(Received for publication, March 13, 1997, and in revised form, May 16, 1997)
From the Departments of Molecular and Human Genetics
and § Pediatrics, Baylor College of Medicine,
Houston, Texas 77030
Voltage-dependent anion channels
(VDACs) are pore-forming proteins found in the outer mitochondrial
membrane of all eucaryotes. VDACs are the binding sites for several
cytosolic enzymes, including the isoforms of hexokinase and glycerol
kinase. VDACs have recently been shown to conduct ATP when in the open
state, allowing bound kinases preferential access to mitochondrial ATP
and providing a possible mechanism for the regulation of adenine
nucleotide flux. Two human VDAC cDNAs have been described
previously, and we recently reported the isolation of mouse VDAC1 and
VDAC2 cDNAs, as well as a third novel VDAC cDNA, designated
VDAC3. In this report we describe the structural organization of each
mouse VDAC gene and demonstrate that, based on conserved exon/intron
boundaries, the three VDAC isoforms belong to a single gene family. The
5-flanking region of each VDAC gene was shown to have transcription
promoter activity by transient expression in cultured cells. The
promoter region of each VDAC isoform lacks a canonical TATA box, but
all are G+C-rich, a characteristic of housekeeping gene promoters. To
examine the conservation of VDAC function, each mouse VDAC was
expressed in yeast lacking the endogenous VDAC gene. Both VDAC1 and
VDAC2 are able to complement the phenotypic defect associated with the
mutant yeast strain. VDAC3, however, is only able to partially
complement the mutant phenotype, suggesting an alternative physiologic
function for the VDAC3 protein.
Voltage-dependent anion channels (VDACs,1 also known as mitochondrial porins) are 30-35-kilodalton (kDa) pore-forming proteins found in the outer mitochondrial membrane of eucaryotes (reviewed in Ref. 1). VDACs play a role in the regulated flux of metabolites across the outer mitochondrial membrane, but their exact cellular role is not well understood. VDACs from a variety of organisms have remarkably similar electrophysiological properties (1). Gating of the channel depends upon the transmembrane potential, while its voltage sensitivity is modulated by an intermembrane protein (2). VDACs are "open" at low transmembrane potentials, with a preference for anions such as phosphate, chloride, and adenine nucleotides. At higher transmembrane potentials, VDACs are in a "closed" configuration and more selective for cations (2, 3). VDACs have been shown to reversibly bind several cytosolic kinases, including glycerol kinase and the hexokinase isoforms I-IV (reviewed in Ref. 4). This interaction is believed to allow bound kinases preferential access to mitochondrial ATP derived from oxidative phosphorylation (5, 6). VDACs have also been associated with the adenine nucleotide translocator of the inner mitochondrial membrane and octomeric creatine kinase of the intermembrane space (6-8). It has been suggested that this complex has properties resembling the permeability transition pore (9).
A direct demonstration of voltage-gated ATP flux through VDAC was
recently reported (10). Physiological concentrations of NADH also
affect VDAC permeability, suggesting one possible mechanism for the
observed ability of glycolysis to suppress oxidative phosphorylation (the Crabtree effect; Refs. 11 and 12). VDACs have been identified as a
component of the peripheral benzodiazepine receptor complex (13), which
is linked to steroid biosynthesis (14). Finally, VDACs have been shown
to co-purify with the brain -aminobutyric acid subunit A receptor
complex (15).
cDNAs encoding two human VDAC isoforms were reported by Blachly-Dyson et al. (16). We have previously described the isolation of mouse orthologues for VDAC1 and VDAC2, as well as a novel mouse VDAC termed VDAC3 (17, 18). Each isoform is 65-70% identical to the other isoforms. Phylogenetic analysis indicates that VDAC3 is the more primordial of the vertebrate VDAC genes, suggesting that if the multiple isoforms arose from a gene duplication and divergence event VDAC3 diverged from the primordial VDAC first, with VDAC1 and VDAC2 arising more recently (18). The three mouse genes have been mapped to separate autosomal loci (17, 18).
To determine whether the genomic organization can be correlated with existing structural information about the VDAC protein, and whether the VDAC isoforms arose by gene duplication and divergence or evolutionary convergence, we have characterized the gene structure for each VDAC locus. In addition, we have examined the promoter regions of each VDAC gene by DNA sequence analysis and expression studies to discern any regulatory similarities. Furthermore, to begin to examine the functional properties of VDAC3, the cDNA was expressed in a VDAC-deficient yeast strain and tested for its ability to complement the temperature-sensitive growth phenotype of a VDAC-deficient yeast.
