria, and no binding of FLAG antibodies was detected in plasma
membrane regions or other cellular compartments above background levels
observed in untransfected cells. Similar results were obtained when
cells transfected with constructs encoding FLAG-tagged HVDAC2 and
HA-tagged forms of HVDAC1 and HVDAC2 were examined (not shown). The
resolution in these frozen thin sections and uncertainty of the plane
of section in any one sample make it difficult; however, to
unambiguously associate all of the labeling observed exclusively with
the outer mitochondrial membrane although no staining of mitochondrial
cisternae is present in images where these structures can be
identified. However, each VDAC isoform does not appear to be uniformly
distributed within an individual mitochondrion but appears to be
clustered at discrete locations on the mitochondrial surface as
reflected in the uneven distribution of gold particles. This result is
consistent with the apparent uneven distribution of HVDAC molecules
within an individual mitochondria observed in light level studies
(Fig. 6).
Figure 9:
Immunoelectron microscopic localization of
epitope-tagged HVDAC molecules in transfected COS7 cells. Frozen
sections were prepared from cells transfected with HVDAC1-FLAG
constructs and sections incubated with mouse anti-FLAG monoclonal
antibodies. Antibody binding was detected with goat anti-mouse
antibodies conjugated to 10 nm gold particles (a). Frozen
sections were prepared from cells co-transfected with HVDAC1-HA and
HVDAC2-FLAG constructs. Sections werethen incubated with rabbit anti-HA
polyclonal antibodies and mouse anti-FLAG monoclonal antibodies.
Binding of rabbit antibodies (HVDAC1-HA) was detected using goat
anti-rabbit antibodies conjugated to 15 nm gold particles and binding
of mouse monoclonal antibodies (HVDAC2-FLAG) detected using goat
anti-mouse antibodies conjugated to 5 nm gold particles (b).
m, mitochondria; pm, plasma membrane. bar = 0.5 µm.
As demonstrated above at the light level
(Fig. 8), an individual mitochondria can contain at least two
different VDAC isoforms. This conclusion was confirmed at the electron
microscopic level by preparing frozen thin sections from co-transfected
cells and processing sections for immunodetection of each epitope using
appropriate secondary antibodies conjugated to colliodal gold particles
of different diameters. As shown in Fig. 9for cells expressing
HA-tagged HVDAC1 and FLAG-tagged HVDAC2, the two VDAC isoforms are
expressed within overlapping mitochondrial domains. As was the case for
cells expressing a single, epitope-tagged HVDAC molecule, areas
containing both isoforms are not uniformly distributed and are located
in discrete patches along the mitochondrial surface. Similar results
were obtained with cells co-transfected with FLAG-tagged HVDAC1 and
HA-tagged HVDAC2 or when the HA epitope was identified with a mouse
monoclonal antibody and the FLAG epitope identified with a polyclonal
antibody (not shown).
-terminal extensions in HVDAC2 and HVDAC2`, a
splice variant of HVDAC2, relative to HVDAC1 (14, 15). Since
amino-terminal sequences have been demonstrated to target many proteins
to appropriate subcellular compartments, this observation raises the
possibility that the NH
-terminal differences found in HVDAC
isoforms may lead to targeting of each protein to a different cellular
locations. Consistent with this hypothesis, a large number of recent
reports have provided evidence consistent with the notion that HVDAC1
and its homolog in related mammalian species may specifically be
present in the plasma membrane of a number of different mammalian cell
types (19-23). To determine the subcellular location of HVDAC1
and define the cellular compartments containing other members of the
VDAC family of proteins, cDNAs encoding these proteins were modified to
incorporate sequences directing the insertion of either of two epitopes
(HA or FLAG) at the extreme COOH terminus of each protein. The
inclusion of either epitope appeared not to affect the function of
these molecules since each is efficiently targeted to the yeast
mitochondria and is capable of complementing growth defects associated
with the elimination of the endogenous yeast VDAC gene, as are the
unmodified forms of these human proteins. Since specific antibodies
only for HVDAC1 have been described, this system provides a convenient
way to individually tag each HVDAC isoform and follow the expression of
these molecules in a variety of cell types.
-terminal
signal sequences present in precursors of proteins targeted to the
mitochondrial matrix, inner membrane, or intermembrane space (37, 38).
In cases where targeting sequences have been identified for
mitochondrial outer membrane proteins, the targeting sequences have
been found at the amino terminus (37, 38). These
NH
-terminal targeting domains lack apparent homology and
appear not to share conserved structural features. Since each HVDAC
expressed in yeast is targeted to mitochondria, it is reasonable to
assume that the distinct NH
-terminal domains present in
each HVDAC directs these molecules to the mitochondria in both yeast
and mammalian cells, although this remains to be demonstrated directly.
