Mitochondrial Telomere-binding Protein from Candida parapsilosis Suggests an Evolutionary Adaptation of a Nonspecific Single-stranded DNA-binding Protein*

Jozef NosekDagger §, L'ubomír Tomáska, Blanka PagácováDagger , and Hiroshi Fukuharaparallel

From the Departments of Dagger  Biochemistry and  Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina Dagger  CH-1 and  B-1, 842 15 Bratislava, Slovakia and parallel  Institut Curie, Section de Recherche, Centre Universitaire Paris XI, 91405 Orsay, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomáska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Terminal structures (telomeres) of eukaryotic chromosomes and telomerases (specialized nucleoprotein enzymes that maintain the telomere length) are involved in several important cellular processes as senescence, immortalization, and carcinogenesis. Telomerase activation appears to be critical for cell immortalization and represents a promising target for cancer therapy. However, several studies have demonstrated that cells lacking functional telomerase utilize an alternative mechanism to elongate the chromosome ends, suggesting that some tumor cells may survive following treatment with telomerase inhibitors (1). Therefore a more detailed understanding of both telomerase-dependent and -independent replication mechanisms is crucial for cancer therapy and the design of therapeutic agents capable of specifically blocking telomere replication. Furthermore, the study of various linear genophores might shed some light on alternative solutions to the end-replication problem.

In contrast to the generally held belief that mitochondrial genomes are circular molecules, a large number of organisms contain linear mitochondrial DNA (mtDNA) molecules possessing a homogeneous terminal structure. Sequence analysis of mitochondrial telomeres from various organisms revealed that they do not conform to a single consensus sequence or structural motif (2). According to their terminal structures, two types of linear mtDNA have been identified in yeasts. Linear mtDNA of the yeast species in closely related genera Williopsis and Pichia terminates at both ends with an inverted terminal repeat possessing a covalently closed single-stranded hairpin loop resembling the structure of the vaccinia virus genome (3, 4). The terminal structures of type 2 linear mtDNA of a pathogenic yeast Candida parapsilosis are represented by inverted terminal repetitions consisting of tandem arrays of a 738-bp1-long repetitive unit. This structure remotely resembles the organization of telomeres of nuclear chromosomes, although their repetitive unit is considerably shorter (5-8 bp). The variable number of tandem units generates a population of mtDNA molecules of heterogeneous size where the shortest molecules containing only incomplete repetitive unit predominate. A more detailed analysis of C. parapsilosis mitochondrial telomeres revealed that mtDNA molecules terminate at a defined position within a repetitive unit, thus generating a 5' single-stranded extension of about 110 nucleotides (5). This unique telomeric structure has raised several important questions: (i) how the mitochondrial telomere is stabilized, (ii) how the 5' single-stranded extension is generated, (iii) why DNA polymerase does not fill the protruding 5' overhang, and (iv) how the shortest mtDNA molecules that do not possess a complete tandem unit restore the missing sequence.

Several proteins that specifically bind either double-stranded or single-stranded DNA of telomeres of nuclear chromosomes have been identified. These proteins mediate telomere functions such as capping the ends of chromosomes, preventing nucleolytic degradation and end-to-end fusions, promoting the formation of telomere chromatin structure and nuclear architecture, participating in the replication and regulation of telomere length, etc. (6). To decipher the role of mitochondrial telomeres, we have initiated a search for proteins interacting with terminal sequence of linear mtDNA. Recently, we identified the first protein specifically recognizing the terminal structure of linear mtDNA from C. parapsilosis. Mitochondrial telomere-binding protein (mtTBP) is a heat- and protease-resistant protein that specifically recognizes the synthetic oligonucleotide identical to the terminal 51 nucleotides of the 5' single-stranded overhang of C. parapsilosis mitochondrial telomere and protects it from various DNA modifying enzymes. Affinity-purified mtTBP exhibits a molecular mass of 15 kDa in its monomeric state under denaturing conditions but forms homo-oligomers under native conditions (7). The properties of mtTBP suggested that it may play an important role in the stabilization and/or replication of linear mtDNA of C. parapsilosis.

