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(The FASEB Journal. 2001;15:635-644.)
© 2001 FASEB

Direct influence of the p53 tumor suppressor on mitochondrial biogenesis and function

RONALD J. DONAHUE, MARJANEH RAZMARA, JAN B. HOEK and THOMAS B. KNUDSEN1

Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA

1Correspondence: Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust St., Philadelphia PA 19107, USA. E-mail: Thomas.Knudsen{at}mail.tju.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial localization of p53 has been observed in several cell systems, but an understanding of its organelle-based physiological activity remains incomplete. The purpose of the present study was to investigate the mitochondrial DNA genomic response to dominant-negative p53 mutant miniprotein (p53DD) fused to a mitochondrial import signal. Constructs were generated to express mitochondrial targeted enhanced green fluorescent protein (mEGFP) or dominant-negative mutant p53 miniprotein (m53DD) by in-frame fusion to the signal peptide sequence of murine Cox8l. Control cytosolic vectors (cEGFP, c53DD) had the signal sequence placed in antisense orientation. NIH 3T3 cells were transiently transfected with these vectors in various combinations. Mitochondrial 16S ribosomal RNA (16S rRNA) expression and fluorochrome staining with Mitotracker Red CMXRos ({Delta}{Psi}m) were decreased in cells expressing m53DD. Both alterations were specific for mitochondrial import competence (e.g., m53DD vs. c53DD) as well as the passenger protein (e.g., m53DD vs. mEGFP). The normal functional state of mitochondria was restored with PK11195, a specific ligand of the mitochondrial peripheral-type benzodiazepine receptor. Negative dominance of m53DD on 16S rRNA expression and CMXRos staining, and rescue of these parameters with PK11195, imply a direct positive effect of p53 on mitochondrial biogenesis and function.—Donahue, R. J., Razmara, M., Hoek, J. B., Knudsen, T. B. Direct influence of the p53 tumor suppressor on mitochondrial biogenesis and function.


Key Words: 16S rRNA • PK11195 • peripheral benzodiazepine receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE P53 TUMOR suppressor is a homotetrameric transcription factor induced by DNA damage (1 , 2) , hypoxia (3 , 4) , and ribonucleotide perturbation (5 , 6) . These stress imbalances can trigger p53-dependent cell cycle arrest or programmed cell death (apoptosis) in a variety of cell types (7 8 9) . Although most of the p53-dependent cellular changes are considered to be nuclear based (10 11 12 13) , mitochondrial localization of p53 protein has also been detected (14 15 16) , raising the possibility of organelle-based effects of this tumor suppressor.

Several lines of evidence support a connection between p53 and the mitochondrion. One pertains to the mitochondrial mechanism of apoptosis. Cells from p53(-/-) mice resist some stress-induced perturbations that invoke apoptosis through collapse of the mitochondrial inner membrane potential ({Delta}{Psi}m) and release of cytochrome c. This implies a failure of p53-deficient cells to engage apoptosis at the level of the mitochondrion (17) . Mitochondrial perturbations observed during p53-mediated apoptosis were preceded by trans-activation of various oxidoreductases (13) and reactive oxygen species (ROS) production (18 , 19) . Oxidative damage and associated protective mechanisms to repair this damage are critical to the mitochondrion and its genome due to oxygen metabolism and ROS production. Several genes coding for mitochondrial proteins have p53 response elements: pyruvate dehydrogenase (12) and Bax (20) are notable examples. By up-regulating Bax expression, p53 may facilitate the proapoptotic state of cells. Mitochondrial localization of p53 was recently correlated with early changes leading to p53-mediated apoptosis in diversified cell types stimulated by a range of stressors, including DNA damage and hypoxia (16) . In that study, redirecting the wild-type p53 protein to the mitochondrion revealed an enhancer pathway for stress-induced apoptosis involving the direct action of p53 on the organelle. Finally, several genes encoded by mitochondrial DNA (mtDNA) express at levels related to p53 functional activity. Early mouse embryos homozygous for a p53 null mutation showed secondary deficiency of mitochondrial 16S ribosomal RNA transcripts at a stage in development when the embryo normally switches from an anaerobic (glycolytic) to aerobic (oxidative) metabolism (21) . This implies a dependence of mitochondrial biogenesis on normal p53 functional activity. At least two other studies reported a positive correlation between p53 activity and expression of mtDNA-coded genes (22 , 23) .