To generate
isoform-specific probes, the polymerase chain reaction (PCR) was used
to amplify the 3-untranslated region of each mouse VDAC isoform (from
nucleotides 959-1277 for VDAC1, 942-1276 for VDAC2, and 1022-1428
for VDAC3 (17, 18). Each PCR product was used to generate
[
-32P]dCTP (NEN Life Science Products) random
primer-labeled probes. The three probes were used to screen a 129/sv
FixII genomic library (Stratagene, La Jolla, CA). Hybridizations
were carried out in Blotto (1.5 × SSPE, 1% SDS, 0.5% nonfat
dried milk) at 65 °C. Purified phage were isolated by three rounds
of sequential purification, and restriction mapping was performed using
standard methods.
Genomic fragments that hybridized to
the full-length cDNAs were subcloned into pBluescript KS vectors
using established protocols. The fragments were sequenced using
Universal and Reverse primers on an Applied Biosystems model 373A
automated fluorescent DNA sequencer. When necessary, complementary
oligonucleotides were used to complete the sequencing of all VDAC
exons. When the genomic clone did not contain the entire genomic
structure of the VDAC isoform, a 5 intronic fragment from the genomic
clone was used to screen the genomic library as before. The new genomic
clone isolated was then hybridized with both cDNA and 5
-RACE
probes until the entire genomic structure of each isoform was
characterized. Nucleotide sequence analysis was performed using the GCG
software package (51).
Total RNA was extracted using
guanidinium isothiocyanate from mouse tissues as described (19). The
RNA was used in the 3-RACE (rapid amplification of cDNA ends)
system (Life Technologies, Inc.) following the manufacturer's
protocol. For VDAC1, PCR amplification of the first-strand cDNA was
performed using two sense nested primers (5
-CTAGCCTGATTGGCTTAGGG-3
and 5
-CCTGCTCGATGGCAAGAAC-3
, respectively). For VDAC2, two sense
nested primers were also used to amplify the 3
-RACE product from
first-strand cDNA (5
-CATTGCAGCTAAATACCA-3
and
5
-GGTACCAACTGCACTCGTTTTG-3
, respectively). The amplified PCR products
were subcloned into a plasmid T-vector (20) and sequenced.
Total RNA was
extracted using guanidinium isothiocyanate from embryonic stem (ES)
cells as described above. RNA (~20 µg/lane) was fractionated on a
1% agarose/formaldehyde gel and transferred to Hybond N+ membranes
(Amersham Life Science). To prevent probe cross-hybridization the
membranes were probed with the 3-untranslated regions of each VDAC
isoform labeled as before. Hybridizations were carried out overnight at
65 °C in Blotto. The membranes were washed twice in 2 × standard saline citrate, 0.1% SDS for 10 min and exposed for 16-24 h
at
80 °C.
Ten micrograms of genomic DNA from the
species listed in Fig. 2 were digested with EcoRI, separated
by electrophoresis on a 0.7% agarose gel, and Southern-blotted onto a
Hybond N+ membrane (Amersham Life Science). Each full-length VDAC
cDNA was labeled as before and used to probe the blot.
Hybridization was carried out in Blotto at 55 °C overnight. After
hybridization the filter was washed several times in 3 × SSC,
0.1% SDS at 37 °C and set to expose for several days. The blot was
stripped after each hybridization by washing in 0.1 × SSC, 0.1%
SDS at 65 °C.
