Mitochondrial Telomere-binding Protein from Candida
parapsilosis Suggests an Evolutionary Adaptation of a
Nonspecific Single-stranded DNA-binding Protein*
Jozef
Nosek
§,
L'ubomír
Tomá
ka¶,
Blanka
Pagá
ová
, and
Hiroshi
Fukuhara
From the Departments of
Biochemistry and
¶ Genetics, Faculty of Natural Sciences, Comenius University,
Mlynská dolina
CH-1 and ¶ B-1, 842 15 Bratislava, Slovakia and
Institut Curie, Section de
Recherche, Centre Universitaire Paris XI, 91405 Orsay, France
 |
ABSTRACT |
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
(Tomá
ka, 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 |
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 |
Strains--
C. parapsilosis SR23 (CBS 7157) is a
laboratory strain from the collection of the Department of Biochemistry
(Comenius University, Bratislava, Slovakia). Escherichia
coli DH5
(deoR, endA1, gyrA96, hsdR17
(rk
,
mk+), recA1, relA1, supE44, thi-1,
(lac-argFV169),
80
lacZ
M15, F
,

) and Saccharomyces cerevisiae S150-2B
(MATa, leu2-3, 112, his3-
, trp1-289, ura3-52) strains
were used in cloning experiments. S. cerevisiae strains
EV
RIM1-8b (MAT
, rim1::URA3, ade2-1, leu2-3, 112, his3-11, 15, trp1-1, ura3-1, can1-100,
0)
and aDBY754 ×
EVRT-7b (MATa/MAT
,
RIM1/rim1::URA3, leu2/leu2, ura3/ura3,
+) were kindly provided by F. Foury
(Université Catholique de Louvain, Belgium). S. cerevisiae D22 strain (MATa, ade2,
+)
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-
-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 [
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 [
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 |
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.

View larger version (50K):
[in this window]
[in a new window]
|
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).

View larger version (47K):
[in this window]
[in a new window]
|
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).

View larger version (43K):
[in this window]
[in a new window]
|
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.
|
|
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).

View larger version (28K):
[in this window]
[in a new window]
|
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.

View larger version (50K):
[in this window]
[in a new window]
|
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).

View larger version (86K):
[in this window]
[in a new window]
|
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).

View larger version (79K):
[in this window]
[in a new window]
|
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).

