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Am J Physiol Regul Integr Comp Physiol 284: R153-R163, 2003. First published September 12, 2002; doi:10.1152/ajpregu.00182.2002
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Vol. 284, Issue 1, R153-R163, January 2003

Changes of carp FoF1-ATPase in association with temperature acclimation

Shiro Itoi, Shigeharu Kinoshita, Kiyoshi Kikuchi, and Shugo Watabe

Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113 - 8657, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously we have shown, using two-dimensional electrophoresis, that mitochondrial ATP synthase (FoF1-ATPase) beta -subunit is the 55-kDa protein increased in cold-acclimated carp Cyprinus carpio (Kikuchi K, Itoi S, and Watabe S. Fisheries Sci 65: 629-636, 1999). To clarify the coordinate expression in various subunits of carp FoF1-ATPase with temperature acclimation, we examined the differences in mRNA levels of mitochondrial proteins encoded by both nuclear and mitochondrial genes in fast muscle of carp acclimated to 10 and 30°C. The mRNA levels of nuclear genes per unit weight of total RNA were nearly twofold higher in the 10°C- than 30°C-acclimated carp. However, the transcripts of mitochondrial genes for the 10°C-acclimated carp in terms of the same comparing unit were six to seven times as much as those for the 30°C-acclimated carp. The FoF1-ATPase activities measured at 10, 25, and 30°C were nearly twofold higher for the cold-acclimated fish than their warm-acclimated counterparts. Such quantitative and qualitative changes in carp FoF1-ATPase may contribute to extra ATP production required to compensate for energy balance at suboptimal temperatures.

mitochondria; fast muscle; temperature changes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN EURYTHERMAL TEMPERATE FISH such as carp Cyprinus carpio and goldfish Carassius auratus, temperature acclimation leads to an array of adaptational physiological changes to compensate for the effect of temperature variation on metabolic processes (13). Such changes are manifested in swimming behavior, for example, where the maximum cruising speed of several fish species is increased at low temperatures after cold acclimation for several weeks (11, 21). In a recent experiment, Wakeling et al. (43) showed that temperature acclimation had profound effects on fast-start behaviors in carp. Another example of consequences accompanying temperature changes is the proliferation of mitochondria as reflected by enhanced mitochondrial volume density in tissues of temporarily cold-acclimated as well as permanently cold-adapted fish (10, 22-24, 41).

Several lines of evidence have shown that the increase in the number of mitochondria in cells is usually associated with an increase in tissue specific activity of cytochrome-c oxidase: low temperatures provoke a compensatory increase of cytochrome-c oxidase activity in fish tissues (6, 12, 42, 51). Cytochrome-c oxidase, the terminal enzyme in the electron transport chain, is located in the inner mitochondrial membrane. While it catalyzes the oxidation of cytochrome c using molecular oxygen, the energy released in this reaction is utilized to translocate protons across the inner mitochondrial membrane. The mammalian cytochrome-c oxidase enzyme complex consists of 13 subunits: 3 subunits are encoded by mitochondrial genes, and the remaining 10 subunits are encoded by nuclear genes (45). Therefore, the increase in mitochondrial volume density must be achieved by coordinate expression of associated proteins encoded by mitochondrial and nuclear genes (12).

We have recently shown that the content of the mitochondrial ATP synthase (FoF1-ATPase) beta -subunit (beta -F1-ATPase) was about twofold higher in carp acclimated to 10°C than in fish acclimated to 30°C both at protein and transcriptional levels (25, 47). FoF1- ATPase harnesses the potential energy of the proton gradient produced by electron transport chain complexes including cytochrome-c oxidase to synthesize ATP from ADP and Pi (36). Thus the increase of mitochondrial volume density in fish would compensate for the decrease of ATP production at low temperatures by means of increasing quantities of mitochondrial protein components in the respiratory chain (25). However, there is no evidence available on the correlation between mitochondrial content and FoF1-ATPase in association with temperature changes.

The eukaryotic FoF1-ATPase is a supramolecule consisting of two functional components that are structurally well defined: a hydrophilic F1 component containing catalytic sites for ATP synthesis, and a proton channel, Fo, embedded in the mitochondrial inner membrane (36). F1 is further composed of alpha -, beta -, gamma -, delta -, and epsilon -subunits, whereas Fo contains more subunits called a, b, c, d, e, F6, OSCP, and A6L (35). The a- and A6L-subunits of the Fo domain that are also collectively called ATPase 6-8 are encoded by mitochondrial genes, whereas all other subunits are distinctly encoded by nuclear genes in vertebrates (2). Therefore, the increase in ATP production must be achieved by coordinate expression of associated proteins encoded by mitochondrial and nuclear genes, as in the case of cytochrome-c oxidase.

The objective of the present study was to compare changes in expression levels of carp FoF1-ATPase subunits encoded by nuclear and mitochondrial genes after temperature acclimation. We observed that the mRNA levels of those encoded by nuclear genes were about twofold higher in the 10°C- than 30°C-acclimated carp. However, the levels of subunits encoded by mitochondrial genes in the former fish were six to seven times higher than those in the latter one. Oligomycin sensitive FoF1-ATPase activity per mitochondrial protein weight in the 10°C-acclimated carp was about twofold higher than that in the 30°C-acclimated fish at 10, 25, and 30°C. Such quantitative and qualitative changes of FoF1-ATPase after temperature acclimation are discussed in terms of energy compensation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fish. Carp (88-238 g) were acclimated in laboratory aquariums to either 10 or 30°C for a minimum of 5 wk (14). All fish were fed commercial pellets daily ad libitum before being killed. Fish were killed by pithing and transection of the spinal cord.