The notion that p53 affects mitochondrial function directly merits attention; however, the only study that has tested this hypothesis dealt with a biological response independent of mtDNA genetic activity, e.g., stress-induced apoptosis (16) . The purpose of the present study was to investigate the mitochondrial genetic response to dominant-negative p53 mutant miniprotein (p53DD) engineered for import to this organelle. Miniprotein p53DD is a deletion mutant containing the first 13 codons of murine p53, followed by codons 302–390 (24) . It is competent for oligomerization with wild-type p53 but lacks the core domain in which sequence specific DNA binding functions reside, and thus acts as a transdominant-negative suppressor of p53 trans-activation (25) . Mitochondrial localization of p53DD was directed with the cleavable amino-terminal signal peptide from Cox8l, a nuclear gene coding for subunit VIII-L precursor protein of cytochrome c oxidase. This leader sequence has been proved to contain all genetic information necessary for specific, efficient mitochondrial import of green fluorescent protein in various cell lines (26) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mitoplast preparation
Mitoplasts are submitochondrial particles collected by differential centrifugation and liberation of the outer mitochondrial membrane with digitonin to remove nuclear, cytosolic, and microsomal proteins. Tissues for mitoplast preparation were harvested from the liver of adult CD-1 mice and embryos on day 10 of gestation. Mitoplast isolation used differential centrifugation techniques in low-salt buffer and digitonin (27) . Briefly, tissues were homogenized in 2 mM HEPES buffer, pH 7.4, containing 0.3M mannitol. The homogenate was centrifuged at 460 g and the supernatant was centrifuged (4°C) stepwise at 3000 g, 6000 g, and 8000 g. The final pellet was resuspended in 0.25 ml mannitol-HEPES to yield the mitochondrial fraction. Protein was measured by dye binding assay (Bio-Rad, Hercules, Calif.). To obtain mitoplasts (e.g., mitochondria whose inner membrane had been exposed), the outer membrane was disrupted with 0.11 mg digitonin/mg protein and incubation for 5–10 min on ice. Mitoplasts were collected by centrifugation at 8000 g and suspended in mannitol-HEPES containing 1 mM EDTA and 0.01 mg/ml proteolytic inhibitors: aprotinin, leupeptin, pepstatin A, and phenylmethylsulfonylfluoride. Final solubilization of this fraction was accomplished with 2% Triton X-100 in mannitol-HEPES buffer and clarification at high speed (100,000 g). In some cases, mitoplasts were solubilized in RIPA buffer (Tris-HCl, pH 7.4, containing 1% Nonidet P-40, 0.25% deoxycholic acid, and 1 mM EGTA (28) .

Immunochemical analysis
Straight Western blotting used 20 to 40 µg protein loaded per lane, as indicated. For immunoprecipitation, ~1 mg protein was incubated with monoclonal antibody overnight. To this was added a slurry of protein A/G-Sepharose (Calbiochem, San Diego, Calif.). After 2 h the beads were collected by centrifugation, rinsed with RIPA containing proteolytic inhibitors, denatured, and reduced. These samples were used for Western blotting. Protein was electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) mini-gels under reducing conditions (28) . Rainbow markers (Amersham, Arlington Heights, Ill.) provided size calibration. Human recombinant p53 protein (rp53) isolated from baculovirus was from Dr. Fatah Kashanchi (UMDNJ-Newark, N.J.). Proteins were electroblotted to nitrocellulose at 4°C. Filters were washed with 5% Tween-20 in phosphate-buffered saline, blocked with 4% non-fat dry milk, and incubated with the primary antibody overnight at 4°C. Primary antibodies included: anti-p53 monoclonal antibodies PAb421, PAb1801, PAb240, or PAb246 from Oncogene Sciences (Gaithersburg, Md.) used at 1:500 dilution; anti-COI monoclonal antibody from Molecular Probes (Eugene, Oreg.) used at 1:500 dilution; and FL393 rabbit antiserum to GST-p53 fusion protein (Santa Cruz, Santa Cruz, Calif.) used at 1:1000 to 1:5000 dilutions. After appropriate secondary antibody steps and rinsing, the blots were developed with enhanced chemiluminescence detection (ECL, Amersham).