In this report we describe the isolation and characterization of a nuclear gene, MTP1, that encodes the mtTBP of the yeast C. parapsilosis. Surprisingly, the sequence analysis of mtTBP revealed a striking homology to a family of bacterial and mitochondrial single-stranded DNA binding (SSB) proteins. Although other members of the SSB protein family bind with high affinity to single-stranded DNA without apparent sequence specificity, mtTBP preferentially binds the terminal 5' single-stranded overhang of the mitochondrial telomere. It has been proposed that the evolutionary appearance of linear mtDNA led to adaptation of the replication machinery to ensure complete replication of the linear genophore. In mitochondria of C. parapsilosis, such adaptation might have forced the conversion of a sequence-nonspecific mitochondrial SSB protein to a telomere binding factor.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains-- C. parapsilosis SR23 (CBS 7157) is a laboratory strain from the collection of the Department of Biochemistry (Comenius University, Bratislava, Slovakia). Escherichia coli DH5alpha (deoR, endA1, gyrA96, hsdR17 (rk-, mk+), recA1, relA1, supE44, thi-1, Delta (lac-argFV169), phi 80delta lacZDelta M15, F-, lambda -) and Saccharomyces cerevisiae S150-2B (MATa, leu2-3, 112, his3-Delta , trp1-289, ura3-52) strains were used in cloning experiments. S. cerevisiae strains alpha EVDelta RIM1-8b (MATalpha , rim1::URA3, ade2-1, leu2-3, 112, his3-11, 15, trp1-1, ura3-1, can1-100, rho 0) and aDBY754 × alpha EVRT-7b (MATa/MATalpha , RIM1/rim1::URA3, leu2/leu2, ura3/ura3, rho +) were kindly provided by F. Foury (Université Catholique de Louvain, Belgium). S. cerevisiae D22 strain (MATa, ade2, omega +) was used as a source for mitochondrial protein extracts.

Preparation of Mitochondrial Protein Extracts and Purification of mtTBP and Protein Sequence Analysis-- Mitochondrial protein extracts from C. parapsilosis and S. cerevisiae D22 were prepared as described previously (7). mtTBP was purified using an affinity chromatography step (7), except that the protein was concentrated by binding to Fast Q-Sepharose (Amersham Pharmacia Biotech), eluted by 1 M NaCl, dialyzed against 20 mM HEPES-NaOH, pH 7, and stored in 100-µl aliquots at -70 °C. Protein concentration was determined by a method of Bradford (8). The sequence of three peptides (HAEIVQWGK, YSLAVNK, and LDKFEDP, respectively) was determined by the Harvard Microchem (Cambridge, MA).

Isolation of MTP1 by Polymerase Chain Reaction (PCR)-- The sequences of oligonucleotides (synthesized by Genset, France) used in this study are shown in Table I. Oligonucleotide I was used for priming the synthesis of cDNA on C. parapsilosis total RNA template using the First-strand cDNA synthesis kit (Amersham Pharmacia Biotech) according to the manufacturer instructions. The sequence of peptide HAEIVQWGK was used to design MTP1-specific degenerate oligonucleotide II. The PCR reaction for amplification of the 3' end of MTP1 cDNA contained 50 mM KCl, 10 mM Tris-HCl, pH 9.0 (at 25 °C), 0.1% Triton X-100, 1.25 mM MgCl2, 0.2 mM dNTP, 2.5 units of Taq DNA polymerase, single-stranded cDNA prepared as described above as the template and 1 µM oligonucleotide I and 5 µM oligonucleotide II as the upstream and downstream primer, respectively. Amplification was performed in DNA Thermal Cycler 480 (Perkin-Elmer) with initial denaturation at 95 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 1 min, and final polymerization at 72 °C for 5 min. The 125-bp PCR product was gel-purified and sequenced. The DNA sequence upstream of the stop codon of putative open reading frame was used to design downstream MTP1-specific primer (oligonucleotide III). The 5' end of MTP1 was amplified using linker-ligation-mediated PCR strategy (9). Briefly, total genomic DNA of C. parapsilosis was digested with AvaII endonuclease, denatured, annealed with MTP1 specific downstream primer, then extended with Vent(exo-) DNA polymerase (New England Biolabs), followed by ligation of the primer extension products with a synthetic linker. The MTP1 sequence was then PCR-amplified using MTP1-specific downstream (oligonucleotide III) and 25-nucleotide-linker primers. The DNA sequence analysis of 600-bp PCR product revealed a putative 399-bp open reading frame containing the sequences corresponding to three known peptides. The integrity of MTP1 gene was confirmed by sequencing of several independent PCR products obtained using MTP1 oligonucleotides IV and V on C. parapsilosis genomic DNA and cDNA as the template, respectively.

Construction of Plasmid Clones-- The plasmid pGFP-C-FUS-mtTBP containing the whole MTP1 open reading frame (lacking stop codon) fused with green fluorescent protein was prepared by ligation of PCR product amplified using 5'-ATGTTGCGAGCATTCACTAGATCA-3' and 5'-TTCTGTAGCTTCGGCTCTATCCTCA-3' primers into SmaI-digested pGFP-C-FUS vector (10, provided by J. H. Hegemann, Justus-Liebig University, Giessen, Germany). The expression of fusion protein in this construct is driven by S. cerevisiae MET25 promoter. The plasmid YEplac181-PMET-mtTBP-tCYC1 was constructed as follows. First, the plasmid pGFP-C-FUS was digested with SalI to remove green fluorescent protein (GFP) sequences. The termini were filled with a Klenow enzyme and then ligated with the MTP1 PCR product amplified using 5'-ATGTTGCGAGCATTCACTAGATCA-3' and oligonucleotide V (Table I) primers. Subsequently, the KpnI-SacI restriction fragment containing a cassette MET25 promoter, MTP1 open reading frame, CYC1 terminator was blunt-ended by T4 DNA polymerase and then ligated into SmaI site of YEplac181 vector (11). pGEX-2T-mTBP designed for production of recombinant mtTBP in E. coli was constructed by insertion of Sau3A fragment of MTP1 gene into the BamHI site of pGEX-2T expression vector (Amersham Pharmacia Biotech). The MTP1 sequence in the constructs was verified by DNA sequencing.