PCR amplification and cDNA cloning. Genomic DNA was prepared from carp fast muscle according to Ausubel et al. (5), and PCR amplification was performed as follows. The reaction mixture for nuclear genome-encoded proteins contained carp fast muscle cDNA library (19) or genomic DNA as a template, 2 µl of 10× Ex Taq buffer supplied with the kit, 0.2 µl of 20 µM primers, 0.2 µl of 20 mM dNTP, and one unit of Takara Ex Taq DNA polymerase (Takara), and the total volume was brought up to 20 µl with sterilized water. PCR consisted of 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. The conditions of PCR for mitochondrial genome-encoded subunits were the same as described above except for annealing at 60°C. PCR products were cloned into the TA site of pT7Blue T-vector (Novagen) according to Marchuk et al. (30) using Escherichia coli strain JM109 as a host bacterium.

Sequencing was performed for both strands with an Applied Biosystems DNA sequencer model 373S using Dye Deoxy terminator cycle sequencing kits (Applied Biosystems). Homology search was carried out using the DDBJ/EMBL/GenBank databases. Comparison of the deduced amino acid sequences for alpha -, gamma -, and c-subunits of carp FoF1-ATPase (hereafter referred to as alpha -F1-ATPase, gamma -F1-ATPase, and c-Fo-ATPase, respectively) and mitochondrial genome-encoded subunits with those appearing in the DDBJ/EMBL/GenBank databases was performed by BLAST search (1). The alignment for deduced amino acid sequences was carried out by CLUSTAL W (39).

Isolation of total RNA and Northern blot analysis. Total RNA for Northern blot analysis was extracted from carp fast muscle using a RNA extraction solution (Isogen, Nippon Gene) according to the manufacturer's protocol. Ten micrograms of total RNA prepared were denatured at 65°C for 15 min in 50% formamide, subjected to electrophoresis on 0.9% agarose gel in 0.2 M MOPS (pH 7.0) containing 2.2 M formamide, 0.05 M sodium acetate, and 5 mM EDTA, and transferred to a Hybond N+ nylon membrane (Amersham Pharmacia Biotech). The membrane was air-dried, baked at 80°C for 15 min, and prehybridized at 65°C for 5 min in 0.5 M phosphate buffer (pH 7.2) containing 1 mM EDTA and 7% SDS (8). Hybridization was performed at 65°C for 21 h in the same solution containing a probe, which was randomly primed in the presence of [alpha -32P]dCTP. The membrane filter was washed twice for 15 min each with 2× SSC (SSC is 15 mM sodium citrate, pH 7.0, 0.15 M NaCl) containing 0.1% SDS and 1× SSC containing 0.1% SDS at 65°C and subjected to autoradiography on an X-ray film with intensifying screens at -80°C for 1 wk (alpha -, beta -, and gamma -F1-ATPase, and c-Fo-ATPase) or at room temperature for 24 h (others). The hybridized membrane was scanned with a Fujix BAS 1000 computerized densitometer scanner and quantified using a recommended scanning program.

Southern blot analysis. Total DNAs including those of mitochondria and genome were prepared from carp fast muscle according to Ausubel et al. (5), and the aliquots (10 µg) were digested with HindIII. The digests were size-fractionated by electrophoresis in a 0.9% agarose gel and transferred to a nylon membrane. Subsequent procedures were the same as in the case of Northern blot analysis.

Isolation of mitochondria. Mitochondria were isolated from carp fast muscle according to Toth et al. (40). The fast muscle (39-61 g) was excised and minced into ~1-mm cubes in 2 vol of an ice-cold isolation medium (pH 7.4) containing 210 mM mannitol, 70 mM sucrose, 5 mM HEPES, and 0.2% BSA. The mince was washed three times with the isolation medium to remove blood and free lipid, and resuspended in 10 vol of the isolation medium to which collagenase from Clostridium histolyticum (Wako) was added at a final concentration of 0.07%. After 15-min incubation in ice-cold water, the tissues were gently homogenized using a Potter-Elvejhem type homogenizer (2 passes with a loosely fitting teflon pestle followed by 1 pass with a tightly fitting pestle). The homogenate was allowed to stand on ice for 10 min, and EGTA was added to a final concentration of 1 mM to halt collagenolysis and prevent mitochondrial uptake of Ca2+. The homogenate was centrifuged at 900 g for 10 min, and the resultant supernatant was centrifuged at 8,400 g for 12 min. The pellet was washed by centrifuging at 17,500 g for 12 min and resuspended in the isolation medium containing 1 mM EGTA. This washing procedure was repeated again, and the resultant mitochondrial pellet was washed twice by decantation using 5 ml of a suspending medium (pH 7.4), including 250 mM sucrose, 5 mM KH2PO4, 2 mM HEPES, and 10 mM EGTA. The mitochondrial pellet thus obtained was resuspended in a small volume of the suspending medium and adjusted to 20-30 mg mitochondrial protein/ml. Mitochondrial protein levels of isolated mitochondria were approximately 0.25 and 0.12 mg/g tissue in 10°C- and 30°C-acclimated carp fast muscle, respectively.