cEGFP and mEGFP expression plasmids
RNA from a male CD-1 mouse liver was reverse-transcribed with Superscript II RNase H- reverse transcriptase (Gibco BRL, Grand Island, N.Y.) to complementary DNA (cDNA). A polymerase chain reaction (PCR) strategy was used to amplify the cDNA sequence coding for the cleavable amino-terminal peptide of cytochrome c oxidase subunit VIII-L (Cox8l) precursor protein (26 , 29) . Sequence information for murine Cox8l was retrieved from GenBank (accession number U37721). Specific primers designed for the first amplification were 5'-caa ggt cgt tcc gcg ccg tca-3' (upper) and 5'-gga ccc agc ccg cag gca gaa-3' (lower). Thirty cycles of PCR (95°C 1 min, 63.9°C 2 min, 72°C 1 min) generated the predicted 195 base pair (bp) product. This product was used for PCR with nested primers designed to selectively amplify the first 24 amino acid residues of the signal peptide and simultaneously convert both ends to ApaI. Nested primers were 5'-ggg ccc atg tct gtc ctg acg cca ctg ctg-3' (upper) and 5'-ggg ccc agc ccg cgg cac cat gag ccg ccg-3' (lower). The reaction mixture was fractionated by GenPak FAX high-performance liquid chromatography (HPLC) at 50°C, yielding a peak at ~84 bp for collection and purification on a Nensorb 20 cartridge (NEN Life Science Products, Boston, Mass.) (21) . This PCR product was T/A cloned into pGEM-T Easy for transformation of JM 109 high-efficiency competent bacterial cells (Promega, Madison, Wis.). Plasmid from transformants was digested with ApaI and fractionated by GenPak FAX HPLC. Isolation of the peak containing the 84 bp ApaI fragment yielded cDNA that was purified and ligated into the ApaI restriction site of plasmid pEGFP-N2 (Clontech Laboratories, Palo Alto, Calif.), thus deriving pmit-EGFP for expression of recombinant mitochondrial protein mEGFP. Bacterial transformants were selected with 30 µg/ml kanamycin. Automated DNA sequencing confirmed the amino-terminal signal peptide sequence of Cox8l fused in-frame with EGFP. Clones in which the leader sequence was in the reverse direction were designated pcyt-EGFP for expression of recombinant cytosolic protein cEGFP.

c53DD and m53DD expression plasmids
Plasmid pSPp53DD was a gift from Dr. Moshe Oren of the Weizmann Institute (Rehovot, Israel). Bacterial transformants were selected with ampicillin. Plasmid digested with XbaI was subjected to PCR to amplify the p53DD cDNA and to add SmaI and NotI restriction sites immediately upstream and downstream of p53DD start and stop codons, respectively. The primers were 5'-ccc ggg atg act gcc atg gag gag tca cag-3' (upper) and 5'-gcg gcc gct cag tct gag tca ggc ccc aca aa-3' (lower). The resulting ~0.32 kilobase (kb) PCR product was T/A cloned into pGEM-T Easy. The SmaI-NotI fragment harboring p53DD was excised and purified by HPLC. Double digestion of pcyt-EGFP and pmit-EGFP with these enzymes removed the EGFP sequence and opened the SmaI-NotI site for insertion of the SmaI-p53DD-NotI cDNA. Ligation and cloning thus derived plasmids pcyt-53DD and pmit-53DD for expression of recombinant proteins c53DD and m53DD in the cytosol and mitochondrion, respectively. Automated DNA sequencing confirmed the presence of the cleavable amino-terminal leader sequence of Cox8l joined by glycine-proline-glycine codons to the p53DD cDNA sequence.

Cells and transfection
NIH 3T3 cells were purchased from ATCC (Manassas Va.). This continuous cell line was established from NIH Swiss mouse embryo cultures. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 0.5% gentamicin on 75 cm2 T-flasks. At ~75% confluence cultures were transfected with SuperFect Transfection Reagent (Qiagen, Chatsworth, Calif.). Plasmid DNA (6 µg) was suspended in 0.15 ml DMEM, mixed with SuperFect reagent, transfected for 3 h, and washed according to the manufacturer’s recommended protocol. Sham (control) cultures were carried through the transfection procedure using nonexpressing vector DNA in place of expression plasmid. Transfected cells were cultured in complete medium for 24 h, producing expression of cEGFP or mEGFP in 20–40% cells in the monolayer. This transfection efficiency compared favorably to the 20–30% reported for HeLa cells under similar conditions (26) . Sham transfection controls produced no fluorescence. In some experiments, cells received a 4 h exposure to 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195), purchased from RBI (Natick, Mass.).

Mitochondrial fluorochroming and confocal microscopy
Transfected NIH 3T3 cell monolayers were coverslipped and imaged with a Bio-Rad MRC 600 Laser Scanning Confocal Imaging System. The system consisted of a Krypton-Argon mixed gas laser that allows image visualization at the 488, 568 and 647 nm wavelengths. The microscope itself, a Zeiss Axiovert 100 inverted fluorescence microscope, was equipped with 10x, 40x, and 63x (oil immersion) objectives. To stain mitochondria, cells were incubated for 15 min at 37°C in DMEM medium containing 10 nM MitoTracker Red CMXRos (Molecular Probes, Eugene, Oreg.) and chased 15 min with DMEM. Images from EGFP (green) and CMXRos (red) fluorescence patterns of the cells were processed as one-color images or two-color overlays as indicated. Neither signal showed significant photobleaching during the time frame required for analysis. The findings reported here were replicated in three independent experiments.