Purification of Recombinant mtTBP and Rabbit Antisera Preparation-- Recombinant mtTBP was purified according to the instructions of the supplier of the expression vector (Amersham Pharmacia Biotech). Briefly, 20 ml of an overnight bacterial culture was inoculated into 1 liter of Superbroth media (3.2% tryptone (Difco), 2% yeast extract (Difco), 0.5% NaCl) and grown at 37 °C to a final A600 = 0.7. The culture was then induced for 3 h at 30 °C with 1 mM isopropyl-1-thio-beta -D-galactopyranoside (Sigma). Cells were washed once with ice-cold double-distilled water, resuspended in 20 ml of buffer G (20 mM HEPES-NaOH, pH 8.0, 500 mM NaCl, 0.1% Triton X-100, 0.1 mM EDTA, protease inhibitor mixture (CompleteTM, Boehringer Mannheim), sonicated, and centrifuged at 25,000 × g for 30 min at 4 °C to remove insoluble material. The supernatant was mixed with 0.3 ml of glutathione-agarose (Sigma) prewashed with buffer G and incubated for 60 min on ice with occasional mixing. Beads were loaded onto a 5-ml column, washed with 100 ml of buffer G (without protease inhibitor mixture) followed by 50 ml of ice-cold phosphate-buffered saline (PBS, 137 mM NaCl, 2.6 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4). The washed beads were resuspended in 0.45 ml of PBS containing thrombin (0.05 units/µl, Amersham Pharmacia Biotech) and incubated overnight at 4 °C. The cleaved mtTBP was eluted in 3 × 0.5 ml of PBS and stored at -70 °C. Rabbit polyclonal antisera was raised against 100 µg of purified recombinant thrombin-cleaved mtTBP and purified by Protein A-Sepharose (Eurogentec, Belgium).

Immunoblotting-- C. parapsilosis SR23 was grown until the late logarithmic phase in YP medium (1% yeast extract, 1% peptone) supplemented with glucose (2% w/v), glycerol (3% w/v), or galactose (2% w/v), and cells (0.1 ml of the culture) were lysed as described by Horváth and Riezman (12). Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13). Resolved proteins were transferred to nitrocellulose filters in the transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) using a semidry electroblotter system (Owl Scientific) for 45 min at 250 mA. Filters were blocked for 2 h at room temperature with blocking solution (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2% (w/v) skim milk (Difco)) and then incubated overnight at 4 °C in the blocking solution containing anti-mtTBP antibody (1:200). Membranes were washed four times with rinsing buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), one time with rinsing buffer without Tween 20, and then incubated with blocking solution containing goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma, 1:3,000) for 2 h at room temperature. Blots were washed as described above and developed by incubating with 0.3 mg/ml p-nitro blue tetrazolium chloride (Sigma) and 0.15 mg/ml 5-bromo-4-chloro-3-indolylphosphate toluidine salt (Sigma) in alkaline phosphatase buffer (100 mM NaHCO3, 1 mM MgCl2, pH 9.8) for 5-20 min at room temperature.

Binding of mtTBP to a Native Mitochondrial Telomere and Immunoprecipitation of the mtTBP-DNA Complex-- Purified mitochondrial DNA (3 µg) from C. parapsilosis was digested with BglII (30 units) in a final volume of 250 µl, followed by heat-inactivation of the restriction enzyme by incubation of the sample for 10 min at 65 °C. Digested mtDNA (100 µl) was mixed with 5 µg of recombinant mtTBP in a final volume of 250 µl of the DNA binding buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl) and incubated for 30 min at room temperature.

To immunoprecipitate mtTBP-DNA complexes, the binding reaction was combined with 50 µl of anti-mtTBP antibody for 2 h at 4 °C. Immune complexes were immobilized by incubation with Protein A-Sepharose 4B (Sigma) for an additional 2 h at 4 °C and centrifuged for 5 s in a microcentrifuge at maximal speed, and the supernatant was removed and placed on ice. The pellet was washed 5 times with 1 ml of PBS containing 200 mM NaCl. The immunoprecipitated complexes were separated from the beads by boiling for 5 min in 300 µl of PBS containing 0.1% SDS. This step was repeated two more times, and the resulting fractions were saved as eluates 1, 2, and 3, respectively.

