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J. Biol. Chem., Vol. 277, Issue 31, 27801-27808, August 2, 2002
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From the Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, 80-822 Gdansk, Kladki 24, Poland
Received for publication, February 21, 2002, and in revised form, April 29, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Mitochondrial DNA synthesis is a thermosensitive
process in the yeast Saccharomyces cerevisiae.
We found that restoration of mtDNA synthesis following heat treatment
of cells is dependent on reactivation of the mtDNA polymerase Mip1p
through the action of a mitochondrial bichaperone system consisting of
the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was
inefficiently restored after heat shock in yeast lacking either
functional component of the bichaperone system. Furthermore, the
activity of purified Mip1p was also thermosensitive; however, the
purified components of the mitochondrial bichaperone system (Ssc1p,
Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under
moderate heat shock conditions as well as to reactivate thermally
inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p
contributed more significantly to the restoration of mtDNA synthesis
than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of
mitochondrial chaperones and the propagation of mitochondrial
genomes under ever-changing environmental conditions.
Dividing eukaryotic cells must faithfully replicate and transmit
their mtDNA to ensure the respiratory competence of their progeny.
Although the mitochondrial genomes of most organisms contain multiple
copies of mtDNA, they encode only a small number of proteins that are
essential for mitochondrial function. In the yeast Saccharomyces
cerevisiae, mtDNA encodes eight major proteins. All other
mitochondrial proteins, including those functioning in replication and
maintenance of mtDNA, are encoded by the nuclear genome, synthesized in
the cytosol, and imported into mitochondria.
Maintenance of mtDNA in S. cerevisiae cells is not essential
for survival; and under laboratory conditions, cells lacking mitochondrial genomes are viable as long as they are provided with a fermentable carbon source (e.g. glucose, galactose).
However, these cells are unable to respire, grow slowly, and are not
able to compete with cells harboring intact mtDNA in their natural environment.
Biochemical mechanisms of replication and maintenance of mtDNA are not
well understood (for review, see Ref. 1); however, extensive genetic
and biochemical studies have revealed a number of proteins that are
important for inheritance of mitochondrial genomes. These
include proteins directly involved in synthesis of mtDNA: the mtDNA
polymerase Mip1p (2); a single-stranded DNA-binding protein, Rim1p (3);
the mitochondrial RNA polymerase Rpo41 (4); as well as other proteins
with yet unknown functions in mtDNA propagation. Mitochondrial
chaperones belong to this latter group.
The involvement of chaperones in the process of DNA replication is well
known (for review, see Ref. 5). Generally speaking, chaperones either
can play very specific roles in triggering replication machineries or
can fulfill their general chaperone function by protecting proteins
participating in DNA synthesis against stress-dependent inactivation. For example, DnaK, DnaJ, and GrpE are required for activation of a bacteriophage One of the first indications that general chaperone function might be
important for genome propagation in eukaryotes came from studies of
yeast mitochondria. It was recently shown that inactivation of Mdj1p,
the mitochondrial DnaJ homolog, results in loss of mitochondrial
genomes from cells grown at high temperature (10, 11). Mdj1p is a
cochaperone in the mitochondrial Hsp70 system, consisting of Ssc1p, a
homolog of DnaK, and Mge1p, a homolog of bacterial GrpE. The Hsp70
system is known to play an important role in mitochondria biogenesis,
participating in protein folding, protein degradation, and in the
protection of proteins against heat stress (12-16). Another
mitochondrial chaperone protein, termed Hsp78p, is a member of the
Clp/Hsp100 class of proteins that exerts chaperone functions that
overlap with those of the mitochondrial Hsp70 system (17). Recent
evidence suggested that Hsp78p is required for the maintenance of
mitochondrial genome integrity under severe temperature stress (18).
Biochemical studies using purified proteins indicated cooperation
between the Hsp70 system and Hsp78p, as refolding of a model substrate
protein in the presence of the bichaperone Hsp70-Hsp78 system is more
efficient than in either system alone (19). However, very little is
known about the physiological function of this mitochondrial
bichaperone system.
