From the Center for Molecular Medicine, Emory University School for Medicine, Atlanta, Georgia 30322
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice deficient in the heart/muscle specific
isoform of the adenine nucleotide translocator (ANT1) exhibit many of
the hallmarks of human oxidative phosphorylation (OXPHOS) disease,
including a dramatic proliferation of skeletal muscle mitochondria.
Because many of the genes necessary for mitochondrial biosynthesis,
OXPHOS function, and response to OXPHOS disease might be
expected to be up-regulated in the
Ant1 The mitochondria generate most of the cells' energy via the
process oxidative phosphorylation
(OXPHOS).1 OXPHOS is
catalyzed by five multi-subunit enzyme complexes (complexes I-V)
located within the inner mitochondrial membrane. The protein subunits
necessary for oxidative phosphorylation are encoded by both the nuclear
and mitochondrial genome. The mitochondrial genome encodes 13 polypeptides involved in oxidative phosphorylation, including seven
subunits of complex I (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6), one
subunit (cytochrome b) of complex III, three subunits of
complex IV (COI, COII, and COIII), and two subunits of complex V
(ATPases 6 and 8) along with 22 tRNAs and 2 rRNA subunits necessary for
translation of these polypeptides. Transcription of the mtDNA initiates
at two sites, yielding polycistronic messages in which regions coding
for protein are interspersed by regions coding for tRNA (1). The mature
mRNAs, rRNAs, and tRNAs are released by cleavage. The nuclear
genome encodes all of the remaining genes for mitochondrial biogenesis.
These include the remaining subunits for OXPHOS; all of the proteins
for mitochondrial regulation, transcription, and translation; and all
proteins for mitochondrial intermediary metabolism (2).
Mutations in both the mitochondrial DNA and the nuclear DNA have been
shown to cause OXPHOS disease. OXPHOS diseases have a highly variable
clinical spectrum, showing the progressive defects in tissues most
reliant on cellular energy, with central nervous system, skeletal
muscle, and heart frequently being affected. Mitochondrial myopathies
are characterized pathologically by degeneration of the contractile
elements and the proliferation of subsarcolemmal mitochondria,
resulting in ragged red muscle fibers as revealed by the modified
Gomori-trichrome stain. The proliferation of mitochondria appears to be
a common compensatory response to energy deficiency in muscle, whether
caused by OXPHOS disease or endurance training in athletes (3). The
induction of the proliferation of mitochondria is a complex task,
requiring coordinate regulation of hundreds of genes dispersed between
the nuclear and mitochondrial genomes. The coordinate up-regulation of
nuclear and mtDNA OXPHOS gene transcripts has been documented in the
skeletal muscle of patients with three mitochondrial DNA mutation
diseases: Kearns-Sayre syndrome; myoclonic epilepsy associated with
ragged red fibers; and myopathy encephalopathy, lactic
acidosis, and stroke-like episodes (4, 24).2
An animal model of mitochondrial myopathy and cardiomyopathy has
recently been created by genetically inactivating mouse Ant1 (6). ANT1 exchanges mitochondrial matrix ATP for cytosolic ADP across
the mitochondrial inner membrane. Knock-out mice deficient in ANT1
exhibit many of the hallmarks of human OXPHOS disease, including a
defect in coupled respiration, lactic acidosis, exercise intolerance,
and a dramatic proliferation of skeletal muscle mitochondria.
The dramatic increase in mitochondrial number and volume in the muscle
of Ant1-deficient mice suggests that there might be a
coordinate up-regulation of genes required for energy production. In an
effort to identify the up-regulated genes, we have analyzed Ant1-deficient mouse muscle RNA using differential display
RT-PCR technology (7). This has resulted in the identification of 17 genes up-regulated in mutant mouse muscle. Among these are genes known
to be involved in OXPHOS, as well as genes previously unknown to be
involved in mitochondrial function.