Gel-mobility Shift Assay-- Oligonucleotides were 5' end-labeled using [gamma 32P]ATP by T4 polynucleotide kinase and separated from nonincorporated nucleotides by chromatography on a BioSpin-6 column (Bio-Rad). 10 ng of purified recombinant mtTBP was incubated with 1 ng of labeled oligonucleotide in 10 µl of DNA binding buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl) containing 1 mg/ml poly(dI:dC) for 10 min at room temperature. DNA-protein complexes were resolved by electrophoresis on 4% polyacrylamide gel in 0.5× TBE (4.5 mM Tris borate, 1 mM EDTA) at 10 V/cm for 90 min. Gels were dried under vacuum at 80 °C and exposed to Kodak X-Omat film at -70 °C between intensifying screens.

In Vitro Oligomerization Assay-- 1 µg of recombinant mtTBP was incubated with various concentrations of glutaraldehyde in 10 µl of PBS for 10 min at room temperature. The cross-linking reaction was stopped by heating the samples in 1× Laemmli sample buffer for 5 min at 95 °C. Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13) and transferred to nitrocellulose filters, and mtTBP was detected by immunoblot analysis as described above.

Alternatively, cAMP-dependent protein kinase assays were performed as described previously (14, 15). Briefly, reaction mixtures (10 µl) contained 10 mM HEPES-NaOH, pH 6.5, 4 mM MgCl2, 20 mM dithiothreitol, 1 µg of recombinant mtTBP, 50 ng of the purified catalytic subunit of protein kinase A (kindly provided by E. Fisher, Seattle, WA), and 250 µM [gamma 32P]ATP (500 cpm/pmol) and were incubated for 240 min at 30 °C. After the phosphorylation of mtTBP, glutaraldehyde and bis(sulfosuccinimidyl)suberate were added to the corresponding samples to final concentrations indicated in legend to Fig. 4. After a 10-min incubation at room temperature, cross-linking reactions were stopped by the addition of an equal volume of 2× SDS-polyacrylamide gel electrophoresis Laemmli sample buffer. Proteins were separated by 13% SDS-polyacrylamide gel electrophoresis by a method of Laemmli (13). Gels were heat-dried under vacuum and exposed to Kodak X-Omat film at -70 °C between intensifying screens.

Pulsed Field Gel Electrophoresis-- DNA samples were prepared as described previously (16). Briefly, yeast cells were lysed in agarose blocks by sequential treatment with zymolyase and proteinase K. Samples were electrophoretically separated in a 0.8% agarose gel in 0.5× TBE (45 mM Tris borate, 1 mM EDTA) buffer in a Pulsaphor apparatus (LKB) in contour-clamped homogeneous electric field configuration. Pulse switching involved three steps of linear interpolation as follows: (i) 10 to 200 s for 48 h, (ii) 200 to 400 s for 16 h, (iii) 400 to 600 s for 48 h, at 100 V and 9 °C throughout.

DNA Manipulations and Analysis-- C. parapsilosis genomic DNA was isolated using a protocol described for S. cerevisiae (17). Restriction and DNA modification enzymes were from New England Biolabs and used according to manufacturer instructions. Southern and Northern blotting, DNA hybridization, DNA cloning, and sequencing were performed essentially as described in Sambrook et al. (18). Intact cells of S. cerevisiae were transformed by standard lithium acetate/ssDNA/polyethylene glycol protocol (19).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mitochondrial Telomere-binding Protein, Encoded by a Nuclear Gene MTP1, Is a Member of the SSB Protein Family-- Previously we reported the purification of a 15-kDa protein designated mtTBP that selectively bound to an affinity matrix containing the terminal 51 nucleotides of the 5' overhang of mitochondrial telomere from C. parapsilosis (7). The amino acid sequence of three peptides were determined (HAEIVQWGK, YSLAVNK, and LDKFEDP), and the corresponding DNA sequence of the peptide HAEIVQWGK was used to design a degenerated oligonucleotide primer for polymerase chain reaction. The entire coding sequence of mtTBP was isolated using two strategies: (i) amplification of the 3' end of the cDNA by reverse transcription-PCR followed by (ii) amplification of the 5' end by a linker ligation-mediated PCR approach. The full-length PCR product was obtained, and its DNA sequence was verified by hybridization to total genomic DNA digested with several restriction enzymes (data not shown). The sequences of several PCR products were determined, and the computer sequence analysis identified a 399-bp open reading frame containing regions corresponding to all three peptides (Fig. 1A). The gene coding for mtTBP was assigned MTP1 (mitochondrial telomere protein 1). A GenBankTM data base search revealed that mtTBP displays significant homology to a family of bacterial and mitochondrial SSB proteins. Its amino acid sequence exhibits 33.8% identity and 62.4% homology to the mitochondrial SSB protein encoded by the S. cerevisiae nuclear gene RIM1 (Fig. 1B). Although the RIM1 gene contains an intron, we were unable to detect any intervening sequences in the C. parapsilosis gene when we compared the sequence of MTP1 PCR products amplified from both a cDNA and genomic DNA template.