Studies conducted on homologous systems in bacteria (DnaK-ClpB) and in
the yeast cytosol (Hsp70-Hsp104) provided some hints regarding the
possible functions of the bichaperone system in mitochondria. DnaK and
ClpB play key roles both in preventing and reversing aggregation of the
bulk of bacterial proteins under severe stress conditions (20-23).
Biochemical experiments have also confirmed the requirement for both
DnaK and ClpB in the refolding of thermally denatured protein
substrates (24-26). Similarly, in the yeast cytosol, protein
aggregates formed under heat stress conditions are rapidly eliminated
in an Hsp104-dependent manner during recovery under
moderate temperature (27). Furthermore, purified components of the
bichaperone Hsp70-Hsp104 system are able to efficiently reactivate
denatured luciferase (28). Thus, bichaperone systems seem to be
responsible for the refolding of aggregation-prone proteins. However,
the contribution of chaperone-mediated protein refolding in the
recovery of metabolic processes, inactivated under heat shock
conditions, is still unclear.
In this study, we investigated the role of the mitochondrial
bichaperone Hsp70-Hsp78 system in the maintenance and restoration of
mtDNA synthesis under heat shock conditions in S. cerevisiae. We found that mtDNA synthesis was a thermosensitive
process and that its restoration, following severe heat shock, was
dependent on chaperone-mediated reactivation of Mip1p polymerase. To
elucidate the biochemical mechanism of protection and reactivation of
mtDNA polymerase, we purified Mip1p and four mitochondrial chaperones (Ssc1p, Mdj1p, Mge1p, and Hsp78p). We found that Mip1p activity was
thermosensitive and that the mitochondrial bichaperone Hsp70-Hsp78 system was able to protect its activity under moderate heat shock conditions. In addition, these chaperones were able to reactivate thermally inactivated Mip1p.
Strains, Growth Conditions, and Isolation of
Mitochondria--
Standard genetic techniques were used for the growth
and manipulation of yeast strains (29). The conditional
mdj1-5 mutant strain (30), the
For construction of the pYesMIP1 plasmid, the entire MIP1
coding region was cloned into the pYes2.0 vector (Invitrogen) using a
procedure described previously (31) and plasmid YEpT7-3 (kindly provided by F. Foury, Université Catholique de Louvain, Louvain, Belgium) (32). Mitochondria were isolated as described (33).
Purification of mtDNA Polymerase Mip1p--
Yeast cells
harboring the MIP1-overexpressing plasmid pYesMIP1
were grown to stationary phase in 400 ml of 2% glucose-containing synthetic complete medium without uracil (29). This culture was diluted
40-fold into 10 liters of 2% galactose-containing synthetic complete
medium to induce the expression of Mip1p. When the culture reached
A600 = 4, the cells were harvested (~100 g of
cells). Spheroplasts were generated by incubation of the cells with
zymolyase 20T (ICN) as described (33). Spheroplasts were isolated by
centrifugation and resuspended in 100 ml of cold lysis buffer (40 mM HEPES-KOH (pH 8.0), 15% glycerol, 2 mM
EDTA, 2 mM 2-mercaptoethanol, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 12 µg/ml pepstatin, and 7 µg/ml leupeptin). Spheroplasts were lysed using a French press (Aminco) at 16,000 p.s.i.
After a clarifying spin (15,000 × g, 20 min), proteins were precipitated with ammonium sulfate (0.28 g/ml), resuspended, dialyzed against 1 liter of buffer A (20 mM HEPES-KOH (pH
8.0), 20% glycerol, 5 mM EDTA, 2 mM
2-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride) containing 0.15 M NaCl, and loaded onto a
DEAE-Sephacel column (Amersham Biosciences). Proteins were eluted with
a linear salt gradient (0.15-0.6 M NaCl). Fractions
containing mtDNA polymerase activity were applied to a heparin-agarose
column (Amersham Biosciences) equilibrated with buffer A containing
0.35 M NaCl. Mip1p was eluted with a linear salt gradient
(0.35-1.2 M NaCl), dialyzed against buffer A with 0.2 M NaCl, and loaded onto a 2-ml double-stranded DNA-agarose
column (Amersham Biosciences). The protein was eluted with a linear
salt gradient (0.2-1 M NaCl). Fractions containing active
Mip1p were pooled, dialyzed against buffer A containing 0.15 M NaCl, and stored at Purification of Other Proteins--
Published protocols were
used for purification of Hsp78p (19) and Ssc1p (34). Purified Mdj1p and
Mge1p were kindly provided by Frank King (University of Gdansk).