Expression PCR
Total cellular RNA digested with RNase-free DNase was reverse-transcribed with SuperScript II RNase H- reverse transcriptase (Gibco BRL). PCR primers designed to amplify target (16S rRNA) and control (ß-actin) transcripts were as follows: 5'-aga gct aga aac ccc gaa ac-3' (upper) and 5'-aag ata aga gac agt tgg ac-3' (lower) for murine 16S rRNA (785 bp product); and 5'-tac cac agg cat tgt gat gg-3' (upper) and 5'-aat agt gat gac ctg gcc gt-3' (lower) for murine ß-actin (310 bp product) (21) . PCR conditions after hot start at 95°C for 5 min were 24 cycles of 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min. Initial PCR used different cycle numbers to determine the optimal number and sample dilution for quantitative amplification. PCR products were resolved on a nondenaturing 8% polyacrylamide gel with ethidium staining.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of p53 immunoreactivity in mitoplasts
Based on evidence for the mitochondrial localization of p53 immunoreactivity (15) , we assayed for this protein in the mitoplast-enriched fraction of the adult mouse liver. The liver was chosen for a study model because hepatocytes have an active mitochondrial cycle (27) . Mitoplasts were used over intact mitochondria (16) to emphasize the link between p53 and mitochondrial genetic activity. Straight Western blotting yielded equivocal demonstration of p53 immunoreactivity in as much as 40 µg mitoplast protein loaded per lane (Fig. 1A , B , C , D , E ). Several cytosol bands reacted with secondary anti-mouse IgG antibody; these nonspecific bands were negative in mitoplasts, thus clearing the fraction of cross-contamination from cytosol (e.g., Fig. 1B ). Essentially the same result was obtained with PAb246, a monoclonal antibody that recognizes an epitope of wild-type p53 abated by denaturing conditions of SDS-PAGE (Fig. 1C ). In contrast PAb240, a monoclonal antibody that will generally react with denatured p53, recognized two robust bands (67 and 120 kDa) and a weak 53 kDa band in overexposed blots (Fig. 1C ). Too many bands appeared in the 50–60 kDa range for confirmation of p53 (30) .



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  		Figure 1. Detection of p53 protein in the mitoplast fraction of murine tissues. A) Calibration standards fractionated with 10% SDS-PAGE minigels under reducing conditions; M, protein marker stained with Coomassie blue; rp53, recombinant human p53 protein from baculovirus immunostained with FL393 antiserum and ECL detection. B–E) Western blotting of the cytosol (cyt) and mitoplast (mtp) fractions of the liver from adult male CD-1 mice; 40 µg protein loaded per lane. B) Negative controls omitting the primary antibody (NFA) showed nonspecific staining in the cytosol fraction primarily. Absence of these nonspecific bands from the mitoplast demonstrates the clarity of fractionation. C) Samples immunostained with PAb246 showed the nonspecific cytosolic bands only; this antibody recognizes wild-type p53 under native conditions. D) PAb240 recognized two undetermined mitoplast-specific bands at 67 and 120 kDa and several weaker bands, including one that migrated with an apparent molecular mass of 53 kDa (arrow) evident when blots were overexposed; this antibody recognizes denatured p53. E) Fractions immunostained with antibodies to subunit I of cytochrome c oxidase (anti-COI) revealed specificity to the mitoplast. F, G) Immunoprecipitation (IP) of 1 mg mitoplast protein with PAb246 or PAb240, as indicated, followed by Western blotting (WB) with FL393 antiserum. Mitoplasts were isolated from the liver of a pregnant CD-1 female mouse (F) and her embryos on day 10 of gestation (G). A negative control is provided omitting FL393 (NFA); the arrow indicates migration of p53 in the rp53 marker lane.

Partitioning of p53 with the mitoplast was demonstrable when 1 mg of protein from this fraction was immunoprecipitated with conformation-specific antibodies and Western blotted with FL393 antiserum (Fig. 1F , G ). Immunoprecipitation-Western blotting detected native (PAb246-reactive) and denatured (PAb240-reactive) p53 in the mitoplast isolated from the liver of a pregnant female CD-1 mouse (Fig. 1F ). Her embryos on day 10 of gestation showed both forms of p53 in the mitoplast (Fig. 1G ); however, the assay showed a greater presence of native p53 in liver than day 10 embryos whereas both samples had denatured p53. Partitioning of p53 with the digitonin-resistant fraction of the mitochondrion confirms that a small percentage of cellular p53 is imported to the organelle in proliferating (embryo) and nonproliferating (liver) tissues (15 , 16) .

Cellular expression of import-competent recombinant proteins mEGFP and m53DD
Most mitochondrial proteins are synthesized as precursor proteins on cytosolic ribosomes and imported to the organelle for processing and sorting. The best-characterized mitochondrial targeting signals are cleavable amino-terminal signal sequences (31) . We used a PCR strategy to clone the cDNA sequence coding for the first 24 amino acids of the 26 residue cleavable amino-terminal peptide of murine cytochrome c oxidase subunit VIII-L precursor protein (29) . The signal sequence was subsequently in-frame fused to the NH2 terminus of enhanced red-shifted variant of wild-type green fluorescent protein (EGFP) in Clontech expression plasmid pEGFP-N2 (Fig. 2 ). This yielded two plasmids: pmit-EGFP, which expresses EGFP fused with the signal peptide (mEGFP); and pcyt-EGFP, which contains the antisense leader sequence and thus does not express a functional signal peptide (cEGFP). DNA sequence was confirmed for the engineered region of plasmids pcyt-EGFP and pmit-EGFP. These plasmids were then converted to pcyt-53DD and pmit-53DD to express recombinant c53DD and m53DD, respectively, by replacing the SmaI-NotI restriction fragment harboring EGFP cDNA with murine p53DD cDNA (Fig. 2) .