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Fig. 1.   A, the sequence of MTP1 gene and its corresponding protein. Amino acid sequences of three peptides determined by protein sequencing are shadowed. Arrows indicate the position of the oligonucleotides used in the PCR amplification of MTP1 (see "Experimental Procedures"). B, comparison of the amino acid sequences of human mitochondrial SSB protein (HsmtSSB), S. cerevisiae Rim1p, and C. parapsilosis mtTBP. Alignment was constructed using CLUSTAL X program (34). The sequences corresponding to the SSB protein family signatures are shadowed. The mitochondrial import signal for HsmtSSB (35) and Rim1p (20) is underlined.

To rule out the possibility that MTP1 is encoded by the mitochondrial genome, we examined restriction enzyme-digested mtDNA and pulsed field gel electrophoresis-separated C. parapsilosis chromosomes, respectively, by Southern blot analysis. Results indicated that MTP1 is localized on the 2.2 Mbp chromosome, thereby confirming its nuclear localization (Fig. 2). In addition, the sequence analysis predicted putative mitochondrial import signal at the N terminus of deduced MTP1 protein product. To determine whether C. parapsilosis does not contain homologous sequences that might encode additional mitochondrial SSB protein(s), we performed the Southern hybridization of restriction enzyme-digested genomic DNA with MTP1 probe at both high and low stringency conditions. The results revealed that MTP1 represents a single copy gene (data not shown).


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Fig. 2.   MTP1 gene localizes on 2.2 Mbp chromosome. Chromosomal DNA of C. parapsilosis SR23 was separated using pulsed field gel electrophoresis (see "Experimental Procedures") and stained with ethidium bromide (left panel), then transferred onto a nylon membrane, subsequently hybridized with a radioactively labeled MTP1 probe, and subjected to autoradiography (right panel).

mtTBP Binds Mitochondrial Telomeres in a Reconstitution Experiment-- To study mtTBP in vitro,we used an E. coli expression system to produce sufficient quantities of recombinant protein as described under "Experimental Procedures." Using the pGEX-2T vector, mtTBP lacking the first seven amino acid residues was fused to glutathione S-transferase containing a thrombin cleavage site. Treatment with protease releases a soluble mtTBP protein containing one additional glycine residue at its N terminus. To assess the specificity of the recombinant mtTBP for the telomeric sequence, an oligonucleotide identical to the terminal 31 nucleotides (TEL31) from the 5' end of mitochondrial telomere was used as a probe in a gel-retardation assay (Fig. 3). Mixing of TEL31 with purified recombinant mtTBP resulted in a DNA-protein complex that migrated slower than the free probe alone (Fig. 3, lanes 1 and 2). A 3- to 300-fold molar excess of unlabeled TEL31 quantitatively competed with the labeled probe for complex formation (Fig. 3, lanes 3-5). In contrast, oligonucleotide OLI31 (see Table I) derived from another part of C. parapsilosis mtDNA did not compete with labeled TEL31 as effectively as homologous competitor under identical conditions (Fig. 3, lanes 6-8). These data demonstrate that the preference of recombinant mtTBP for the telomeric sequence parallels the behavior of the natural mtTBP isolated from C. parapsilosis mitochondria (7).


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Fig. 3.   Recombinant mtTBP specifically recognizes telomeric sequence in vitro. Gel retardation assay (see "Experimental Procedures") was performed without (lane 1) or with (lanes 2-8) 10 ng of recombinant mtTBP using terminally labeled TEL31 as a probe. The assay was done in the absence (lanes 1 and 2) or presence (lanes 3-8) of various molar excesses of the oligonucleotide competitors that are indicated above the lanes.

                              
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Table I
The sequences of synthetic oligonucleotides

Recombinant mtTBP was used to raise the rabbit polyclonal antisera, which ultimately recognized a single 15-kDa protein under denaturing conditions in cell extracts prepared from C. parapsilosis. No similar size protein has been detected in several other yeast species from various genera including Candida, Williopsis, Pichia, Saccharomyces, and Kluyveromyces. It was shown previously that mtTBP forms homo-oligomeric complexes in its native state. Using two chemical cross-linkers, glutaraldehyde and bis(sulfosuccinimidyl)suberate, we now demonstrate that recombinant mtTBP also undergoes homo-oligomerization in vitro. Dimers were the predominant oligomeric form in the presence of either cross-linker (Fig. 4).


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Fig. 4.   mtTBP oligomerizes in vitro. Chemical cross-linking of mtTBP was performed as described under "Experimental Procedures." Concentrations of glutaraldehyde and bis(sulfosuccinimidyl)suberate (BS3), respectively, are indicated above each lane. The oligomeric forms of mtTBP were visualized either by immunoblot using anti-mtTBP antisera (A) or by autoradiography of mtTBP phoshorylated by the cAMP-dependent protein kinase before cross-linking (B).