Preparation of muscle extracts. Muscle extracts were prepared essentially according to Weber and Osborn (48). Briefly, muscle tissues (0.2 g) were minced and incubated at room temperature for 24 h in 10 vol of an extraction solution containing 8 M urea, 1% SDS, 1% 2-mercaptoethanol, and 5 mM EDTA. After incubation the mixture was dialyzed at 4°C for 14 h against 500 ml of 0.125 M Tris · HCl buffer (pH 6.8) containing 0.1% SDS and 0.1% 2-mercaptoethanol. The dialyzed muscle extracts were centrifuged at 16,000 g for 5 min, and the resulting supernatant was used for protein content determination and SDS-PAGE analysis.

Determination of protein concentration. The protein concentration of the mitochondrial suspension and muscle extract were determined by the bicinchoninic acid (Pierce) method using BSA as the standard.

Electrophoresis and protein identification. SDS-PAGE was carried out by the method of Laemmli (27) using 7.5-20% polyacrylamide gradient slab gels containing 0.1% SDS. Gels were stained with 0.1% Coomassie brilliant blue R250 after electrophoresis. The content of proteins on the SDS-PAGE gel was quantified using an image analysis program, Scion Image version Beta 4.0.2 (Scion).

alpha - and beta -F1-ATPases were identified by immunoblotting and NH2-terminal amino acid sequencing, respectively. The NH2-terminal amino acid sequence was determined by the method of Matsudaira (33). Briefly, the part of the membrane carrying the blotted protein on an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore) was cut out with a clean razor. Several membranes bearing the same protein were placed together on the teflon seal of the cartridge block in an Applied Biosystems protein sequencer (model 476A) with an online system (model 120A).

Immunoblotting was carried out as follows. Proteins in SDS-PAGE gels were electrophoretically transferred to an Immobilon PVDF membrane. The membrane was blocked with 2% BSA in 50 mM Tris · HCl (pH 8.0) containing 150 mM NaCl and then incubated with the anti-bovine heart alpha -F1-ATPase monoclonal antibody (Molecular Probes). Goat anti-mouse IgG-horseradish peroxidase conjugate (Kirkegaard and Perry Laboratories) was used as the second antibody and visualized using 0.2 mg/ml 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2.

Measurement of ATPase activity. ATPase activity was measured essentially as described by Stiggall et al. (37) using a Jasco Model V-560 spectrophotometer at a wavelength of 340 nm with a Jasco Model PSC-498T temperature controller. The FoF1-ATPase was only measured in the direction of ATP hydrolysis. The reaction mixture in 2 ml at pH 8.2 contained 25 mM KHCO3-Tris, 300 mM sucrose, 2 mM MgCl2, 1.5 mM phosphoenolpyruvate, 0.25 mM NADH, 1.25 mM ATP, 10 U of pyruvate kinase (Roche Molecular Biochemicals), and 10 U of lactate dehydrogenase (Roche Molecular Biochemicals). Assays were performed at 10, 25, and 30°C in the presence of 100 µg mitochondrial protein. Oligomycin sensitivity of ATPase activity was determined by the addition of 10 µg oligomycin. ATPase activity was calculated using the stable kinetic phase of ATP hydrolysis by measuring the absorbance change caused by the addition of 10 µl of 0.665 mM ADP as a standard.

Statistical analysis. Statistical analysis was performed between 10°C- and 30°C-acclimated groups of fish using the parametric Student's t-test. Data are given as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of carp alpha -F1-ATPase cDNA. Oligonucleotide primers, CATPAF1 and CATPAR1, were designed from well-conserved regions of alpha -F1-ATPase from rat (GenBank database accession no. J05266), mouse (L01062), human (D14710), bovine (M22465), and Xenopus laevis (M16259) (Table 1). SK and KS were universal primers in pBluescript SK(-). PCR using the synthesized primers and carp muscle cDNA library as a template yielded cDNA fragments of approximately 1.6 kbp (CATPAF1 and KS) and 1 kbp (SK and CATPAR1), and these two fragments were just overlapped in the DNA nucleotide sequence. To obtain a cDNA encoding a full length of carp alpha -F1-ATPase, oligonucleotide primers alpha5'term2 and alpha3'term1 were designed referring to the sequences obtained (Table 1). PCR with these primers yielded a cDNA of 1,857 bp containing putative initiation and termination codons and the coding region of 1,659 nucleotides for 552 amino acids. The first methionine was followed by a short polypeptide of 43 amino acids that probably serves as a signal peptide for transporting alpha -F1-ATPase across mitochondrial membranes. The predicted molecular mass and isoelectric point (pI) of mature protein were 55,079 Da and 8.46, respectively. The 3'-noncoding region of 147 bp contained a putative polyadenylation signal, AATAAA, at 14 bp upstream of the poly(A) tail. The nucleotide sequence of alpha -F1-ATPase appears in the DDBJ/EMBL/GenBank databases with accession no. AB042437.