View larger version (32K):
[in this window]
[in a new window]
|
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
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 ×
EVRT-7b strain that is heterozygous for
RIM1/
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
rim1. These data indicate that
the expression of MTP1 did not prevent the loss of mtDNA in
a
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 |
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
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.Ková
(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 |
-
Autexier, C.,
and Greider, C. W.
(1996)
Trends Biochem. Sci.
21,
387-391[CrossRef][Medline]
[Order article via Infotrieve]
-
Nosek, J.,
Tomá
ka, L'.,
Fukuhara, H.,
Suyama, Y.,
and Ková
, L.
(1998)
Trends Genet.
14,
184-188[CrossRef][Medline]
[Order article via Infotrieve] -
Fukuhara, H.,
Sor, F.,
Drissi, R.,
Dinouël, N.,
Miyakawa, I.,
Rousset, S.,
and Viola, A. M.
(1993)
Mol. Cell. Biol.
13,
2309-2314[Abstract]
-
Dinouël, N.,
Drissi, R.,
Miyakawa, I.,
Sor, F.,
Rousset, S.,
and Fukuhara, H.
(1993)
Mol. Cell. Biol.
13,
2315-2323[Abstract]
-
Nosek, J.,
Dinouël, N.,
Ková
, L.,
and Fukuhara, H.
(1995)
Mol. Gen. Genet.
247,
61-72[Medline]
[Order article via Infotrieve] -
Fang, G.,
and Cech, T. R.
(1995)
in
Telomeres (Blackburn, E. H., and Greider, C. W., eds), pp. 69-105, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Tomá
ka, L'.,
Nosek, J.,
and Fukuhara, H.
(1997)
J. Biol. Chem.
272,
3049-3056[Abstract/Free Full Text] -
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Garrity, P. A.,
Wold, B.,
and Mueller, P. R.
(1995)
in
PCR 2: A Practical Approach (McPherson, M. J., Hames, B. D., and Taylor, G. R., eds), pp. 309-322, IRL Press at Oxford University Press, Oxford
-
Niedenthal, R. K.,
Riles, L.,
Johnston, M.,
and Hegemann, J. H.
(1996)
Yeast
12,
773-786[CrossRef][Medline]
[Order article via Infotrieve]
-
Gietz, R. D.,
and Sugino, A.
(1988)
Gene
74,
527-534[CrossRef][Medline]
[Order article via Infotrieve]
-
Horváth, A.,
and Riezman, H.
(1994)
Yeast
10,
1305-1310[Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.
(1970)
Nature
277,
680-685
-
Resnick, R. J.,
and Racker, E.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
2474-2478[Abstract]
-
Tomá
ka, L'.
(1998)
Biochem. Biophys. Res. Commun.
242,
457-460[CrossRef][Medline]
[Order article via Infotrieve] -
Nosek, J.,
and Fukuhara, H.
(1994)
J. Bacteriol.
176,
5622-5630[Abstract]
-
Phillippsen, P.,
Stotz, A.,
and Scherf, C.
(1991)
Methods Enzymol.
194,
169-182[Medline]
[Order article via Infotrieve]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Gietz, R. D.,
and Schiestl, R. H.
(1995)
Methods Mol. Cell. Biol.
5,
255-269
-
Van Dyck, E.,
Foury, F.,
Stillman, B.,
and Brill, S. J.
(1992)
EMBO J.
11,
3421-3430[Abstract]
-
Chase, J. W.,
and Williams, K. R.
(1986)
Annu. Rev. Biochem.
55,
103-136[CrossRef][Medline]
[Order article via Infotrieve]
-
Meyer, R. R.,
and Laine, P. S.
(1990)
Microbiol. Rev.
54,
342-380
-
Lohman, T. M.,
and Ferrari, M. E.
(1994)
Annu. Rev. Biochem.
63,
527-570[CrossRef][Medline]
[Order article via Infotrieve]
-
Suck, D.
(1997)
Nat. Struct. Biol.
4,
161-165[Medline]
[Order article via Infotrieve]
-
Yang, C.,
Curth, U.,
Urbanke, C.,
and Kang, C. H.
(1997)
Nat. Struct. Biol.
4,
153-157[Medline]
[Order article via Infotrieve]
-
Raghunathan, S.,
Ricard, C. S.,
Lohman, T. M.,
and Waksman, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6652-6657[Abstract/Free Full Text]
-
Webster, G.,
Genschel, J.,
Curth, U.,
Urbanke, C.,
Kang, C. H.,
and Hilgenfeld, R.
(1997)
FEBS Lett.
411,
313-316[CrossRef][Medline]
[Order article via Infotrieve]
-
Curth, U.,
Urbanke, C.,
Greipel, J.,
Gerberding, H.,
Tiranti, V.,
and Zeviani, M.
(1994)
Eur. J. Biochem.
221,
435-443[Abstract]
-
Edgell, D. R.,
and Doolittle, W. F.
(1997)
Cell
89,
995-998[Medline]
[Order article via Infotrieve]
-
Appel, R. D.,
Bairoch, A.,
and Hochstrasser, D. F.
(1994)
Trends Biochem. Sci.
19,
258-260[CrossRef][Medline]
[Order article via Infotrieve]
-
Casas-Finet, J. R.,
Khamis, M. I.,
Maki, A. W.,
and Chase, J. W.
(1987)
FEBS Lett.
220,
347-352[CrossRef][Medline]
[Order article via Infotrieve]
-
Curth, U.,
Bayer, I.,
Greipel, J.,
Mayer, F.,
Urbanke, C.,
and Maass, G.
(1991)
Eur. J. Biochem.
196,
87-93[Abstract]
-
Bujalowski, W.,
and Lohman, T. M.
(1991)
J. Biol. Chem.
266,
1616-1626[Abstract/Free Full Text]
-
Thompson, J. D.,
Higgins, D. G.,
and Gibson, T. J.
(1994)
Nucleic Acids Res.
22,
4673-4680[Abstract]
-
Tiranti, V.,
Rocchi, M.,
DiDonato, S.,
and Zeviani, M.
(1993)
Gene
126,
219-225[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.