To compare the DNA binding properties of mtTBP with a homologous protein from a yeast with circular mitochondrial DNA, we performed a gel retardation experiment using mitochondrial protein extracts from C. parapsilosis and S. cerevisiae, respectively. Both extracts contained ssDNA binding activity. It was shown previously that the factor responsible for the ssDNA-protein complex formation is mtTBP in C. parapsilosis (7) and Rim1p in S. cerevisiae (20), respectively. However, in contrast to C. parapsilosis, 300-fold excess of OLI31 competed quantitatively with the labeled TEL31 probe for complex formation in mitochondrial lysates of S. cerevisiae (Fig. 5). This suggests that, in addition to general ssDNA binding activity, mtTBP gained a preference for a telomeric sequence.


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Fig. 5.   Comparison of ssDNA binding activity in mitochondrial extracts from C. parapsilosis and S. cerevisiae. Gel retardation assay (see "Experimental Procedures") was performed without (lane 1) or with (lanes 2-15) 5 µg of mitochondrial protein extracts from C. parapsilosis (lanes 2-8) and S. cerevisiae (9-15) using terminally labeled TEL31 as a probe. The assay was done in the absence (lanes 1, 2, and 9) or presence (lanes 3-8 and 10-15) of various molar excesses of the oligonucleotide competitors that are indicated above the lanes.

Next we tested whether mtTBP was capable of binding natural mitochondrial telomeres in a reconstitution experiment. (Fig. 6). Recombinant mtTBP was incubated with BglII-digested mtDNA of C. parapsilosis, and the DNA-protein complexes were immunoprecipitated with anti-mtTBP and Protein A-Sepharose. DNA-mtTBP complexes were eluted from the beads, and the fractions were examined by Southern blot analysis following hybridization to either a specific 738-bp EcoRI fragment representing the telomere repeat unit or to a control 2.9-kilobase BglII fragment derived from an internal region of mtDNA. The results shown in Fig. 6 demonstrate that anti-mtTBP antiserum immunoprecipitated mtTBP complexed to terminal mtDNA fragments, producing a ladder pattern typical of telomeric DNA (5).


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Fig. 6.   mtTBP recognizes a native mitochondrial telomere of C. parapsilosis. Binding of mtTBP to a native mitochondrial telomere and immunoprecipitation were performed as described under "Experimental Procedures." The original sample containing the mtTBP-DNA complex plus antisera (lane 1), three subsequent eluates (lanes 2-4), and supernatant after immunoprecipitation (lane 5) were subjected to agarose gel electrophoresis, transferred to a nylon membrane, hybridized with a 738-bp EcoRI fragment representing telomere repeat unit (left panel) and a 2.9-kilobase BglII fragment as an internal mtDNA probe (right panel), respectively.

MTP1 Is Constitutively Expressed, and Its Protein Product Localizes to Mitochondria-- Next we examined the expression of the MTP1 gene under different growth condition. C. parapsilosis cells were grown to late logarithmic phase in YP medium (1% yeast extract, 1% peptone) supplemented with either glucose, glycerol, or galactose. Northern blot analysis indicated that MTP1 RNA was constitutively transcribed regardless of the carbon source present. Similar results were observed at the translational level when the mtTBP was assayed by immunoblotting in extracts from cells grown under identical conditions (Fig. 7).


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Fig. 7.   Constitutive expression of MTP1. Total RNA was isolated from C. parapsilosis cells grown on various carbon sources as indicated, electrophoresed in a denaturing agarose gel, and stained with ethidium bromide (A). Northern blots were probed with radioactively labeled MTP1 (B). Immunoblot analysis of mtTBP from C. parapsilosis grown under identical conditions was performed as described under "Experimental Procedures" (C).

Although cell fractionation experiments suggest that mtTBP is enriched in mitochondria (7), it was important to verify these results in intact cells. Because a system for the genetic transformation of C. parapsilosis is not available, we took advantage of S. cerevisiae as a heterologous expression system for these experiments. We constructed a plasmid expressing GFP fused to the C terminus of mtTBP (see "Experimental Procedures"). Visualization of cells harboring this plasmid by fluorescent microscopy showed that mtTBP-GFP was restricted to mitochondria and appeared to co-localize with mitochondrial DNA, as judged by 4',6-diamidino-2-phenyl-indol staining. In contrast, cells expressing GFP alone displayed a diffuse fluorescence throughout the cell. Together these data confirm the specific mitochondrial localization of mtTBP (Fig. 8).


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Fig. 8.   A mtTBP-GFP fusion protein localizes to mitochondria. Fluorescence micrographs of S. cerevisiae cells expressing GFP (A) and mtTBP-GFP fusion (B), respectively. Visualization of nuclear and mitochondrial DNAs by 4',6-diamidino-2-phenyl-indol staining showed that fusion protein colocalizes with 4',6-diamidino-2-phenyl-indol staining of mitochondrial DNA (C).