Differential Display--
Total RNA was isolated from
gastrocnemius muscle of two wild type and two Ant1-deficient
mice using TRIZOL Reagent (Life Technologies, Inc.). DNA contamination
was removed with the MessageClean kit, and the mRNA was
reverse-transcribed and amplified using the differential display
RNAimage Kits 1-4 (GenHunter Corporation, Nashville, TN). The
resulting 33P-labeled RT-PCR products were run on 6%
LongRanger polyacrylamide gels. Putative differentially regulated bands
were cut from the gel, eluted, and reamplified.
Differential display (DD) products were identified by the direct
sequencing of reamplified products (8) using the ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) and
the ABI 377 DNA sequencer. The resulting sequence information was
compared with GenBankTM and EST data bases at the National
Center for Biomedical Information via the BLAST computer program.
Northern and Reverse Northern Analysis--
Northern
hybridization analysis was performed using glyoxal and
Me2SO to denature 10 µg of total RNA, followed by
electrophoresis and transfer to Hybond N membrane (Amersham Pharmacia
Biotech). To verify the differential expression of the genes identified by differential display, DD products were cloned into the TA vector (Invitrogen, Carlsbad, CA), and the resulting cDNAs were used as
probes for Northern and reverse Northern hybridization. First, Northern
blot strips that contained only skeletal muscle RNA were probed with
the corresponding cloned and reamplified DD product. If this Northern
blot showed up-regulation of the gene of interest, then RNA samples
from the tissues containing various ratios of Ant1/Ant2 mRNA (skeletal muscle, heart, and
brain) were blotted and probed with the corresponding cloned and
reamplified DD product. Probes for evaluation of mitchondrial DNA
transcripts were created by amplification of nt 3351-7570 and nt
8861-14549 of the mitochondrial DNA from mouse genomic DNA using
mitochondrial DNA specific primers. The Rediprime DNA labeling system
(Amersham Pharmacia Biotech) was used to label probes with
[
For reverse Northern blots, DD PCR products were immobilized on
membrane and hybridized to probe synthesized by reverse transcription of RNA from either wild type or Ant1-deficient mice. One
µg of each DD PCR product was applied to duplicate Hybond N membranes via a dot blot apparatus (Millipore). Probes were made by reverse transcription of 20 µg of total RNA derived from gastrocnemius muscle
of either wild type or Ant1-deficient mice using the
ReversePrime kit (GenHunter Corporation).
Rapid Amplification of cDNA Ends (RACE)--
The 5' ends of
cDNAs identified by differential display were isolated using the
RACE technique. Template for amplification was mouse skeletal muscle
Marathon-Ready cDNA (CLONTECH, Palo Alto, CA).
Gene-specific primers designed from the differential display product
sequence were combined with linker-specific primers of the cDNA and
the Advantage cDNA polymerase mix (CLONTECH), and the 5' end of the gene was amplified. The products were sequenced directly using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) and analyzed using an ABI 377 DNA sequencer. The products were also cloned into the TA vector (Invitrogen).
DD RT-PCR techniques (7) were used to identify genes up-regulated
in Ant1/
mouse, we used differential
display reverse transcription-polymerase chain reaction techniques in
an effort to identify these genes. 17 genes were identified as
up-regulated in Ant1-deficient mice, and they fall into
four categories: 1) nuclear and mitochondrial genes encoding OXPHOS
components, 2) mitochondrial tRNA and rRNA genes, 3) genes involved in
intermediary metabolism, and 4) an eclectic group of other genes. Among
the latter genes, we identified the gene encoding anti-apoptotic
Mcl-1, the Skd3 gene, and the WS-3 gene, which
were previously unknown to be related to mitochondrial function. These
results indicate that identification of genes up-regulated in the
skeletal muscle of the Ant1-deficient mouse provides a
novel method for identifying mammalian genes required for
mitochondrial biogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP. The intensity of bands was quantitated
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice. Total RNA from the
gastrocnemius muscle of a 3-month-old littermate
Ant1+/+ and
/
mice was
extracted, reverse transcribed, and subjected to differential display
PCR using various arbitrary primers (exemplified in Fig. 1). Using 96 primer pairs that displayed
approximately 7,500 cDNA PCR products, 84 PCR products were
initially identified as differentially expressed. These DD products
were then reamplified, sequenced, and cloned into a plasmid vector.