CAT Reporter Gene Constructs
CAT (chloramphenicol
acetyltransferase) reporter plasmid constructs of the 5-flanking
region of each mouse VDAC isoform were prepared by standard cloning
techniques. The VDAC1 5
-flanking 918-base pair (bp) fragment was
generated by PCR using a sense oligonucleotide upstream of the first
exon and an antisense oligonucleotide from the 5
-untranslated region
(5
-GCTTGATATCGAATTCTCCTCG-3
and 5
-GGGAGCAGCGGCAGCTAC-3
,
respectively; 94 °C for 1 min, 55 °C for 1 min, and 72 °C for
1 min, 30 cycles). The PCR product was subcloned into a plasmid
T-vector (20), and the restriction enzymes XbaI and
AccI were used to subclone the DNA in both orientations in
the pCAT Basic and Enhancer vectors (Promega, Madison, WI) using
adapter oligonucleotides. The same strategy was used to subclone a
smaller 513-bp VDAC1 5
-flanking fragment using a different upstream
sense oligonucleotide (5
-GCCGCTCTGACCTATACAGC-3
) and the same
5
-untranslated antisense oligonucleotide. A VDAC2 1014-bp EcoRI/PvuII fragment was isolated from a genomic
phage clone and subcloned into pKS. The restriction enzymes
XbaI and SalI were used to subclone the DNA in
both orientations in the pCAT Basic vector using adapter
oligonucleotides. A 4-kilobase pair (kb) upstream EcoRI
VDAC2 fragment was subcloned into the EcoRI site of the
1014-bp DNA in the correct orientation, and also inserted into the pCAT
Basic vector as before. Since the most 5
VDAC3 genomic clone isolated
only contained an additional 529 bp of upstream genomic sequence, a
549-bp XbaI/TaqI fragment was subcloned into pKS,
and the restriction enzymes XbaI and AccI were
used to subclone the DNA in both orientations in the pCAT Basic and Enhancer vectors using adapter oligonucleotides. The pCAT Basic vector,
which lacks a promoter and enhancer, and the pCAT Enhancer vector,
which lacks a promoter, were used as negative controls, while the pCAT
Control vector containing both the promoter and enhancer of simian
virus 40 (SV40) was used as a positive control. DNA was prepared using
a Qiagen plasmid miniprep kit (Qiagen, Santa Clarita, CA).
NIH3T3 cells were cultured in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum, and
cells were plated at a density of 1.5 × 105 cells/60
mm plate. The following day, transfections were carried out using 1 µg of CAT construct DNA, 8 µl of LipofectAMINE (Life Technologies,
Inc.), and 0.2 µg of the -galactosidase expression vector pCMV
(Promega, Madison, WI) in serum-free medium (Opti-MEM, Life
Technologies, Inc.) following the manufacturer's recommended protocol.
The cells were harvested 48 h after transfection, and lysed by
three freeze-thaw cycles. Cell extracts (20 µl) were heated for 10 min at 65 °C and assayed for CAT activity, as described by Sambrook
et al. (49). CAT assay results were quantified using a
Betascope Betascanner (IntelliGenetics, Cambridge, MA). CAT activities
are expressed as a percentage of the pCAT control.
For each of the three
VDAC isoforms oligonucleotide-directed mutagenesis was used to create
NcoI sites at the start codon for both VDAC1 and VDAC2, and
an AflIII site for VDAC3. The same strategy was used to
generate NsiI sites in the 3-untranslated region of each
gene. This allowed for the complete open reading frame of each VDAC
cDNA to precisely replace the yeast VDAC gene previously cloned
into a yeast single-copy shuttle vector (kindly provided by M. Forte,
Oregon Health Sciences University, Portland, OR; Ref. 22). The
oligonucleotides for each gene were used to amplify the cDNA
inserts, the products were digested with the appropriate restriction
enzymes, and the fragments were subcloned to replace the yeast VDAC
gene between the NcoI and NsiI sites. From the
starting ATG codon, the length of each cDNA insert was 1111 bp for
VDAC1, 1079 bp for VDAC2, and 1014 bp for VDAC3. The three constructs
were then introduced into the VDAC-deficient yeast strain M22-2 by
lithium acetate transformation (23). The yeast were streaked onto media
containing 2% glycerol as the sole carbon source and incubated at
30 °C or 37 °C for 6 days.
The importance of VDAC proteins for normal metabolic homeostasis was recently emphasized by the report of a child with a mitochondrial myopathy who was shown by Western analysis to have a partial deficiency of VDAC1 protein (24). The disorder appeared to be somewhat tissue-specific, with a greater deficiency of VDAC1 in skeletal muscle than fibroblasts. The patient's skeletal muscle mitochondria also exhibited reduced rates of pyruvate oxidation and ATP production. Since VDACs are expressed in nearly all tissues and have been shown to bind various kinases in liver, fat, and brain tissues and in several tumor cell lines, a global role for VDACs in metabolic homeostasis has been suggested (4). However, despite the extensive electrophysiological characterization of the VDAC channels from numerous organisms, the functional roles of the VDAC isoforms in metabolic homeostasis are not well understood. To begin to understand the genetic basis for VDAC function we have determined the structural and regulatory regions of the murine genes encoding the three VDAC isoforms.