Protein concentrations were determined with the Bio-Rad Bradford assay
system using bovine serum albumin as a standard. Molar concentrations
are given on the basis of a hexameric structure for Hsp78p (19) and of
a monomeric structure for the other proteins.
Measurements of mtDNA Polymerase Activity--
The activity of
purified Mip1p was measured in 25 µl of buffer B (20 mM
HEPES-KOH (pH 8.0), 150 mM NaCl, 2 mM
2-mercaptoethanol, 0.1 mM EDTA (pH 8.0), 10 mM
MgCl2, 5 mM ATP, 10 mM creatinine phosphate, and 100 µg/ml creatinine kinase) supplemented with 25 µM concentrations each of dATP, dCTP, dTTP, dGTP, and
[methyl-3H]dTTP (100-140 cpm/pmol dNTP).
Either salmon sperm double-stranded DNA activated by DNase I treatment
(prepared as described (32) at 0.13 mg/ml) or M13mp18(+)
single-stranded DNA (Amersham Biosciences) primed with M13 sequencing
primer ( Separation of Monomeric and Aggregated Mip1p by
Centrifugation--
Purified Mip1p was incubated for 10 min at
42 °C in 25 µl of buffer B. Samples were then layered onto 50 µl
of buffer B supplemented with 25% (w/v) sucrose in 500-µl
microcentrifuge tubes. The samples were centrifuged in a Ti-50
rotor (Beckman Instruments) at 30,000 × g for 1 h
at 4 °C. 25 µl of the upper layer was removed, and the remaining
liquid was discarded. The protein pelleted at the bottom was
resuspended in 25 µl of Laemmli sample buffer. An aliquot of the top
fraction was used to measure replication activity. Both top and bottom
fractions were subjected to SDS-PAGE analysis.
Measurements of mtDNA Synthesis in Isolated
Mitochondria--
For analysis of mtDNA synthesis in
organelle, isolated mitochondria (50 µg of total protein) were
incubated in 25 µl of medium containing 50 mM HEPES-KOH
(pH 7.2), 500 mM sorbitol, 4 mM
KH2PO4 (pH 7.2), 80 mM KCl, 10 mM MgCl2, 3% (w/v) bovine serum albumin, 10 mM creatinine phosphate, 100 µg/ml creatinine kinase, 2 mM ATP, 10 µM GTP, 10 µM CTP,
10 µM UTP, 25 µM dATP, 25 µM
dCTP, 25 µM dTTP, 25 µM dGTP, 25 µM [methyl-3H]dTTP (100-140
cpm/pmol dNTP), and 50 µg/ml aphidicolin (Sigma). The rate of mtDNA
synthesis was measured after incubation for 30 min at 30 °C by
counting the radioactivity present in acid-insoluble material as
described above.
The Bichaperone Hsp70-Hsp78 System Is Responsible for Restoration
of mtDNA Synthesis following Severe Heat Shock--
Our goal was to
investigate a possible role of the mitochondrial bichaperone
Hsp70-Hsp78 system in the protection and reactivation of mitochondrial
DNA synthesis under heat shock conditions. To perform such a study, we
first had to establish conditions under which the activity of mtDNA
synthesis was severely reduced, but the survival rate of the cells was
high, to avoid isolation of mitochondria from dead cells. We found that
incubation of cells at 39 °C for 30 min (to induce thermotolerance),
followed by an increase in temperature to 46 °C for another 30 min,
did not affect the survival rate, as 90% of the cells gave rise to
colony-forming units on plates (data not shown). However, this
treatment resulted in a significant decrease in mtDNA synthesis. In
mitochondria isolated from the wild-type cells immediately after the
heat shock, we observed an 85% decrease in the rate of incorporation
of radioactive deoxynucleotides into mtDNA (Fig.