                              
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  		Table 1.   Primers used for amplifying carp FoF1-ATPase subunits, cDNA, and mitochondrial genes

Cloning of carp gamma -F1-ATPase cDNA. The oligonucleotide primer CATPGR1 was designed from a well-conserved region of gamma -F1-ATPase from human (D16563), bovine (M22463), and rat (L19927) (Table 1). PCR with carp muscle cDNA library as a template and with SK and CATPGR1 primers yielded a cDNA fragment of ~800 bp. DNA sequencing revealed that the fragment encoded a part of carp gamma -F1-ATPase. Following a series of PCR with oligonucleotide primers, CATPGF2 and KS (Table 1), a cDNA of ~450 bp encoding a COOH-terminal region of carp gamma -F1-ATPase was cloned. These two DNA fragments, one amplified by primers SK and CATPGR1 and the other by primers CATPGF2 and SK, were overlapped in 109 bp. PCR using gamma5'term1 and gamma3'term1 primers (Table 1), which were designed from the above two cDNA fragments, yielded a cDNA of 1,113 bp encoding a full length of carp gamma -F1-ATPase containing putative initiation and termination codons. The coding region of 879 nucleotides for 292 amino acids included a predicted short signal polypeptide of 20 amino acids. A molecular mass of a mature protein was calculated to be 29,946 Da, and its predicted pI was 9.64. The 3'-noncoding region of 212 bp contained two putative polyadenylation signals, AATAAA, at 14 and 106 bp upstream of the poly(A) tail. The nucleotide sequence of gamma -F1-ATPase appears in the DDBJ/EMBL/GenBank databases with accession no. AB042438.

Cloning of carp c-Fo-ATPase cDNA. Oligonucleotide primers c-Fo/P3F1 and c-Fo/P3R1 were designed from the highly conserved region of c-Fo-ATPase P3-isoform from human (U09813), rat (AF315374), zebrafish (AF311603), and Fugu (genomic scaffold_293; http://www.jgi.doe.gov/index.html) (Table 1). PCR using c-Fo/P3F1 and SK primers and carp muscle cDNA library as a template yielded a cDNA fragment of ~600 bp. Following a series of PCR with oligonucleotide primers KS and c-Fo/P3R1 (Table 1), a cDNA of ~300 bp encoding an NH2-terminal region of carp c-Fo-ATPase containing the putative initiation codon was cloned. These two fragments were completely overlapped in the DNA nucleotide sequence. The coding region of 423 nucleotides for 140 amino acids included a predicted signal polypeptide of 65 amino acids. The molecular mass of a mature protein was calculated to be 7,633 Da and its predicted pI was 8.82. The 3'-noncoding region of 212 bp contained a putative polyadenylation signal, AATAAA, at 19 bp upstream of the poly(A) tail. The nucleotide sequence of c-Fo-ATPase appears in the DDBJ/EMBL/GenBank databases with accession no. AB078926.

Comparison of deduced amino acid sequences of carp alpha - and gamma -F1-ATPase and c-Fo-ATPase. Comparison of the amino acid sequence for carp alpha -F1-ATPase precursor protein with those appearing in the DDBJ/EMBL/GenBank databases revealed that the carp protein was 91, 90, 89, and 67% identical to those of bovine (M22465), human (D14710), X. laevis (M16259), and Saccharomyces cerevisiae (D37948), respectively. The overall structure of alpha -F1-ATPase was highly conserved among eukaryotes except for approximately 50 and 60 residues from the NH2 and COOH termini, respectively, of the precursor proteins. Walker A motif and ATP synthase alpha - and beta -subunit signature sites were completely conserved among eukaryotes (data not shown). On the other hand, gamma -F1-ATPase precursor protein was 75, 75, 74, and 39% identical to those of human (D16563), bovine (M22463), mouse (AK007063), and S. cerevisiae (U09305), respectively. The overall structure of gamma -F1-ATPase was conserved among vertebrates including carp, whereas the sequence of ATP synthase gamma -subunit signature site was identical among vertebrates. The carp gamma -F1-ATPase appears to be of muscle isoforms as there was no aspartate residue at its COOH terminus (Fig. 1A). Carp c-Fo-ATPase precursor protein was 96, 78, 77, 75, and 75% identical to those of zebrafish P3 (AF311603), mouse P3 (P56384), human P3 (U09813), and mouse P2 (AK007747) and P1 (AK008191) isoforms of c-Fo-ATPase, respectively. The overall structure of c-Fo-ATPase was highly conserved among vertebrates irrespective of isoforms, especially in the region of the predicted mature protein (Fig. 1B).


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  		Fig. 1.   Deduced amino acid sequences of carp gamma -F1-ATPase (A) and c-Fo- ATPase (B) precursor proteins and the mammalian isoforms. The M- and L-types in human and bovine gamma -F1- ATPase represent the muscle- and liver (nonmuscle)-type isoforms of gamma -F1- ATPase, respectively. The additional aspartate residues at the COOH terminus of gamma -F1-ATPase L-type are gray shaded. The P1, P2, and P3 in zebrafish, human, and mouse of c-Fo- ATPase represent the P1, P2, and P3 isoforms of c-Fo-ATPase, respectively. Proteins cited are c-Fo-ATPase from zebrafish P3 (GenBank accession no. AF311603), human P3 (GenBank accession no. U09813), and mouse P3 (GenBank accession no. P56384), P2 (GenBank accession no. AK007747), and P1 (GenBank accession no. AK008191), and gamma -F1-ATPase from human M-type (GenBank accession no. D16563) and L-type (GenBank accession no. D16562) and bovine M-type (GenBank accession no. M22463) and L-type (Swissprot accession no. P05631). Numbers start from the putative NH2-terminal amino acid of the premature proteins. Identical and gapped amino acids are shown by periods and dashed lines, respectively. Predicted mature protein sequences are boxed.