Structure of the Mouse VDAC GenesThe VDAC1 gene spans
approximately 28 kb, and is made up of 9 exons (Fig. 2). The predicted
start codon is located 4 bp into the second exon. Sequencing of the
first 5-untranslated exon of VDAC1 identified 41 bp of cDNA
sequence previously obtained by 5
-RACE (17). Since high promoter
activity is detected in the region immediately 5
to this sequence,
transcription initiation is predicted to occur at or close to this
sequence. In addition to the spliced functional gene, a total of five
distinct VDAC1 processed pseudogenes, each without an open reading
frame, were isolated and sequenced from the genomic library (data not
shown).
The VDAC2 gene, unlike VDAC1 or VDAC3, is encoded by 10 exons, with the
additional exon constituting part of the 5-untranslated region. VDAC2
spans approximately 12 kb, with the 132-bp 5
-untranslated region (17)
encoded in the first two exons. The predicted start codon of VDAC2 is
located 26 bp into the second exon. A single VDAC2 processed pseudogene
lacking an open reading frame was also isolated (data not shown).
The VDAC3 gene is encoded by 9 exons and spans approximately 16 kb. The
predicted start codon is found 3 bp into the second exon, and the first
and second exons contain the entire 78 bp of 5-untranslated sequence
predicted by 5
-RACE (18). A single VDAC3 processed pseudogene lacking
an open reading frame was isolated, as well as an intronless VDAC3-like
sequence with a complete open reading frame (data not shown; GenBankTM
U89990). This sequence differs from the VDAC3 cDNA sequence at only
eight nucleotide positions, five of which are in the predicted coding
region, leading to three silent codon changes and two amino acid
substitutions; a glycine to glutamic acid substitution and tryptophan
to glycine substitution. Despite the presence of an open reading frame,
the lack of conserved amino acid substitutions suggests it is a
processed pseudogene under no evolutionary selection.
The exon/intron junctions were
sequenced for each gene, and the size of all introns was determined by
DNA sequencing, restriction site mapping, and/or PCR amplification
(Table I). All exon/intron splice junctions follow the
GT-AG rule and conform to the established consensus exon boundary
sequences (48). The VDAC exons generally correspond to the
transmembrane regions proposed for the human VDAC protein structure
(Fig. 1; Ref. 25). It may be argued that the
introduction of introns into the VDAC gene defines a modular organization to the different transmembrane regions of the VDAC protein, however it may also simply be a chance occurrence. The genomic
conservation of all coding exon/intron boundaries is indicative of a
gene family, and strongly suggests gene duplications and divergence as
the origin of the three VDAC isoforms. The 5-untranslated region
exon/intron boundaries and the intron sizes are not conserved, suggesting the gene duplications were ancient evolutionary events that
followed the introduction of introns, and that these sequences may have
evolved a new function after the duplication of the genes, or
alternatively are under no functional constraints. The splice acceptor
boundaries of the third exon of VDAC2 and the second exon of VDAC3 are
conserved. In VDAC3 the site is located within the 5
-untranslated
sequence, while in VDAC2 it is located in the coding region. This
demonstrates a single instance of coding and non-coding splice site
conservation between the VDAC isoforms. The same boundary site in the
5
-untranslated region of the VDAC1 gene differs by a single nucleotide
(Table I).
|
VDAC Polyadenylation Signals
The human VDAC1 gene was
reported to contain 854 bp of 3-untranslated sequence with a consensus
polyadenylation site (16). Since the original mouse VDAC1 cDNA did
not contain a consensus polyadenylation site (17), 3
-RACE was used to
identify the polyadenylation site(s). Using this technique, a 873-bp
3
-untranslated region was sequenced, and, in contrast to the human
VDAC1 gene, two consensus polyadenylation signals (AATAAA) at
nucleotides 1737 and 1748 were identified. The latter signal is most
likely the primary polyadenylation signal used, both because of its
proximity to the poly(A) tract and because the signal at nucleotide
1737 is not conserved in the human 3
-untranslated sequence. The mouse VDAC1 3
-untranslated region has approximately 70% DNA sequence conservation with the human VDAC1 cDNA. This region is 20 bp longer than the human VDAC1 3
-untranslated sequence, but the high degree of
sequence similarity between the two regions further supports that these
genes are orthologues.