Seventeen genes were verified as up-regulated in the Ant1
knock-out mouse, and they fell into four categories: 1) nuclear and
mitochondrial genes encoding OXPHOS components, 2) mitochondrial tRNA
and rRNA genes, 3) other genes involved in intermediary metabolism, and
4) an eclectic group of other genes.
View larger version (85K):
[in a new window]
Fig. 1.
Examples of differential display gels.
Total RNA extracted from two Ant1+/+ mice
and two Ant1 /
mice was subjected to DD
RT-PCR in the presence of [
-33P]dATP. Reactions were
run on 6% denaturing polyacrylamide gels, with the reactions from
/
mice run in the inner two lanes, and those from +/+ mice run in
the outer two lanes. Bands identified as putatively differentially
expressed are marked by arrowheads.
OXPHOS Genes Up-regulated in Mice Deficient for Ant1-- The sequences of ten of the DD products (Table I) revealed genes encoding subunits of the electron transport chain. Six of these are mitochondrially encoded and include the complex I subunit genes ND1, -2, -4, -5, -6, and the complex IV subunit gene COI (Fig. 2). Northern analysis revealed that all six of these mRNAs, and three others tested (ND4L, COII, and COIII) were up-regulated in the skeletal muscle of the ANT1-deficient mice (Fig. 3). All mitochondrial mRNAs tested were up-regulated between 2.5-3.5-fold. Similar increases are seen for patients with mitochondrial myopathies (4).
|
|
|
The remaining four up-regulated OXPHOS transcripts are derived from the
nuclear DNA and encode components of complexes I and IV. The complex IV
subunit genes encode two subunits of cytochrome c oxidase,
COXVa (9) and COXVb (10). The complex I subunits are homologues of the
bovine genes encoding CI-B8 and the 18-kDa Fe-S protein (11). Because
these genes had not been cloned from mouse, RACE was used to isolate,
clone, and sequence the mouse CI-B8 and CI-18k genes
(GenBankTM accession numbers AF124786 and AF124785,
respectively). Northern blots of all four of these subunits has
confirmed that they are up-regulated in the
Ant1/
skeletal muscle (Fig. 4 and data
not shown).
mtDNA Transcripts Involved in Mitochondrial Protein Synthesis-- Two of the DD RT-PCR products were identified by sequence analysis to correspond to mtDNA genes encoding structural RNAs, the mitochondrial 16 S rRNA (DD57) and a cluster of three tRNAs encompassed in nucleotides 4821-5171 (DD65). The differential display product DD65 contains the 3' end of ND1, in addition to the three contiguous tRNA genes. The association in one RNA of these continuous genes suggests that this is a processing intermediate of the polycistronic H-strand transcript.
Intermediary Metabolism Genes--
Sequence analysis of two
differential display products revealed that they encode the
intermediary metabolism proteins malate dehydrogenase and glycogen
phosphorylase. The sequence of DD67 is identical to the 3' region of
the mouse gene for the mitochondrial isoform of malate dehydrogenase
(12). Malate dehydrogenase catalyzes the final reaction of the citric
acid cycle, the regeneration of oxaloacetate. The sequence of DD21
reveals that it is the mouse homologue of the gene for the rat muscle
isozyme of glycogen phosphorylase (13). Glycogen phosphorylase
catalyzes the breakdown of glycogen to yield glucose-1-phosphate. The
mouse homologue of the glycogen phosphorylase gene was isolated by RACE
(GenBankTM accession number AF124787). The RNA levels of
both malate dehydrogenase and glycogen phosphorylase genes were shown
to be up-regulated in Ant1 knock-out mice (Fig.