Effects of acclimation temperatures on the levels of carp FoF1-ATPase mRNAs. Northern blot analysis was performed for investigating the changes in the mRNA levels of carp FoF1-ATPase subunits in the fast muscle of carp after acclimation to cold and warm temperatures. cDNA probes for carp alpha -, beta -, and gamma -F1-ATPases and c-Fo-ATPase were constructed by PCR with oligonucleotide primers CATPAF1 and CATPAR1 for alpha -F1-ATPase, CATPBF4 and CATPBR3-2 for beta -F1-ATPase (25), gamma5'term1 and CATPGR1 for gamma -F1-ATPase, and c-Fo/P3F1 and c-Fo/allR1 for c-Fo-ATPase from the clones in the plasmids, pT7Blue (alpha - and gamma -F1-ATPases), pBluescript II (beta -F1-ATPase) and pGEM (c-Fo-ATPase). Relative mRNA levels of these nuclear genes were calculated using that of 18S rRNA as an internal standard. The cDNA probe of carp 18S rRNA was obtained by PCR using specific primers referring to the nucleotide sequence of carp (U87963) (Table 1). As shown in Fig. 2, the alpha -, beta - and gamma -F1-ATPase and c-Fo-ATPase transcripts in the 10°C-acclimated carp were about twofold higher than those in the 30°C-acclimated fish. The differences were significant at the levels of P < 0.05 for alpha - and gamma -F1-ATPase, P < 0.01 for c-Fo-ATPase, and P < 0.005 for beta -F1-ATPase (Fig. 2).


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  		Fig. 2.   mRNA levels of FoF1-ATPase subunits and other mitochondrial gene-encoded in fast muscle of carp acclimated to 10 and 30°C. A: Northern blot analysis for 3 individuals each from the 10°C- and 30°C-acclimated groups. Lanes 1-3 contain 10 µg of total RNAs from the 10°C-acclimated fish, whereas lanes 4-6 contain those from the 30°C-acclimated fish. The homogeneity and integrity of the loaded samples were verified by ethidium bromide staining of the gel (A, bottom). B: relative mRNA levels of carp FoF1- ATPase subunits and other mitochondrial gene-encoded proteins in fast muscle were determined using 18S rRNA as an internal control. RNA blots were quantified using a computerized densitometer. The average value of mRNA levels for the 30°C-acclimated carp was taken as 100. Bars represent means ± SD. Student's t-test was employed for statistical comparison (* P < 0.05, ** P < 0.01, *** P < 0.005). Cyt-c oxidase subunit II, cytochrome-c oxidase subunit II.

Three pairs of oligonucleotide primers, mtATPaseF1 and mtATPaseR1 for ATPase 6-8, CCOXIIF1 and CCOXIIR1 for cytochrome-c oxidase subunit II, and CCytbF1 and CCytbR1 for cytochrome b, were synthesized referring to the nucleotide sequence of carp mitochondrial genome (X61010) (Table 1). PCR with synthesized primers and carp fast muscle total DNA as a template amplified cDNA fragments of 895 bp for ATPase 6-8 and 680 bp for both cytochrome-c oxidase subunit II and cytochrome b. These cDNA fragments were used as probes for Northern blot analysis to investigate the changes in the accumulated mRNA levels in carp fast muscle after temperature acclimation. The transcripts of these mitochondrial genes were 6.7-fold higher on average in the 10°C- than 30°C-acclimated carp, their differences being significant at the levels of P < 0.01 for ATPase 6-8 and P < 0.05 for cytochrome-c oxidase subunit II and cytochrome b (Fig. 2, Table 2). The intensity of detected signals from transcripts encoded by mtDNA was 5- to 10-fold higher than those encoded by nuclear genes.

                              
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  		Table 2.   Comparison of the accumulated levels of mRNAs encoding mitochondrial proteins of carp acclimated to 10°C and 30°C

The different accumulation levels between mRNAs encoded by nuclear and mitochondrial genome could be due to enhanced copy numbers of mitochondrial genome. To address such ambiguity, the ratio of the quantities of mitochondrial to those of nuclear DNA was examined by Southern blot analysis on total DNA extracted from the fast muscle of carp acclimated to 10 and 30°C. The probe for mitochondrial DNA (mtDNA) was prepared by PCR with oligonucleotide primers, mtATPaseF2 and mtATPaseR2 (Table 1), from the clone in plasmid pT7Blue, where no recognition site was present for HindIII used for digestion of total DNAs. The probe for nuclear gene was amplified from the alpha -F1-ATPase gene with primers CATPAF1 and CATPAR1. Relative mtDNA levels in carp fast muscle were calculated using those of nuclear genome-encoded alpha -F1-ATPase as an internal control. As shown in Fig. 3, carp exhibited no significant changes in the ratio of the mitochondrial to nuclear genome content after temperature acclimation.


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  		Fig. 3.   The content of mitochondrial DNA (mtDNA) in fast muscle of carp acclimated to 10°C and 30°C. A: Southern blot analysis for 5 individuals each from the 10°C- and 30°C-acclimated groups. Lanes 1-5 contain 10 µg of total DNAs from the 10°C-acclimated fish, whereas lanes 6-10 contain those from the 30°C-acclimated fish. Arrows at top and bottom indicate mtDNA fragment of 624 bp and nuclear DNA fragment of alpha -F1-ATPase gene, respectively. Total DNAs were digested with HindIII. B: relative mtDNA levels in carp fast muscle were determined using the nuclear genome encoding alpha -F1-ATPase as an internal control. mtDNA blots were quantified using a computerized densitometer. The average value of mtDNA levels for the 30°C-acclimated carp was taken as 100. Bars represent means ± SD.