MTP1 Does Not Rescue S. cerevisiae Delta rim1-- Nuclear gene RIM1, which encodes the mitochondrial SSB protein in S. cerevisiae, was originally isolated as a multicopy suppressor of a mutation in the gene PIF1 encoding the mitochondrial DNA helicase (20). A functional RIM1 gene is essential for replication and maintenance of mtDNA, because its disruption results in the complete loss of mtDNA. Because mtTBP is approximately 62% homologous to Rimlp, it was of interest to determine whether MTP1 could complement the disruption of RIM1 in S. cerevisiae. The cells of aDBY754 × alpha EVRT-7b strain that is heterozygous for RIM1/Delta rim1 were transformed using a plasmid YEplac181-PMET-mtTBP-tCYC1 and selected for leucine prototrophy. Subsequently, the transformants were sporulated, and the tetrades were dissected. Only the spores that retained both the plasmid expressing mtTBP and a functional copy of RIM1 gene were able to grow on media lacking leucine and containing glycerol as the sole carbon source. Further analysis confirmed that the loss of mtDNA cosegregated with Delta rim1. These data indicate that the expression of MTP1 did not prevent the loss of mtDNA in a Delta rim1 mutant. Because the expression of MTP1 was driven by the S. cerevisiae MET25 promoter and the fusion protein mtTBP-GFP, driven by the same promoter, appropriately localized to mitochondria, we conclude that despite homology between RIM1 and MTP1, the latter is not able to functionally replace mitochondrial SSB in S. cerevisiae.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

DNA replication, recombination, and repair require an essential accessory protein that binds with high affinity to single-stranded DNA and protects the polynucleotide chain from refolding and nucleolytic degradation (21-23). Single-stranded DNA-binding proteins can be regarded as one big family of proteins sharing common functional, structural, and mechanistic features. However, the sequence comparison can distinguish several separate classes having little in common except their ability to bind the single-stranded nucleic acids (24). Although the replication of nuclear DNA in yeast and human requires heterotrimeric replication protein A protein, the nuclear genome encodes yet another SSB protein, which is imported into mitochondria. Mitochondrial SSB proteins isolated from several sources (human, rat, Xenopus, Drosophila, and bakers' yeasts) are homologous to bacterial SSB proteins. The N-terminal domain of E. coli SSB, which has been implicated in ssDNA binding, shares extensive homology with human mitochondrial SSB protein. The crystal structures of both proteins revealed that the polynucleotide chain wraps around the SSB homotetramer (25-27). These bacterial and mitochondrial proteins also share several common biochemical and physicochemical properties, which suggests a conserved mechanism(s) of binding to single-stranded DNA (27, 28). These data further support the hypothesis that mitochondria may have originated from a bacterial endosymbiont. Interestingly, the genome of the archean Methanococcus jannaschii apparently does not encode a protein homologous to bacterial and mitochondrial SSB proteins (29).

We now report the isolation of MTP1 gene coding for a mitochondrial telomere-binding protein of C. parapsilosis. Sequence analysis of mtTBP revealed a significant homology to a family of mitochondrial and bacterial SSB proteins that possess two motifs defined in the Prosite data base as SSB protein family signatures 1 and 2. Although the ScanProsite (ExPASy molecular biology server, Swiss Institute of Bioinformatics (30)) did not detect SSB signatures in mtTBP, a more rigorous examination of the sequence revealed that patterns corresponding to both motifs are present, albeit SSB 1 is weakly conserved (Fig. 1B). In addition, amino acids Trp-54 and Phe-60 in E. coli SSB protein, which were found to be involved in ssDNA-binding (31), are conserved in mtTBP and correspond to Trp-61 and Phe-67, respectively. In the E. coli SSB protein, histidine in position 55 has been found to play a central role in tetramerization, and its replacement to tyrosine in E. coli ssb-1 mutant displays temperature-sensitive DNA replication defect and causes the dissociation of tetramers to dimers and monomers. The mutant protein is still minimally capable of forming tetramers at high protein concentration, and the effects of ssb-1 mutation can be suppressed by the overexpression of ssb-1 gene product (32, 33). His-55 of E. coli SSB is equivalent to His-69 in human mitochondrial SSB (24), but it is apparently replaced by tyrosine in both S. cerevisiae (Tyr-61 in Rim1p) and C. parapsilosis homologues (Tyr-62 in mtTBP). Because the mitochondrial SSB protein from bakers' yeast forms tetramers in solution (20), it was of interest to determine whether mtTBP behaves similarly. Previous results from UV cross-linking and gel filtration experiments suggest that mtTBP forms homo-oligomers in its native state (7). We now show that recombinant mtTBP also forms tetrameric complexes in vitro, although dimers were the predominant oligomeric form under the conditions used in this study.