1), indicating that mtDNA synthesis is a
thermosensitive process.
Next, we investigated whether mtDNA synthesis could be restored when
cells were allowed to recover for 2 h at 25 °C following the
heat treatment. However, this restoration could be due to either
reactivation of endogenous mitochondrial proteins or the import of
newly synthesized proteins from the cytosol. To distinguish between
these possibilities, we set up experimental conditions under which
protein synthesis was inhibited. Cycloheximide, a cytosolic protein
synthesis inhibitor, was added just before the recovery phase to
prevent import of newly synthesized proteins into mitochondria (18).
Under these conditions, the level of mtDNA synthesis could be restored,
as mitochondria isolated from the wild-type cells demonstrated 57% of
the activity measured before the heat shock (Fig. 1). This result
indicates that reactivation of thermolabile endogenous mitochondrial
proteins involved in DNA synthesis occurs during the recovery phase
after heat shock.
It is well documented that chaperones are able to protect proteins
against heat-induced denaturation, but are also capable of dissociating
protein aggregates and catalyzing protein refolding. To study the
involvement of the bichaperone Hsp70-Hsp78 system in the restoration of
mtDNA synthesis, we performed the experiment described above using
mutants that inactivate each component of the system separately. To
inactivate the Hsp70 system, we used the conditional mutant
mdj1-5, which functions as a cochaperone of mitochondrial
Hsp70. It was shown previously that mdj1-5p is inactive at
elevated temperature, both in vivo and in
organelle (10, 30, 35). Cells harboring the
mdj1-5 allele subjected to severe heat stress exhibited a
decrease in mtDNA synthesis in organelle, similar to the
wild-type strain (Fig. 1). However, mdj1-5 cells
demonstrated no significant increase in mtDNA synthesis following the
recovery phase (Fig. 1). From this result, we concluded that active
Mdj1p is essential for the restoration of mtDNA synthesis. Likewise, we
used cells harboring a deletion of the HSP78 gene to
determine the role of Hsp78p in the restoration of mtDNA synthesis. In
mtDNA Polymerase Mip1p Is a Native Substrate for the Mitochondrial
Bichaperone System--
mtDNA propagation is a complex process
dependent on many cellular activities. However, incorporation of
deoxynucleotides into mtDNA is catalyzed by only one enzyme present in
the mitochondrial matrix, Mip1p polymerase (1). We have shown
previously that the activity of Mip1p polymerase under heat stress
conditions depends on the function of Mdj1p, a cochaperone of the Hsp70
system (10). Thus, Mip1p is a likely candidate for a protein substrate that is reactivated during the heat shock recovery phase by the bichaperone Hsp70-Hsp78 system. It is also possible that the rate of
mtDNA synthesis was limited by another protein involved in the
mitochondrial replication complex. This hypothetical protein might also
be inactivated at high temperature and reactivated during the recovery phase.
If inactivation and restoration of mtDNA synthesis are dependent on
Mip1p polymerase activity, then the rate of mtDNA synthesis should be
directly correlated with the cellular levels of Mip1p polymerase.
Therefore, we repeated the experiments described above using cells
harboring a plasmid that overproduces Mip1p (Fig. 2B). We found that 30-fold
overproduction of Mip1p (data not shown) resulted in a 4-fold increase
in the rate of mtDNA synthesis in organelle compared with an
isogenic strain without the plasmid (note the different levels of mtDNA
synthesis in Fig. 2, A versus B).