5).
|
Other Genes Up-regulated in the Ant1 Knockout Mouse--
Skd3.
Northern blots probed with DD product DD24 revealed that this message
is up-regulated in both heart and skeletal muscle of the
Ant1/
mice (Fig.
6A). The DNA sequence of DD24
is identical to the previously cloned gene Skd3.
Skd3 was isolated from a mouse cDNA library based on its
ability to suppress the growth defect of a yeast K+
transporter mutant (14). The predicted Skd3 gene product is a 76-kDa protein consisting of a C-terminal domain with 57-64% similarity with members of the Clp/HSP104 family of proteins (Fig. 5B). SKD3, unlike other members of the Clp/HSP104 family,
contains four ankyrin-like repeats in its N-terminal domain. SKD3 also contains a hydrophilic region at its amino-terminal that is predicted by the computer program PSORT (15) to target the protein to the
mitochondrion (Fig. 6B).
|
|
WS-3-- The sequence of DD product DD25 proved to be identical to several mouse expressed sequence tags and homologous to the human WS-3 gene. WS-3 was cloned and characterized in humans by virtue of its proximity to the gene responsible for Werner syndrome (WS) (16). Mouse WS-3 (DD25) was shown by Northern blot analysis to be up-regulated in the skeletal muscle of the Ant1 knock-out mouse (Fig. 5B). The putative WS-3 gene product is a 20.7-kDa hydrophilic protein of unknown function. However, BLAST searches performed with the WS-3 predicted protein sequence reveal a similarity to the third enzyme in the enterbacterial lipid A biosynthesis pathway (17, 18). The mouse homologue of human WS-3 was isolated by RACE (GenBankTM accession number AF124788).
Mcl-1--
The sequence of the DD26 product is identical to the 3'
end of the Mcl1 gene. Mcl-1, a member of Bcl-2 family of
anti-apoptotic proteins, is located at least partially in the
mitochondrion (19). Northern analysis verified that Mcl1 is
up-regulated in the skeletal muscle of mice deficient in ANT1 (Fig. 5).
In addition to being up-regulated in the ANT1 knock-out mouse,
Mcl1 mRNA also appears to have an altered mobility on
Northern blots.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the absence of the adenine nucleotide translocator, ATP
synthesized in the mitochondrion is not transported into the cytoplasm, and an energy deficient state is created. The cell recognizes this
deficiency, and sends an as yet unidentified signal to the nucleus to
increase transcription of mitochondrial genes. Accordingly, most of the
genes shown to be up-regulated in Ant1/
mice
should encode products directly involved in the synthesis of ATP,
including components of the mitochondrial energy generating apparatus,
such as the electron transport chain and the citric acid cycle,
machinery for mitochondrial biogenesis, including the mitochondrial
rRNAs and tRNAs, and functionally related proteins such as glycogen
phosphorylase and malate dehydrogenase.
Previous studies2 have shown that human glycogen
phosphorylase mRNA is up-regulated in mitochondrial myopathy patients
and that the gene contains a 5' REBOX binding site. The 8-base pair REBOX sequence motif was identified as a specific protein binding site
in Ant1 and ATP synthetase subunit promoters by
electrophoretic mobility shift assays (20). This binding is sensitive
to NADH and thyroxine, suggesting that it may modulate OXPHOS gene
expression in response to environmental and hormonal changes (20).
Several of the genes shown to be up-regulated are not likely to be
directly involved in ATP synthesis. Up-regulation of Mcl-1, a Bcl-2
homologue, is more likely a response to oxidative stress in the cell.
Mcl1 has been shown to be up-regulated in response to
reactive oxygen species (21), which have shown to be at higher levels
in Ant1/
mice.3 The finding that
Mcl1 is up-regulated in this animal model for mitochondrial
myopathy suggests that apoptosis plays a role in the pathogenesis of
mitochondrial mutations in human patients.