In contrast to other SSB proteins that bind essentially any single-stranded DNA, mtTBP was identified by virtue of its selective interaction with the synthetic oligonucleotide derived from the single-stranded overhang of the mitochondrial telomere using both gel-retardation and UV cross-linking assays (7). We have now expanded our observation to show that recombinant mtTBP binds not only synthetic telomeric oligonucleotide with a specificity similar to the endogenous protein, but more importantly, binds the natural mitochondrial telomere from C. parapsilosis in a reconstitution experiment.

Subcellular fractionation experiments have shown that mitochondria from C. parapsilosis were enriched in mtTBP and DNA binding activity selective for the terminal sequences of mitochondrial telomeres (7). In vivo analysis of MTP1 is complicated by the lack of tools necessary for the genetic manipulation of C. parapsilosis (i.e. absence of sexual state, diploid or aneuploid cells, no suitable mutant strains, no system for genetic transformation, etc.). Therefore we exploited the S. cerevisiae system in an attempt to partially circumvent this problem. Because the heterologous expression of the mtTBP-GFP fusion protein resulted in its localization to mitochondria of S. cerevisiae, we were tempted to test if the expression of MTP1 can prevent the loss of mtDNA in Delta rim1 cells. The inability of mtTBP to replace Rim1p implies that these proteins differ in their DNA binding properties and/or their capacity to interact with other components of mtDNA replication machinery.

The results of Northern and Western analysis of C. parapsilosis grown in media containing either glucose, glycerol, or galactose as the sole carbon source suggested that MTP1 gene is constitutively expressed. Previous studies indicate that DNA binding activity, but not oligomerization, is affected by the phosphorylation of mtTBP by the cAMP-dependent protein kinase, thereby suggesting a potential mechanism for the regulation of mitochondrial telomere replication (15). Although mtTBP contains several putative protein kinase A phosphorylation sites, their physiological relevance also awaits the generation of an appropriate genetic transformation system for C. parapsilosis.

The study of linear mitochondrial genomes evokes several questions with regard to the evolutionary origin of linear and circular mitochondrial DNAs and the mechanisms that lead to the generation of the linear form. Several lines of evidence suggest that the linear and circular forms of mitochondrial DNA neither have an independent origin nor represent a radical difference in their life styles. Rather, the conversion from one form to another may have occurred accidentally through a relatively simple mechanism during evolution. This means that the cell with linearized mitochondrial genome may mobilize a pre-existing set of proteins to ensure its replication (2). The nature of mtTBP from C. parapsilosis supports this idea and has prompted us to speculate that it might be derived from an SSB protein that lacks sequence specificity. Although mtTBP had retained some features common to other mitochondrial and bacterial SSB proteins, it has gained a preference for binding the terminal 5' single-stranded overhang of the mitochondrial telomere. This suggests that evolutionary emergence of linear mtDNA has been accompanied by the adaptation of the component(s) of the replication machinery to complete the replication and stabilization of a linear genophore. According to our data, it does not seem that C. parapsilosis genome encodes for another, more conventional copy of mitochondrial SSB protein. Therefore it is likely that mtTBP retained its role as a nonspecific SSB protein in the replication of mtDNA, and simultaneously, it gained an additional function in the maintenance of mitochondrial telomeres.

Development of a genetic system for C. parapsilosis is of utmost importance to unravel the physiological role of mtTBP in the maintenance of mitochondrial telomeres. Moreover, this should facilitate the identification of ancillary proteins, which in concert with mtTBP, may help to reveal novel features involving the replication of the linear mitochondrial genome.

    ACKNOWLEDGEMENTS

We thank L.Kovác (Comenius University, Bratislava, Slovakia) for continuous support, helpful discussions, and comments, J.Kolarov (Comenius University, Bratislava) for reading the manuscript, and M. Slaninová and L. Adamíková (Comenius University, Bratislava) for technical assistance. We also thank R. Resnick (Cornell University, Ithaca, NY) for reading the manuscript and for valuable editorial advice.

    FOOTNOTES

* This work was supported in part by Comenius University Grants 3856/98 and 3877/98, Slovak Grant Agency Grants 1/4164/97, 1/4159/97, and 1/6168/99, Howard Hughes Medical Institute Grant 75195-547301, and European Community Grant (BIO4-CT96-0003).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) Y17006 (MTP1).

§ Supported by postdoctoral fellowship from the Ministry of Education of French government. To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovakia. Tel.: 421-7-60296-536; Fax: 421-7-65429-064; E-mail: nosek{at}fns.uniba.sk.

    ABBREVIATIONS

The abbreviations used are: bp, base pair(s); mtTBP, mitochondrial telomere-binding protein; SSB protein, single-stranded DNA-binding protein; PCR, polymerase chain reaction; GFP, green fluorescence protein; ssDNA, single-stranded DNA; PBS, phosphate-buffered saline.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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