Moreover, in yeast cells overproducing Mip1p, the activity of mtDNA
synthesis decreased following heat shock to 20% of the control value
and increased after the recovery phase to 60% of the control value
(Fig. 2B). Thus, regardless of the higher level of mtDNA
synthesis measured in the strain that overproduces Mip1p, the relative
values of inactivation-reactivation remained the same as in the control
strain. We also observed that the steady-state levels of overproduced
Mip1p did not change throughout the experiment (Fig. 2C),
although the synthesis of a new polymerase was completely inhibited
during the recovery phase by the presence of cycloheximide (Fig. 2,
D and E). Thus, we concluded that the pool of
inactive endogenous Mip1p polymerase is a substrate for the
chaperone-mediated reactivation. Furthermore, this result
suggests that Mip1p is the limiting factor for restoration of mtDNA
synthesis and that changes in the levels of mtDNA synthesis in
organelle are good indicators of the changes in Mip1p polymerase
activity in vivo.
Contributions of Reactivation and Import of Newly Synthesized Mip1p
to Recovery of mtDNA Synthesis after Heat Shock--
As shown above,
mtDNA synthesis from heat-treated cells efficiently recovered to 60%
of the value of untreated cells in the absence of new protein
synthesis. Next, we attempted to assess what contribution the import of
newly synthesized Mip1p polymerase would make to the restoration of
mtDNA synthesis following severe heat stress. To evaluate this
contribution, we compared the levels of mtDNA synthesis in mitochondria
isolated from cells that recovered in the presence of the protein
synthesis inhibitor cycloheximide with the analogous values measured in
mitochondria isolated from cells that recovered in the absence of
cycloheximide. Two possible outputs of this experiment were expected:
first, if the contributions of reactivation and import are similar,
then mtDNA synthesis in mitochondria isolated from cells that recovered
in the absence of cycloheximide should be significantly higher than in
mitochondria isolated from cells that recovered in the presence of the
protein synthesis inhibitor; and second, if the contribution of import to restoration of mtDNA synthesis is negligible, then the levels of
mtDNA synthesis in mitochondria isolated from cells that recovered in
either the presence or absence of cycloheximide should be similar.
We found little difference in the levels of mtDNA synthesis in
wild-type cells that recovered in the presence or absence of cycloheximide (Fig. 3), indicating that
import of newly synthesized Mip1p does not significantly contribute to
the recovery process. If the rate of Mip1p synthesis is a limiting
factor for Mip1p import and thus for restoration of mtDNA synthesis,
then increasing the contribution of import by overproducing Mip1p
should increase recovery of mtDNA synthesis. Indeed, when we repeated
the experiments described above using a wild-type strain harboring the
plasmid that overproduces Mip1p, we observed that the level of mtDNA
synthesis was 2-fold higher in the cells that recovered in the absence
of the protein synthesis inhibitor (Fig. 3). These results demonstrate that the contribution of import of newly synthesized Mip1p polymerase to the restoration of mtDNA synthesis can be observed only when the
rate of Mip1p synthesis is significantly increased.
As shown in Fig. 1, cells harboring a deletion of HSP78 are
very inefficient in reactivating Mip1p polymerase. We took advantage of
this fact to further study the contributions of reactivation and import
of Mip1p to recovery of mtDNA synthesis. We compared the levels of
restoration of mtDNA synthesis in mitochondria isolated from the
Mitochondrial Chaperones Protect Mip1p Polymerase in Vitro--
To
directly test whether Mip1p is indeed a thermolabile protein that can
be protected against heat inactivation by the bichaperone Hsp70-Hsp78
system, we purified Mip1p and the mitochondrial chaperones Mdj1p,
Mge1p, Ssc1p, and Hsp78p. The activity of purified Mip1p polymerase was
tested by measuring incorporation of [3H]dTTP into a DNA
template (see "Experimental Procedures" for details). We found that
Mip1p was indeed thermosensitive (Fig. 4). Incubation of purified Mip1p at
elevated temperatures resulted in rapid loss of DNA synthesis activity.