SDS-PAGE and immunoblotting patterns of the mitochondria preparations and muscle extracts. To examine the effect of thermal acclimation on the mitochondrial protein composition, we performed SDS-PAGE and immunoblotting for isolated mitochondria. SDS-PAGE showed no apparent change in the mitochondrial protein composition after temperature acclimation. After SDS-PAGE, the band carrying alpha -F1-ATPase was identified by immunoblotting, although its NH2-terminal amino acid sequence could not be determined. The monoclonal antibody against bovine heart alpha -F1-ATPase reacted specifically with carp alpha -F1-ATPase. The band corresponding to beta -F1-ATPase was determined as VAPAAAAAAAA, which was identical to 40th-49th residues from the NH2 terminus of carp beta -F1-ATPase precursor protein (25). No significant differences were observed for alpha - and beta -F1-ATPase per mitochondrial protein between fish acclimated to 10°C and 30°C (Fig. 4A).


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  		Fig. 4.   Quantitative analysis on alpha - and beta -F1-ATPase in mitochondrial proteins and muscle extract from fast muscle of carp acclimated to 10°C and 30°C. A: analysis on alpha - and beta -F1-ATPase in mitochondria. A, top: immunoblotting patterns for alpha -F1-ATPase and SDS-PAGE patterns for alpha - and beta -F1-ATPase in 10 and 20 µg mitochondrial protein, respectively, from the 10°C-acclimated carp (lanes 1-3) and from the 30°C-acclimated fish (lanes 4-6). A, bottom: relative amount of alpha - and beta -F1-ATPase. B: analysis on alpha -F1-ATPase in muscle extract using immunoblotting. Immunoblotting patterns are shown for 3 individuals each from the 10°C-acclimated (lanes 1-3) and 30°C-acclimated fish (lanes 4-6). Arrow (B, top) indicates alpha -F1-ATPase quantified at B, bottom, which shows relative amount of alpha -F1-ATPase in muscle extract. The contents of alpha - and beta -F1-ATPase in the PVDF membrane and SDS-PAGE gel were quantified using an image analysis program, Scion Image. Average values of alpha - and beta -F1-ATPase levels for the 30°C-acclimated carp were taken as 100. Bars represent means ± SD. Student's t-test was employed for statistical comparison (* Significant at P < 0.05).

To compare the abundancy of alpha -F1-ATPase in the total muscle extracts from the 10°C-acclimated carp with those from 30°C-acclimated fish, we performed SDS-PAGE and immunoblotting. No difference was observed in electrophoretic patterns for the total muscle extracts between the 10°C- and 30°C-acclimated carp. However, comparison of immunoblotting patterns for the muscle extracts from thermally acclimated carp revealed that the abundancy of alpha -F1- ATPase in the 10°C-acclimated carp was 2.1-fold higher than that in the 30°C-acclimated fish (P < 0.05) (Fig. 4B). In this system, a linear relationship between the amount of protein and signal intensity of alpha -F1-ATPase on the PVDF membrane was observed for the muscle extracts from 5 to 40 µg/lane. These observations showed the increase of mitochondrial content in the fast muscle tissue with cold acclimation of carp.

Changes of the oligomycin-sensitive ATPase activity. ATPase activity of FoF1-ATPase was determined at 10, 25, and 30°C by ATP-regenerating system using oligomycin to examine whether functional properties of carp mitochondria changed after temperature acclimation. The specific activity of FoF1-ATPase in the 10°C- and 30°C-acclimated fish mitochondria normalized as nanomoles per minute per milligram mitochondrial protein differed significantly, which was calculated to be 36 ± 9 and 18 ± 3 at 10°C, 155 ± 11 and 67 ± 5 at 25°C, and 152 ± 11 and 65 ± 16 at 30°C, respectively (Fig. 5). Consequently, the oligomycin-sensitive specific FoF1-ATPase activity in the 10°C-acclimated carp at 10°C was about half of that in the 30°C-acclimated fish at 30°C (P < 0.05) (Fig. 5).


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  		Fig. 5.   Effect of temperature acclimation on FoF1-ATPase activity of mitochondria isolated from carp fast skeletal muscle. ATPase activity was determined as described in MATERIALS AND METHODS using an ATP-regenerating system. Filled symbols represent values for the 10°C-acclimated carp, whereas open symbols represent values for 30°C-acclimated fish. Significant differences between carp acclimated to 10°C and 30°C: **** P < 0.0005, *** P < 0.005, * P < 0.05. The activity at 10°C for the 10°C-acclimated carp was about 2 times lower than that at 30°C for the 30°C-acclimated fish (P < 0.05). Bars represent means ± SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that beta -F1-ATPase was about twofold higher in the 10°C- than 30°C-acclimated carp at both translation and transcription levels (25). It is likely that all subunits encoded by both mitochondrial and nuclear genes are coordinately regulated, since FoF1-ATPase is composed of multiple subunits and has to work as one functional unit for ATP synthesis. Thus the increased levels of carp beta -F1-ATPase after cold acclimation suggest that other subunits of FoF1-ATPase would also increase. Such a situation prompted us to clone DNA fragments encoding carp alpha - and gamma -F1- ATPase and c-Fo-ATPase as well as those for other subunits encoded by mitochondrial genes and subsequently to determine the changes in their accumulated mRNA levels for carp acclimated to different temperatures.