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  		Figure 2. Restriction maps for pmit-EGFP and pmit-53DD. pEGFP-N2: Plasmid pEGFP-N2 (Clontech) provided the backbone. The SalI-AflII region of pEGFP-N2 is depicted, encompassing nucleotides 639-1644 of this parental vector (4.7 kb). The EGFP gene codes an enhanced red-shifted variant of wild-type GFP. The multiple cloning site encompasses nucleotides 591–665 and is between the immediate early promoter of CMV (PCMV IE) and the EGFP coding sequences. SV40 polyadenylation signals downstream of the EGFP gene direct proper 3' processing of the mRNA. Nucleotides 676–686 encompass the Kozak consensus translation initiation site. The sequence given below the restriction map corresponds to nucleotide positions 653–686; the ApaI site is underlined and the first two codons of EGFP are upper cased. pmit-EGFP: The leader sequence of the murine cytochrome c oxidase subunit VIII-L gene (MIT), having ends converted to ApaI, was ligated into the multiple cloning site of pEGFP-N2 placing it in the same reading frame as EGFP. The sequence given below the restriction map corresponds to nucleotide positions 653–776; the ApaI sites are underlined, the MIT leader sequence is boldfaced, and the first two codons of EGFP are upper cased as are the intervening linker peptide sequences. A reasonable Kozak sequence was retained. pmit-53DD: Plasmid pmit-53DD was derived by removing the SmaI-NotI restriction fragment from pmit-EGFP and inserting in its place the murine p53DD cDNA sequence having corresponding ends converted to SmaI and NotI; thus, the leader sequence of murine COVIII-L precursor protein is in the same reading frame as p53DD. The sequence given below the restriction map corresponds to nucleotide positions 653–773; the SmaI site is underlined, the MIT leader sequence is boldfaced, and the first eight codons of p53DD are upper cased as is the glycine-proline-glycine linker peptide.

Transfection of NIH 3T3 cells confirmed competence of the signal peptide with respect to selective mitochondrial translocation of passenger protein (e.g., EGFP). Cells expressing cEGFP or mEGFP and stained with MitoTracker Red CMXRos were used for confocal imaging of fluorescence for EGFP (green channel) and CMXRos (red channel) at 24 h after transfection. MitoTracker Red accumulated in response to the membrane potential forms a covalent complex with mitochondrial constituents, which prevents subsequent release of the fluorochrome. Robust green fluorescence was detected in 20–40% cells transfected with pcyt-EGFP or pmit-EGFP. The uniform cellular fluorescence pattern for cEGFP (Fig. 3A ) was clearly distinct from the CMXRos staining pattern of the mitochondria (Fig. 3B ). In contrast, mEGFP showed a punctate cytoplasmic pattern (Fig. 3C ) that colocalized with CMXRos (Fig. 3D ). This observation confirmed that expressed recombinant mEGFP was confined to mitochondria and was strongly fluorescent despite extensive manipulation of its amino terminus and the reducing properties of the mitochondrial matrix (29) . Cells expressing either cEGFP or mEGFP could not be distinguished from nontransfected cells in the red channel indicating normal properties of mitochondrial energization.



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  		Figure 3. Cellular Localization of cEGFP and mEGFP in NIH 3T3 cells. Confocal microscopy of NIH 3T3 cells imaged in the green channel (A, C) or the red channel (B, D). Cells were transfected with expression plasmids pcyt-EGFP (A, B) or pmit-EGFP (C, D), cultured for 24 h, and stained with MitoTracker Red CMXRos. The punctate green fluorescence of mEGFP was specific for the mitochondrion as indicated by colocalization with CMXRos. Magnification = 270x

Western blotting verified m53DD expression (Fig. 4 ). Lacking the core domain yet retaining the carboxyl terminus of p53, the recombinant protein reacts with PAb421 but not PAb246. Whole cell lysates immunoprecipitated with these antibodies displayed the predicted PAb421-positive/PAb246-negative band at ~14 kDa (lanes 1–2 in Fig. 4 ). Cultures expressing mEGFP (lanes 3–4 in Fig. 4 ) or sham (lanes 5–6) transfections failed to display the PAb421-reactive/PAb246-nonreactive band.