After a 10-min incubation at 42 °C, the enzyme lost 98% of its
activity compared with the same amount of protein assayed without the
heat treatment. Next, we tested whether mitochondrial chaperones are
able to protect Mip1p against thermal denaturation. We added purified
mitochondrial chaperones to the reaction mixtures containing Mip1p
protein and incubated them at elevated temperatures. Following this
incubation, DNA synthesis activity was measured at 30 °C as
described under "Experimental Procedures." After testing different
combinations of Mdj1p, Mge1p, Ssc1p, and Hsp78p (data not shown), we
found that the most efficient protection was observed when a mixture of
all four chaperones was present together with Mip1p (Fig. 4) and that
such treatment did not affect processivity of the polymerase (data not
shown). These results indicated that the bichaperone Hsp70-Hsp78 system
was able to protect Mip1p polymerase under moderate heat stress
conditions (42 °C), but was not very efficient when the temperature
was raised to 46 °C (15% of activity) and was completely inactive
at 50 °C (data not shown). To confirm that molecular chaperones play
a specific role in the protection of Mip1p against heat inactivation,
we also tested how the presence of other factors, including DNA
template, bovine serum albumin at concentrations equal to the
concentration of chaperone proteins, and Triton X-100, affects
polymerase activity. We found that none of these factors was able to
protect Mip1p against heat inactivation.
In an effort to understand the mechanism underlying Mip1p inactivation,
we tested whether protein aggregation is responsible for the loss of
Mip1p activity. Following incubation at the elevated temperature, the
reaction mixture containing Mip1p was centrifuged through a 20% (w/v)
sucrose solution to separate the monomeric soluble fraction from the
aggregated proteins that formed a pellet. We found that untreated Mip1p
localized predominately in the fraction containing soluble protein
(top) (Fig. 5A). In contrast,
incubation for 10 min at 42 °C resulted in extensive aggregation, as
most of the Mip1p was found in the pellet. In the presence of the four purified chaperones, Mip1p was distributed equally between the soluble
and aggregated fractions (Fig. 5A). The presence of Mip1p in
the soluble fraction correlated well with its level of DNA synthesis
activity (Fig. 5B), indicating that mitochondrial chaperones are able to maintain mtDNA polymerase in its native conformation under
moderate heat stress conditions.
Bichaperone-mediated Reactivation of Aggregated Mip1p Polymerase in
Vitro--
It is well documented that chaperones are not only able to
protect proteins against heat-induced denaturation, but are also capable of dissociating protein aggregates and catalyzing protein refolding (for review, see Ref. 36). To test whether the mitochondrial bichaperone Hsp70-Hsp78 system is able to dissociate aggregates of
Mip1p and to restore its DNA synthesis activity, purified Mip1p was
incubated for 10 min at 42 °C, and then the purified chaperone proteins, listed above, were added to the reaction mixture in the
presence of ATP. After a recovery incubation for 10 min at 30 °C,
DNA synthesis was measured at 30 °C (see "Experimental Procedures"). The presence of the complete bichaperone Hsp70-Hsp78 system resulted in reactivation of Mip1p to 37% of the value without the heat treatment (Fig. 6). Lack of
either Hsp78p or chaperones of the Hsp70 system resulted in a very
significant decrease in the efficiency of the Mip1p reactivation
reaction. These results correlated well with our observations in
vivo. They also indicated that Mip1p polymerase is a native
substrate for mitochondrial chaperones and that its enzymatic activity
can not only be protected under heat shock conditions, but may be
regained when the temperature drops below the heat shock threshold.
Several lines of evidence indicate that Mip1p is a native
substrate of the mitochondrial bichaperone system. In vitro
experiments show that Mip1p is indeed a thermosensitive protein and
that, under moderate heat stress conditions, mitochondrial chaperones are able to prevent its inactivation. These results correlated well
with in vivo experiments showing that, in a wild-type
strain, moderate heat stress conditions do not affect the level of
mtDNA synthesis in organelle (10). However, in strains
harboring either an mdj1-5 mutant (10) or a deletion of the
HSP78 gene,1
levels of mtDNA synthesis decrease significantly at elevated temperature. These results indicate that the mitochondrial bichaperone system is required to maintain Mip1p activity. The thermosensitivity of
Mip1p polymerase is not surprising considering its large size (143.5 kDa). Analysis of thermolabile proteins in Escherichia coli
reveals that large proteins (>90 kDa) make up 80% of the thermosensitive protein fraction (22). Furthermore, the heat sensitivity of partially purified Mip1p has been previously reported (37).