Blachly-Dyson et al. (16) suggested the existence of
alternative polyadenylation sites in the human VDAC2 gene based upon finding two sets of human cDNA clones with differing
3-untranslated sequence lengths. Four of the human cDNA clones
terminated at a position equivalent to nucleotide 1301 in the mouse
VDAC2 gene. The mouse VDAC2 gene was previously shown to be encoded by
multiple transcripts, based on Northern blot analysis (Ref. 17; Fig. 2). 3
-RACE was used to determine if the multiple VDAC2
transcripts are due to alternative polyadenylation signals. Three
distinct PCR products were generated by 3
-RACE (data not shown), each of which was subcloned and sequenced. The 3
-untranslated region of
VDAC2 contains one aberrant and two canonical polyadenylation signals.
The two longer products can be accounted for by consensus polyadenylation signals found at nucleotides 1462 and 1637, respectively. The shortest product, corresponding to the strongest
signal on a Northern blot of ES cell RNA (Fig. 2), terminates in a 4-bp region from nucleotides 1300 to 1303, with no consensus polyadenylation signal present. This 4-bp region is completely conserved in the human
VDAC2 3
-untranslated region. Less than 5% of mRNAs are generated
by non-consensus polyadenylation signals (26), although the most
proximal alternative polyadenylation signal used by the mouse and human
VDAC2 genes is not apparent. The lack of a consensus polyadenylation
signal in VDAC2 may cause the termination site of the short transcript
to be less precise, thus accounting for the 4-bp region of termination
identified by 3
-RACE. 3
-Untranslated regions have been shown to
control mRNA turnover, translation efficiency, and the subcellular
location of transcripts (27-30); it is possible that the shorter
3
-untranslated region plays a specific role in regulating VDAC2 gene
expression during development. Ha et al. (31) has suggested
the existence of two alternative VDAC2 amino termini in humans based
upon the isolation of variant cDNAs. The possibility that the mouse
VDAC2 gene also generates alternative amino termini has not been
exhaustively addressed, although no alternative VDAC2 cDNAs were
identified in the course of these studies.
The VDAC3 transcript has previously been shown to terminate at a single polyadenylation site (18).
Cross-species ConservationThe evolutionary conservation of
the VDAC genes, suggested by previous electrophysiologic studies of
VDACs isolated from different species (1, 16) is further supported at a
DNA sequence level by the "zoo blot" results (Fig.
3). Each VDAC isoform gives a unique hybridization
pattern (data not shown). These results suggest that each of the mouse
VDAC genes is evolutionarily conserved in a number of distantly related
species, including cow, chicken, sea urchin, Caenorhabditis
elegans, and Drosophila melanogaster. Some of the
hybridizing bands do overlap from the different isoforms, reflecting
sequence identity with more than one mouse VDAC isoform or,
alternatively, a genomic repeat sequence. VDAC1 gives the most
complicated hybridization pattern, perhaps reflecting the abundance of
pseudogenes for this isoform encountered in the mouse. This high level
of sequence conservation and the functional conservation suggested by
the ability of VDACs from other species to complement the yeast
VDAC-deficient phenotype implies that the VDAC isoforms function in
conserved pathways across phyla.
DNA Sequence Analysis of the Transcription Control Regions
Sequence analysis of the 5-flanking region of each VDAC
isoform reveals several characteristic features. The promoter regions of all three VDAC isoforms lack canonical TATA boxes and are G+C-rich, a characteristic of housekeeping gene promoters (32). In these regions,
VDAC1 (493 bp; GenBankTM U89987), VDAC2 (952 bp; GenBankTM U89988), and
VDAC3 (529 bp; GenBankTM U89989), have an average G+C content of
69.8%, 60.6%, and 68.7%, respectively (Fig. 4). The
transcription initiation sites for each VDAC gene were predicted using
the TSSG and TSSW programs.2 For VDAC1 the
transcription initiation site is predicted to be nucleotide 453, 41 bp
upstream of the first confirmed 5
-untranslated exon nucleotide (Fig.