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  		Figure 4. Expression of recombinant m53DD protein in NIH 3T3 cells. Whole cell lysates were immunoprecipitated with PAb421 (upper blot) or PAb246 (lower blot) and Western blotted with FL393 antiserum. Arrowheads mark ~14 kDa position. Cells were transfected with pmit-53DD (lanes 1–2), pmit-EGFP (lanes 3–4), or sham controls (lanes 5–6).

Negative dominance of m53DD on mitochondrial function
Previously we observed under-representation of mtDNA coded 16S rRNA transcripts in early embryos homozygous for a p53 null mutation (21) . To determine whether 16S rRNA levels were sensitive to m53DD expression, relative PCR was run with primers for test (16S rRNA) and reference (ß-actin) genes. Expression PCR data for 3T3/m53DD cells and various controls (3T3/c53DD, 3T3/mEGFP, 3T3/sham) is illustrated in Fig. 5 and summarized in Table 1 . The effect of p53 deficiency (21) was observed as a significant (P=0.015) decline of 16S rRNA levels in 3T3/m53DD cells (Table 1) . The decline was from 2 h through at least 36 h post-transfection, which were the only time points tested here (not shown).



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  		Figure 5. Expression PCR analysis of NIH 3T3 cells. RT-PCR used primers specific for 16S rRNA and ß-actin. NIH 3T3 cells were transfected for 24 h, followed by 4 h incubation in control medium (odd numbered lanes) or medium supplemented with 4 µM PK11195 (even numbered lanes). A) Transfection with pcyt-53DD (lanes 1–2) or sham cultures (lanes 3–4): neither construct perturbed the 16S rRNA levels. B) Transfection with pmit-EGFP (lanes 5–6) or pmit-53DD (lanes 7–8): the latter dropped 16S rRNA levels and this effect was reversed with PK11195.


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  		Table 1. Negative dominance of pmit-53DD on mitochondrial 16S rRNA levelsa

Work in progress showed perturbation of 16S rRNA expression with methyl mercury in early embryos and striking reversal with PK11195 (32) . This isoquinoline carboxamide derivative specifically ligands the mitochondrial peripheral-type benzodiazepine receptor (33) . Since sensitivity to m53DD implies biological activity at the level of the mitochondrion, we asked whether PK11195 might also rescue 16S rRNA levels in 3T3/m53DD cells. Cultures treated with 4 µM PK11195 showed normal 16S rRNA levels at 4.0 h (lanes 7 and 8 in Fig. 5B ). This observation was significant (P=0.005) across four independent experiments (Table 1) . Hence, the decline of 16S rRNA levels in 3T3/m53DD cells was reversed with PK11195.

A decrease in 16S rRNA expression might be expected to reflect in the energized state of mitochondria. To test this prediction, NIH 3T3 cells were cotransfected with pmit-EGFP and pmit-53DD and stained with Mitotracker Red CMXRos. Confocal microscopy and two-color overlay revealed three distinct mitochondrial phenotypes: green, indicating importation of expressed recombinant mEGFP + m53DD; red, indicating energization of the organelle; and yellow, indicating both phenotypes. Cells expressing m53DD tended toward less CMXRos fluorescence than normal cells (Fig. 6A , B , C ). Signal intensity was quantified in the same image for transfected (3T3/m53DD) and nontransfected (3T3) cells. The effect of m53DD was seen as a significant (P=0.001) 2.4-fold decrease in CMXRos signal intensity in 3T3/m53DD cells (61.2±17.0) vs. neighboring 3T3 cells (141.6±10.9). CMXRos staining appeared normal in 3T3/m53DD cells treated with PK11195 (Fig. 6D , E , F ). Signal intensity was 1.4-fold higher in 3T3/m53DD cells (121.5±8.4) vs. neighboring 3T3 cells (88.4±12.0) in cultures treated with 4.0 µM PK11195. Because this assay compared cellular CMXRos staining within the same confocal image, we could not confirm the apparent decline of mitochondrial energization observed in the nontransfected 3T3 cells treated with PK11195.



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  		Figure 6. Impact of m53DD expression on mitochondrial energization. Confocal microscopy of NIH 3T3 cells cotransfected with pmit-EGFP and pmit-53DD and stained with CMXRos. Representative field of four cells from normal cultures (A–C) and cultures incubated 4.0 h in medium containing 4 µM PK11195 (D–F). Similar attenuation and gain settings were used for both sets of images. A, D) Cells imaged in the green channel revealed mEGFP expression; positives were assumed to also express m53DD. PK11195 had no obvious effect. B, E) Same cells imaged in the red channel to reveal energized mitochondria; positives for mEGFP tended toward weaker CMXRos fluorescence than neighboring cells negative for mEGFP. This trend was not evident in cultures treated with PK11195. C, F) Two-color overlay reveals decreased mitochondrial energization associated with m53DD expression and rescue with PK11195; yellow coloration indicates green-red images superimposed.