While the deduced amino acid sequence of carp alpha -F1-ATPase was highly homologous to those of other eukaryotes, especially to the muscle specific type of bovine (44), the sequences of carp gamma -F1-ATPase and c-Fo-ATPase were less conserved compared with the cases of alpha - (in this study) and beta -F1-ATPase (25). The gamma -F1-ATPase, especially its COOH-terminal region, has been reported to be important in regulation of FoF1-ATPase in Escherichia coli (20). The COOH-terminal region of carp gamma -F1-ATPase was also highly conserved compared with those from other organisms so far reported. On the other hand, two tissue-specific isoforms produced by alternative splicing from a single gene of gamma -F1-ATPase have been reported in mammals; the non-muscle type isoform has an additional aspartate residue at the COOH terminus compared with its muscle type counterpart (18, 31, 32). Thus carp gamma -F1-ATPase in this study appears to be of muscle type as there was no aspartate residue at its COOH terminus (see Fig. 1A). The sequence of carp c-Fo-ATPase was highly identical to those of other vertebrates except the signal sequences. Considering sequence diversity in the predicted signal part of the c-Fo-ATPase, it appears that the cloned cDNA of carp in the present study correspond to the P3 isoform of zebrafish c-Fo-ATPase, which showed 96% identity in the amino acid sequence. Although P1 and P2 isoforms have been found in mammalian c-Fo-ATPase (3, 15, 35) and Fugu genome database (Scaffold_1299 for P1 and Scaffold_6377 for P2), such isoforms could not be detected in carp fast muscle in this study. The fact that the mature c-Fo-ATPase irrespective of isoforms was almost completely conserved among vertebrates including carp (see Fig. 1A) suggests that c-Fo-ATPase is one of the housekeeping components of FoF1-ATPase. Although the occurrence of tissue-specific isoforms of these subunits in carp was not investigated, we did not observe any isoforms associated with temperature acclimation.

Changes in the aerobic capacity of muscle tissues with different acclimation temperatures result not only from the varied mitochondrial content, but also from their altered properties. Wodtke (50, 51) has claimed that the acclimation temperature of carp does not affect the amount of cytochrome-c oxidase per milligram mitochondrial protein in slow muscle but induces different molar activities, probably due to the changes in the lipid composition of mitochondrial membranes. Furthermore, St-Pierre et al. (38) have reported that rainbow trout Oncorhynchus mykiss modifies the activities of respiratory enzymes such as cytochrome-c oxidase, citrate synthase, and carnitine palmitoyl transferase, by shifts in enzyme level and surface density of mitochondrial cristae. On the other hand, an increase in mitochondrial volume density induced by cold acclimation has been demonstrated in crucian carp Carassius carassius (24) and goldfish (41).

In mammals, higher mtDNA copy numbers per total genome unit including mitochondrial and nuclear genes lead to increased mitochondrial transcription rates and consequently higher mRNA levels (49). While this copy number per total genome unit may also increase in fishes during cold acclimation due to increases in mitochondrial volume density (10), this has not been proven. However, we demonstrate in the present study that the ratio of mitochondrial to nuclear genome content did not change significantly after temperature acclimation in carp fast skeletal muscle (see Fig. 3). Similarly, Battersby and Moyes (6) showed that changes in mtDNA transcripts could increase without changes in mtDNA copy number of cold-acclimated trout. The notion that transcription does not limit mitochondrial protein synthesis (4), together with the fact that mtDNA is present in excess (7), has led Battersby and Moyes (6) to speculate that mtDNA copy number is not the main determinant of mitochondrial content.

We observed that gene expression of FoF1-ATPase subunits was regulated in a coordinated manner albeit with different magnitude of changes for mitochondrial and nuclear genomes-encoded proteins following temperature acclimation at the transcriptional level (see Fig. 2). Houstek et al. (17) reported that the expression of c-Fo-ATPase is a limiting factor because its level is lower than any other subunits. In this study, carp fast muscle c-Fo-ATPase showed expression changes similar to those of other subunits encoded by nuclear genes. We also showed that cold acclimation induced higher mRNA levels of FoF1-ATPase subunits encoded by mitochondrial genes than those of the subunits encoded by nuclear genes (see Fig. 2). It has been reported that an equimolar upregulation of mitochondrial and nuclear genes encoding cytochrome-c oxidase in brown adipose tissues occurs during biogenesis of mitochondria induced by cold exposure of Djungarian hamsters (26). On the other hand, discrepancy in increasing transcriptional levels of cytochrome-c oxidase subunits between those encoded by nuclear and mitochondrial genomes has been demonstrated when mitochondrial contents were increased in growing 3T3 cells as in the present study (28, 34). Similar differences in transcriptional discrepancy for cytochrome-c oxidase subunits have been reported in cold acclimation of rainbow trout (6) and North Sea eelpout Zoarces viviparus (12), although nuclear genome-encoded subunits were in excess in contrast to those observed for carp in the present study. Such differences in the different transcript levels between two genomes among fish species with cold acclimation might be due to the temperature conditions of cold acclimation. The temperature of cold acclimation for trout (4°C) and eelpout (0°C) would be very low, which may lead to the hibernation for these fish, resulting in significant reduction of their metabolic needs. This may also explain the increase in mitochondrial genome-encoded FoF1-ATPase subunits by a factor of six to seven times in carp, which seems quite high compared with other results in fish. The 10°C acclimation temperature set for carp in the present study was intended to be low enough to trigger physiological responses as is well documented for the onset of several myosin isoforms (19, see Ref. 46). In this regard, raising carp at temperatures as low as 0-4°C would be interesting to have comparable data with other fish species reported so far.