4). VDAC2 transcription initiation sites are predicted to be
nucleotides 656 and 956. The latter site is the fourth nucleotide in
the first 5
-untranslated exon. The VDAC3 transcription initiation site
is predicted to be nucleotide 474, 56 bp upstream of the first
5
-untranslated exon nucleotide.
A data base search for transcription factor binding motifs revealed
multiple sites in the 5-flanking sequences of each VDAC gene,
including a number of Sp1 binding sites. It is common for at least one
Sp1 binding site to be located within the promoter of a housekeeping
gene (32); VDAC1 contains two Sp1 sites, VDAC2 contains 10 Sp1 sites,
while VDAC3 contains five Sp1 sites. VDAC1 and VDAC2 also have a sterol
repressor element 1 (SRE-1) binding site within the predicted promoter
regions. The SRE-1 octanucleotide sequence appears to enhance
transcription in the absence of sterols but is transcriptionally
inactive in the presence of sterols. Therefore this sequence may
contribute to the maintenance of cholesterol homeostasis in cells (33).
VDAC proteins have been identified as a component of the peripheral
benzodiazepine receptor complex, which has been implicated in the
metabolism of cholesterol for steroidogenesis (14). Since it has been
shown previously that in vitro VDAC channel activity
requires the presence of sterols (34), it is noteworthy that a SRE-1
site is located in the promoter region of these two genes, and suggests
a role for sterols in the regulation of VDAC1 and VDAC2 expression, or
perhaps indirectly in the regulation of cholesterol import into
mitochondria.
To determine if the
predicted promoter region of each VDAC gene is capable of directing
transcription, plasmids were constructed containing the different
5-flanking regions of each VDAC gene placed upstream of a
promoter-less CAT gene. A VDAC1 5
-flanking 918-bp fragment containing
19 bp of the first 5
-untranslated exon was found to direct expression
of the CAT gene in both the sense and antisense orientations. A pCAT
Basic vector containing a shorter 513-bp VDAC1 5
-flanking fragment
with 19 bp of the first 5
-untranslated exon was subsequently generated
and found to have greater promoter activity in the sense orientation
than the 918-bp fragment, while the antisense orientation lacked any significant promoter activity (Fig. 5), suggesting there
may be silencer sequences in the first 406 bp of the larger VDAC1
fragment. These results indicate that a minimal VDAC1 promoter fragment is transcriptionally active in only the sense orientation.
It has been reported previously that the promoters of several housekeeping genes demonstrate transcriptional activity in both orientations when linked to a reporter gene in transfection assays (35-38). It is possible that elements of the VDAC1 promoter may also direct the transcription of a second gene aligned in the opposite orientation, as has been demonstrated with other bidirectional promoters (39). Alternatively, the promoter of a second gene may be in a head-to-head configuration with the VDAC1 promoter (40).
A VDAC2 5-flanking 1014-bp fragment containing 62 bp of the first
5
-untranslated exon, as well as a longer 5
-flanking fragment of
approximately 5 kb in length, were subcloned into the pCAT Basic vector
in both orientations. The 1014-bp and 5-kb fragments were essentially
equivalent in activity to the pCAT Control vector that contains the
SV40 early promoter and enhancer. In contrast, when the fragments were
placed in the antisense orientation, no transcriptional activity was
detected.
The genomic clone containing the first 5-untranslated exon of VDAC3
has only an additional 529 bp of upstream genomic sequence, thus
limiting the size of the putative promoter region that was tested. A
549-bp fragment, including 20 bp of the first 5
-untranslated exon, was
subcloned into both the pCAT Basic and Enhancer vectors, and was found
to have promoter activity in the sense orientation. In the pCAT Basic
vector the fragment had approximately 16% of the activity obtained
with the control SV40 early promoter, while the pCAT Enhancer vector
was able to induce this activity by 3-fold (Fig. 4). Again, the
opposite orientation was found to lack significant promoter
activity.