Some 3T3/m53DD cells showed a qualitative effect on CMXRos staining (Fig. 7 ). When examined closely, this subset of 3T3/m53DD cells showed increased heterogeneity of mitochondrial confocal phenotypes vs. the control phenotype of primarily yellow (overlay) mitochondria displayed by 3T3/mEGFP cells. Heterogeneity in some 3T3/m53DD cells reached roughly equal proportion between green, red, and yellow phenotypes (Fig. 7A , B , C ). This heterogeneity was infrequently observed when 3T3/m53DD cells treated with PK11195 (Fig. 7D , F ). Assuming mitochondria do not distinguish between recombinant mEGFP and m53DD proteins, this heterogeneity implies coexistence of three kinds of organelles within the same cell: yellow, representing energized organelles with imported m53DD; red, representing energized organelles naïve for imported m53DD; and green, representing de-energized organelles having imported m53DD.



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  		Figure 7. Local impact of m53DD expression on mitochondrial energization. Confocal microscopy of NIH 3T3 cells cotransfected with pmit-EGFP and pmit-53DD and stained with CMXRos. Representative cell coexpressing mEGFP and m53DD imaged in the green channel (A, D), red channel (B, E), and two-color overlay (C, F). Arrows identify mitochondria having green and red phenotypes; yellow coloration indicates green-red images superimposed. The cell in panels D–F was exposed 4.0 h to 4 µM PK11195 and demonstrates the normal (control) mitochondrial pattern. Both cells were imaged with similar attenuation and gain settings.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of the present study support a direct influence of p53 on mitochondrial biogenesis, thus expanding the emerging role of mitochondrial p53 from apoptosis (16) to intrinsic regulation of mtDNA genomic expression. Mitochondrial p53 was detected in the digitonin-insoluble mitoplast fraction of the murine liver and day 10 embryo, implying its association with the matrix space or inner membrane of the organelle. This detection required immunoprecipitation-Western blotting and showed a greater presence of PAb246 (native) p53 in the liver than embryos while both tissue types had denatured (PAb240-reactive) p53. The requirement of 1 mg protein for detecting mitochondrial p53 in the mitoplast fraction contrasts with 5 to 10 µg protein for intact mitochondrion during stress-induced apoptosis (16) . In that study, a majority of the p53 targeted for mitochondrial import resorted to the outer membrane and/or intermembranous compartment. Since digitonin solubilization would most likely have removed any proteins in these regions of the mitochondrion, the mitoplast-derived p53 detected here may only represent a subset of p53 associated with the organelle.

Most nascent proteins imported to the mitochondrion arrive from the cytosol by a mechanism dependent on the inner mitochondrial membrane potential ({Delta}{Psi}m) and chaperone proteins. Because p53 does not harbor a typical amino-terminal mitochondrial leader sequence, the translocation mechanism is not immediately apparent. An earlier study reported coimmunoprecipitation of p53 with Grp75, a mitochondrion specific heat shock protein (15) . The same mitochondrial chaperone system was implicated in the importation and resorting of PAb246-positive (wild-type) p53 to the mitochondrion during stress-induced apoptosis (16) . Mitochondrial localization of Bax is also observed during apoptosis (34 , 35) . However, the translocation mechanism for p53 may depend on its importation and resorting to the outer membrane. Mitoplast-derived p53 may represent a precursor pool for resorting p53 to the membranous space and/or outer membrane within a small number of cells undergoing p53-mediated apoptosis or it may represent a unique compartmentation of this well-characterized tumor suppressor among a percentage of nonapoptotic cells.

The dominant-negative miniprotein used in the present study (p53DD) retains the carboxyl-terminal oligomerization domain and interferes with sequence-specific DNA binding properties of the active p53 tetramer (24 , 25) . Additional studies are needed to confirm that m53DD assembles with mitoplast-derived p53; however, the properties of m53DD responsible for the decline of 16S rRNA levels and CMXRos staining of mitochondria were dependent on import competence. Evidence for this comes from the observed activity of p53DD when expressed as mitochondrial (m53DD) but not cytosolic (c53DD) protein. Negative dominance was also specific for the passenger protein (p53DD) because 16S rRNA levels and CMXRos staining were normal in 3T3/mEGFP cells. These findings suggest a novel, organelle-based effect of p53 as a direct positive regulator of mitochondrial biogenesis and function. The normal physiological activity of mitoplast-derived p53 may be separate from the mitochondrial mechanism of apoptosis, because {rho}0 cells lacking mtDNA remain highly glycolytic and competent to apoptosis (36) , and because apoptosis involving mitochondrial p53 required neither oligomerization nor sequence-specific DNA binding domains of the p53 protein (16) .

