©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subcellular Localization of Human Voltage-dependent Anion Channel Isoforms (*)

Wei Hong Yu (1) (2), William Wolfgang (1), Michael Forte (1)(§)

From the (1) Vollum Institute for Advanced Biomedical Research and the (2) Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The voltage-dependent anion channel of the mitochondrial outer membrane (VDAC,() 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.

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.


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).

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.


MATERIALS and METHODS

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).

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.

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.

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).

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.

Cells were also processed for light level immunocytochemical analysis by the methods outlined in Dermietzel et al.(19) .

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.

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 MCl 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).

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).

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).


RESULTS

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'').

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 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).


DISCUSSION

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-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.

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-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.

Cloning of cDNAs for HVDAC2 and HVDAC2` indicate that each is expressed with a different NH-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.

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 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.

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.


FOOTNOTES

*
This work was supported by Grant GM35759 from the National Institutes of Health (to M. F.) and Tartar Trust fellowship (to W-H. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vollum Institute, L474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-5454; Fax: 503-494-4976; E mail: forte@ohsu.edu.

The abbreviations used are: VDAC, voltage-dependent anion channel; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

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.


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