-terminal extension relative
to HVDAC1 (14, 15). Consistent with this expectation, the relative
mobility of these proteins on SDS-polyacrylamide gels is retarded by
the amount expected (roughly 11 residues for HVDAC2 and 26 residues for
HVDAC2`) relative to HVDAC1 (Fig. 2, 4, and 5). Since the
NH
terminus of these and essentially all other VDAC
proteins is blocked (39, 40), direct determination of the
NH
-terminal sequence of each protein is not possible.
Results obtained with HVDAC2` expressed in COS7 cells are informative
in this regard. Translation of cDNAs encoding HVDAC2` in vitro results in the production of two proteins that apparently differ
by the alternate use of NH
-terminal methionines for the
initiation of protein synthesis; the first ATG codon present in cDNAs
for HVDAC2` and an internal ATG corresponding to the translational
initiation point for HVDAC1 (15). Since the size of the HVDAC2` protein
expressed in yeast is larger than either HVDAC1 and HVDAC2, this first
ATG codon is apparently used exclusively for translational initiation
in yeast. However in COS7 cells, both start sites are apparently used
with roughly equal frequency although it is also possible that this
NH
-terminal extension may be proteolytically cleaved when
expressed in these cells. HVDAC2` molecules generated by initiation at
the second ATG site or by proteolysis of NH
-terminal
extensions are essentially identical to HVDAC1 in size and
NH
-terminal sequence and might be expected to be targeted
primarily to mitochondria as has been shown here for HVDAC1. However,
since molecules containing the HVDAC2`-specific NH
-terminal
extension (initiation at the first ATG) are equally abundant, both
NH
termini are apparently sufficient for targeting to the
mitochondria since all the epitope marker in cells transfected with
tagged HVDAC2` constructs is exclusively associated with mitochondria.
Thus, it may be that a diverse set of sequences lacking in apparent
sequence or structural homology is responsible for directing proteins
to the mammalian outer mitochondrial membrane. Alternatively,
mitochondrial targeting could be directed by sequences present at the
NH
terminus of HVDAC1 and equivalent positions in HVDAC2
and HVDAC2`, since these regions are essentially identical in each of
the three proteins. It is also possible that other domains within these
highly homologous proteins are responsible for targeting. In this
event, NH
-terminal extensions present in HVDAC2 and HVDAC2`
may function in processes unrelated to mitochondrial targeting.
receptor, or with plasma membrane
specializations such as caveolae (19, 24, 25, 39). In these cases, the
starting material for purification is essentially a total membrane
fraction including mitochondria. Since VDAC is the major protein of
outer mitochondrial membranes, it is easy to imagine that outer
membranes, and therefore VDAC, can contaminate these preparations. VDAC
may then nonspecifically associate with other proteins during
subsequent solubilization and purification. In this light, it is
interesting to note that VDAC can easily be separated from
``purified'' GABA
receptors by subsequent
chromatographic steps (25). Second, a large number of studies reporting
the presence HVDAC1 in the plasma membrane of a variety of cell types
depend primarily on the use of a set of monoclonal antibodies that
recognize the NH
terminus of HVDAC1 (19, 21-23).
Initial reports suggested that these antibodies specifically recognize
plasma membrane VDAC forms (21), although more recent studies indicate
that they also react quite well with mitochondria (41). In these cases,
it is easy to imagine that nonspecific reactivity with plasma membranes
may have been mistaken for the presence of HVDAC molecules in this
cellular compartment. As shown in Fig. 7, antibodies for epitopes
not found in nature (FLAG) can label plasma membranes in specific cell
types, although this labeling is clearly nonspecific. Consistent with
this interpretation, these monoclonal antibodies are only able to
recognize ``plasma membrane'' forms of VDAC if they are added
to living cells prior to fixation (23). In addition, polyclonal
antisera to mammalian VDAC proteins have been generated by a number of
other groups which fail to identify plasma membrane forms of VDAC (42,
43). While it is impossible to exclude the possibility that some very
small fraction of the VDAC is present in the plasma membrane below the
limits of detection of the three techniques used here, our results
indicate that it is unlikely that any significant fraction of VDAC can
be in the plasma membrane and that the vast majority of this protein is
located in mitochondria, regardless of isoform. Finally, a number of
studies report the presence of plasma membrane channels with
physiological characteristics similar to purified VDAC when
incorporated into planar lipid bilayers (17-19). In the majority
of these studies, VDAC-like channels are only observed following
incubation of excised plasma membrane patches in rather unusual ionic
conditions for extended periods of time (many minutes). The results
presented here demonstrate that if the VDAC-like channels identified in
these patch clamping studies exist in the plasma membrane under normal
conditions, they are unlikely to be due to the presence of HVDAC1, as
reported, or HVDAC2 or HVDAC2`. Rather, these channels must be composed
of proteins that remain to be identified or characterized.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.