The promoter region of VDAC1, although imparting significant transcriptional activity, has the least amount of activity of the three isoforms examined. Addition of an enhancer sequence to the VDAC1 and VDAC3 promoter regions (VDAC1-.9E and VDAC3-.5E, respectively in Fig. 5) led to higher transcriptional activity, suggesting that additional regulatory elements for these VDAC genes are not present in the DNA fragments analyzed. Although VDAC2 consistently directed the highest level of activity in the transient transfection assays, by Northern analysis the in vivo expression of the three isoforms is fairly comparable, with the exception of a lack of VDAC1 expression in testes (17). The distribution of these regulatory elements must also vary between the genes, since comparably sized fragments of each VDAC gene were examined, and each gave different levels of promoter activity. Alternatively, in vitro transcription assays may not reflect in vivo regulation, due to altered mRNA stability or the rate of protein turnover. It is also possible that other VDAC promoters exist within each locus, particularly for the VDAC2 gene, which is expressed as multiple sized transcripts. The VDAC2 promoter, because it directs a high level of expression and appears to be expressed ubiquitously in mouse tissues, may be useful as a heterologous promoter in transgenic mouse experiments. The minimal active region and in vivo regulation of this promoter can be analyzed to investigate this possibility.
Expression of Mouse VDACs in YeastTo examine the ability of
the mouse VDAC isoforms to form a functional VDAC protein, each mouse
VDAC cDNA was expressed in yeast lacking the endogenous yeast VDAC
gene (22). Such yeast are viable but have a temperature-sensitive
growth phenotype on media containing glycerol as the sole carbon source
(41-43). The biochemical basis for this observed phenotype is unknown.
Mouse VDAC cDNA constructs were generated which contain the
complete mouse coding region flanked by the yeast VDAC promotor and
5-untranslated region and the yeast 3
-untranslated region. Thus, each
mouse VDAC plasmid differs only within the coding region, with all
relevant control elements in common. The yeast shuttle vector used is a single copy plasmid, and in combination with the yeast VDAC regulatory elements should approximate the endogenous levels of VDAC expression in
yeast. Like their human orthologues (16), introduction of either mouse
VDAC1 or VDAC2 into the mutant yeast eliminated the temperature-sensitive growth defect (Fig. 6). Thus,
VDAC1 and VDAC2 cDNAs appear to encode VDAC proteins that are able
to substitute for the endogenous activity of the yeast VDAC gene.
Phylogenetic analysis indicates that VDAC3 is the more ancient of the three VDAC isoforms. The placement of VDAC3 on a separate branch of a phylogenetic tree also suggests that this protein may have a physiological function distinct from that of VDAC1 and VDAC2 (18). This prediction is indirectly supported by the observation that VDAC3 does not rescue the temperature-sensitive phenotype completely, but generates a lower level of growth under the restrictive conditions. A small number of rapidly growing colonies are seen when yeast expressing VDAC3 are grown at the restrictive temperature. The basis for the vigorous growth found in this subpopulation of VDAC3-expressing yeast is currently under investigation.
Because the molecular basis of the VDAC-deficient yeast phenotype is unknown, the inability of VDAC3 to completely rescue this phenotype is also not understood. It is unlikely that the VDAC3 protein is simply not expressed efficiently in the yeast because the transcriptional elements used for the three isoform constructs are identical and the VDAC3 translation initiation site in the yeast construct matches the yeast consensus translation initiation sequence more closely than the mammalian "Kozak" consensus sequence (44, 47). Since mammalian VDACs are known to interact with various cytosolic kinases, the VDAC3 protein may be unable to interact with certain kinases necessary for full complementation, and therefore only partial complementation is observed. It is worth noting that of the three mouse VDAC isoforms only VDAC3 contains a leucine zipper motif (amino acids 150-171; LAGYQMSLDTAKSKLSQNNFAL), as determined by a protein motif search,3 whereas all other recognized motifs (e.g. PKC or CK2 phosphorylation sites, myristoylation sites) are conserved between the isoforms. This motif is found in a region of the protein predicted to form an exposed cytoplasmic loop (25).
Information gained from defining the gene structure of each VDAC isoform provides insights into the history of VDAC evolution and the reagents necessary to examine whether transcriptional control of VDAC expression occurs. Although the existence of other as yet undiscovered mouse VDAC isoforms has not been completely excluded, screenings of several cDNA and genomic libraries, and data base searches for related ESTs have failed to identify any additional VDAC-like sequences. Data from the experimental work described in this paper can be used to design targeting vectors for generating mutations at each locus to genetically define VDAC function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U89987-U89990.
We thank Michael Edwards and Bill Jones for help with the yeast experiments.