A transdominant negative effect of m53DD on mtDNA genetic activity would be consistent with our understanding of wild-type p53 as a multifunctional transcription factor (7 , 8) . Alternative conformational states of mitoplast-derived p53 (e.g., PAb246-positive and PAb240-positive) could reflect this differentiality with respect to sequence-specific DNA binding activity (37 , 38) . Tetrameric p53 binds avidly to a tandem sequence with the consensus half-site motif 5'-RRRC(A/T)(T/A)GYYY-3', where R and Y are purines and pyrimidines, respectively (39) . Search of the D loop region of mtDNA did not reveal the consensus p53 binding sequence in proximity to transcription and replication control elements (40) . However, several mtDNA sequences show close similarity with at least part of a consensus p53 recognition site. For example, a p53 half-site occurs at coordinates 2434–2444 (5'-AGGCATGCTC-3') in the 16S rRNA locus of the human mtDNA genome and at 10090–10100 (5'-GGACTAGCC-3') in the ND4L locus of the murine mtDNA genome. Although it is not easy to explain how these potential p53 recognition sites might regulate transcription or replication of the mtDNA genome, sequences outside the D loop in the mtDNA genome have been implicated in the direct receptor-mediated actions of thyroid hormone (41) and glucocorticoids (42) on mtDNA gene expression, concomitant with the effects of these hormone receptors on the nuclear genome. Further studies are needed to determine which, if any, mtDNA binding sequences serve as recognition sites for mitochondrial p53 and if this putative binding is a target of the m53DD-induced perturbations described here.

Homoplasmic mutations in mtDNA occur in some rapidly growing tumors, including tumors harboring p53 mutations (43 , 44) . Therefore, it seems plausible that mutation of p53 may be directly or indirectly linked with loss of mtDNA genomic stability. The mtDNA genome is susceptible to spontaneous mutation due to preferential accumulation of carcinogens in mitochondria, its proximity to ROS generation, and low capacity for mtDNA repair (45) . Mitochondrial damage, abnormalities of mtDNA, or dysregulation of mtDNA genetic activity may all contribute to bioenergetic imbalances as it is known that some rapidly growing tumors display alterations of oxidative metabolism and high rates of aerobic glycolysis (46) .

Selective loss of {Delta}{Psi}m among a subpopulation of mitochondria targeted with the expressed recombinant m53DD protein is another unexpected finding of this study. It raises question pertaining to the link between m53DD expression, mtDNA genetic activity, and mitochondrial membrane depolarization. Even with complete loss of mtDNA coded proteins, such as {rho}0 cells, one might still expect mitochondria to retain {Delta}{Psi}m through ATP supply from glycolysis (36) . A critical test would be to add oligomycin A to deenergize all mitochondria that rely on ATP for {Delta}{Psi}m. Using the confocal assay described here, we would be expect oligomycin A to select for cells having mitochondria that were red (e.g., positive for CMXRos and negative for m53DD) or green (e.g., positive for m53DD and negative for CMXRos), but no more overlap (e.g., positive for CMXRos and m53DD). This prediction fits the heterogeneity of red and green phenotypes observed among 3T3/m53DD cells.

It also seems likely that the presence of m53DD, and impairment of intramitochondrial p53 function, would invoke oxidative stress since generation of ROS (36) and down-regulation of 16S rRNA (47) has been affiliated with oxidative stress. PK11195 was an antidote to m53DD at drug concentrations that promoted mitochondrial import of manganese superoxide dismutase in another cell model system (48) . This drug binds selectively and with high affinity to the peripheral-type benzodiazepine receptor. Located on the outer mitochondrial membrane, the peripheral-type benzodiazepine receptor participates in the mitochondrial permeability transition pore complex and, consequently, can influence the mitochondrial mechanism of cell death (33 , 49) . Because we did not investigate apoptosis this contribution to long-term effects of m53DD and PK11195 remain to be studied.

In conclusion, tumor suppressor p53 may be added to the growing list of metabolic enzymes, growth factors, transcription factors, and chaperones (50) that display multicompartmental functions in the cell. Negative dominance of m53DD on mitochondrial biogenesis and function implies a direct positive effect of p53 on the hypothetical ‘mitochondrial cycle’ (51) but needs further insight into the mechanism by which p53 might regulate mtDNA genetic activity and energization. These effects may be seen under normal physiological conditions and thus add to the recent observation of an organelle-based, enhancer pathway during p53-mediated apoptosis (16) . Most likely, the mechanism of m53DD-mediated effects on mitochondrial biogenesis differs fundamentally from the tetramer-independent effect of mitochondrial p53 on apoptosis (16) .


   ACKNOWLEDGMENTS
 
This research was funded by N.I.H. grant RO1 ES09120 from the National Institute of Environmental Health Sciences (T.B.K.). Mr. Donahue was a fellow on Training Grant T32 ES07282 from the National Institute of Environmental Health Sciences.

Received for publication May 17, 2000. Revision received September 13, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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