We assume that differences in the increasing rate of mRNAs between mitochondrial and nuclear genes may be the result of different activation systems in transcriptional regulation and/or different mRNA stabilities between nuclear and mitochondrial gene groups. In this regard, Wu et al. (52) reported that PGC-1, a coactivator of nuclear receptors, could regulate coordinate expression of the subunits of respiratory chain encoded by nuclear and mitochondrial genes through regulation of the nuclear respiratory factors (NRFs). They also showed that PGC-1 binds to and coactivates the transcriptional function of NRF1 on the promoter for mitochondrial transcription factor A (mtTFA), a direct regulator of mtDNA transcription. On the other hand, Mao et al. (29) observed that the mtTFA protein level was elevated without increase of NRFs. Thus the possible imbalance in the integrity of the expression mechanism between nuclear and mitochondrial genes may result in different expression systems for mRNAs encoding FoF1-ATPase subunits in carp fast muscle. Alternatively, the different rates of mRNA accumulation between nuclear and mitochondrial genes may be caused by their different mRNA stabilities (9), because the ratio of mitochondrial to total genome did not change after temperature acclimation in carp fast skeletal muscle as described before.

In accordance with the increased levels of mRNA for FoF1-ATPase subunits, the quantities of alpha -F1-ATPase in the 10°C-acclimated carp were 2.1-fold higher than those in the 30°C-acclimated fish (see Fig. 4B). This increased level was well correlated with that of beta -F1-ATPase, as we have reported previously (25). On the other hand, no change was observed in mitochondrial protein composition including alpha - and beta -F1-ATPase between the 10°C- and 30°C-acclimated fish (see Fig. 4A). Although proteins encoded by mitochondrial genes were not quantified in this study, we assume that most mitochondrial proteins would also be increased, maintaining the same expression level of all transcripts for the FoF1-ATPase subunits in cold acclimation. However, the SDS-PAGE method is not sensitive enough to detect small but relevant changes for weakly stained proteins in such crude preparations. Because the mRNA of mitochondrial compared with nuclear-encoded subunits is upregulated to a much larger extent (see Fig. 2), the question of whether the copy number of these subunits is increased remains open.

In this study, the oligomycin-sensitive ATPase activity per mitochondrial protein in cold-acclimated carp was higher than that in warm-acclimated carp at 10, 25, and 30°C (see Fig. 5). The possible decrease of ATP production in mitochondria at low temperatures could be compensated by the increase of FoF1-ATPase activity. The increase in specific activity of mitochondrial ATPase without change of mitochondrial protein composition in the cold-acclimated fish may be due to the changes in the lipid composition of mitochondrial membrane. Another possibility for the changes in specific activity of FoF1-ATPase is the effect of some other interacting molecules in addition to those of FoF1-ATPase subunits. For example, F1-inhibitor protein called IF1 is known as an inhibitor protein of FoF1-ATPase, although the change of its expression levels after thermal acclimation has not been studied. Eurythermal fish species are able to maintain normal biological activity across a wide range of environmental temperature, which implies that temperature acclimation invokes mechanisms that serve to ameliorate the intrinsic effects of temperature on biochemical reaction rates at the level of enzyme. Two types of compensatory mechanisms can be employed: either through increasing the catalytic ability of an enzyme or by increasing the enzyme concentration or by both (16). This proposition has in fact been well reflected by the result of FoF1-ATPase activity in the present study. While the ATPase activities from cold-acclimated fish were consistently higher than those in their warm-acclimated counterparts under the same reaction temperature, the enzyme activity in cold-acclimated carp measured at 10°C was about half of that in warm-acclimated fish measured at 30°C (see Fig. 5). Thus it seems that despite higher specific activity, at 10°C the cold-acclimated fish has to increase the enzyme concentration to attain the 30°C-activity level to maintain the normal metabolic activity.

We found that the mRNA levels of nuclear genes per unit weight of total RNA were nearly twofold higher in the 10°C- than 30°C-acclimated carp. However, the transcripts of mitochondrial genes for the 10°C-acclimated carp in terms of the same comparing unit were six to seven times as much as those for the 30°C-acclimated carp. Similarly, the FoF1-ATPase activities were nearly twofold higher for the cold-acclimated fish than their warm-acclimated counterparts. Such quantitative and qualitative changes in carp FoF1-ATPase may contribute to extra ATP production required to compensate for energy balance at suboptimal temperatures. These changes further suggest that alterations in FoF1-ATPase, functioning at the final step of the ATP production chain, are the primary issues during temperature acclimation of carp. Further investigation for regulatory mechanisms involved in expression of carp FoF1-ATPase subunits during temperature acclimation will give new insights to understand overall reorganization of energy production of eurythermal fish in association with varied environmental temperatures.


    ACKNOWLEDGEMENTS

We thank Dr. T. Higuti and M. Suenaga, Univ. of Tokushima, for protocols relating to FoF1-ATPase assay. Measurement of ATPase activity was performed at the National Research Institute of Fisheries Science. Fugu genome data have been provided freely by the Fugu Genome Consortium for use in this publication/correspondence only. We thank M. N. Ahsan, The Univ. of Tokyo, and Dr. S. SriKantha, Kyoto Univ., for critical reading of this manuscript.


    FOOTNOTES

This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports, and Technology of Japan, and from the Fisheries Agency of Japan.

Address for reprint requests and other correspondence: S. Watabe, Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The Univ. of Tokyo, Bunkyo, Tokyo 113-8657, Japan (E-mail: awatabe{at}mail.ecc.u-tokyo.ac.jp).

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.

September 12, 2002;10.1152/ajpregu.00182.2002

Received 26 March 2002; accepted in final form 9 September 2002.


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