By analogy with the other genes found to be up-regulated, we
propose that WS-3 and Skd3 are involved in
mitochondrial biogenesis. BLAST searches performed with the predicted
WS-3 protein reveal a similarity to the third enzyme in enterbacterial
lipid A biosynthesis (17, 18). Because mitochondria are believed to
have originated by endosymbiosis of bacteria, and several important
mitochondrial proteins such as DNA polymerase (1) have greater
similarity to bacterial proteins than eukaryotic proteins, it seems
possible that this WS-3 plays a role in mitochondrial lipid synthesis. This hypothesis is strengthened by comparison with the model of the two
families of fatty acid biosynthesis enzymes. In addition to the
previously known eukaryotic (type I) cytosolic enzymes of fatty acid
biosynthesis, bacterial homologues of these enzymes (type II) have been
discovered in various eukaryotic species (22). Some of these enzymes
have been localized to the mitochondria, and deletion of the
corresponding gene leads to defects in respiratory function. It is
possible that prokaryotic type genes involved in lipid synthesis have
also been retained from the endosymbiotic ancestor of the mitochondrion
and that WS-3 is one of these genes. Because
Ant1
/
mice have a dramatic proliferation of
mitochondria, enzymes involved in mitochondrial lipid synthesis would
be expected to be up-regulated in these mice.
Skd3 encodes a predicted protein with a putative mitochondrial targeting signal, ankyrin-like repeats, and similarity to the Clp/HSP104 family of proteins. Interestingly, Saccharomyces cerevisiae Hsp78, another member of the Clp/HSP104 family, appears to cooperate with the mitochondrial matrix protein Hsp70 in maintenance of mitochondrial function (23). In addition, Hsp78 is responsible for compartment-specific thermotolerance in yeast (5). SKD3, unlike other members of the Clp/HSP104 family, contains four ankyrin-like repeats in its N-terminal domain. In erythrocytes, ankyrin is responsible for attachment of spectrin to integral membrane proteins, whereas in nonerythroid cells, ankyrin is thought to be involved in protein/protein or protein/cytoskeleton interactions. Because of these sequence motifs, we hypothesize that the SKD3 protein is localized to the mitochondria, where it may interact with an unknown protein and assist in the assembly of some multi-subunit complex.
In this work, we have identified 17 genes up-regulated in the
Ant1/
mice by DD RT-PCR. Interestingly, 33 other differential display products isolated have been identified by
sequence, but their relative expression cannot be assessed by filter
hybridization techniques, because they are of very low abundance in
muscle (data not shown). Six mRNAs, one tRNA, and one rRNA encoded
by the mitochondrial genome were identified as differentially expressed
by differential display. Because all mitochondrial mRNAs appear to
be up-regulated, differential display methods identified half of all
up-regulated transcripts from the mitochondrial genome. By
extrapolation to the nuclear genome, the 17 mRNAs with levels high
enough to be identified as up-regulated, and the 33 genes with low
level expression identified by differential display, should represent a
significant proportion of the genes up-regulated in
Ant1-deficient mice. We conclude that differential
expression methods provide an effective means to identify genes
involved in mitochondrial biogenesis and function. Experiments using
complementary differential expression methods, such as serial analysis
of gene expression, are in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the contribution of Brett H. Graham.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL45572, NS21328, and AG13154 (to D. C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF124786, AF124785, AF124787, AF124788.
To whom correspondence should be addressed: Center for Molecular
Medicine, 1462 Clifton Rd., N. E., Emory University School for
Medicine, Atlanta, GA 30322. Tel.: 404-727-8368; Fax:
404-727-8367.
2 A. Heddi and D. C. Wallace (1999) manuscript in preparation.
3 L. A. Esposito and D. C. Wallace, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: OXPHOS, oxidative phosphorylation; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; DD, differential display; nt, nucleotide(s); WS, Werner syndrome; RACE, rapid amplification of cDNA ends.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|