However, when the temperature rises above 42 °C, reaching values
characteristic for severe heat stress conditions, purified mitochondrial chaperones are not able to protect Mip1p polymerase against inactivation. A similar effect was observed in vivo,
as the levels of mtDNA synthesis dropped significantly in mitochondria isolated from wild-type cells subjected to severe heat stress. When the
same cells were allowed to recover for 2 h at moderate temperature, the level of mtDNA synthesis was restored up to 60% of
the value measured before heat treatment. Moreover, this restoration was observed when import of mitochondrial proteins from the cytosol was
prevented by incubating cells in the presence of the cytosolic protein
synthesis inhibitor cycloheximide. These results indicate that
restoration of mtDNA synthesis is dependent on the reactivation of
endogenous mitochondrial proteins. Further analysis using cells harboring either mdj1-5 or Because we identified Mip1p polymerase as a native substrate of the
mitochondrial bichaperone system, we were able to assess the
significance of both the reactivation of thermally inactivated endogenous Mip1p and the import of newly synthesized Mip1p to the
restoration of mtDNA synthesis. By manipulating the level of Mip1p
protein synthesis and by inhibiting the reactivation of endogenous
Mip1p using a What is the molecular mechanism of the bichaperone-mediated
reactivation of Mip1p in vivo? One possible scenario is
that, under severe heat stress, Mip1p dissociates from the mtDNA and forms a large protein aggregate. When the temperature drops below the
heat shock threshold, the mitochondrial bichaperone system may
dissociate the aggregate and refold Mip1p. This mechanism is in
agreement with our in vitro observations that Mip1p
incubated at high temperatures forms aggregates large enough to pellet
in 20% sucrose solution and that purified chaperone proteins are able
to restore Mip1p activity. The ability of the mitochondrial bichaperone
Hsp70-Hsp78 system to dissociate protein aggregates was shown
previously using aggregated model substrate proteins (19). Moreover,
formation of protein aggregates under heat stress conditions was
observed both in bacteria (20, 21) and in yeast (27). In these
organisms, molecular chaperones were shown to be responsible for
elimination of protein aggregates upon recovery at moderate temperature.
An alternative scenario is based on the recent observations that
several molecular chaperones are present in the isolated protein-mtDNA
complexes called nucleoids (40). These are believed to be units of
replication and segregation of mtDNA. We have also been able to isolate
nucleoids and to detect the presence of Ssc1p, Mdj1p, Mge1p, and Mip1p
in the protein-mtDNA complex.1 What is the function of
chaperones in the nucleoid? One possibility is that chaperones are
attracted to the nucleoid by the presence of their natural substrates,
like Mip1p, and probably other proteins involved in mtDNA metabolism.
Thus, the presence of chaperones in the nucleoid protein-DNA complex
might explain why reactivation of Mip1p is so efficient. If the thermal
inactivation of mtDNA polymerase occurs within the scaffold of the
nucleoid, then the rapid reactivation by chaperones that are present in
the near vicinity would be possible as soon as the temperature drops
below the heat shock threshold. Indeed, we have observed that preformed complexes of purified Mip1p with primed M13 single-stranded DNA template remained intact during heat shock treatment, even though the
DNA synthesis activity was lost.1 This attractive
hypothesis will be a subject of further research in our laboratory.
Is the role of mitochondrial chaperones in mtDNA synthesis restricted
to their chaperone function? From our work, we can conclude that
mitochondrial chaperones play a critical role in maintaining the
activity of at least one important replication protein; however, this
does not exclude the involvement of chaperones in more specific mechanisms of mtDNA replication. For example, we have shown previously that deletion of the MDJ1 gene results in the transition
from a functional mitochondrial genome (rho+) into
its nonfunctional, deleted form (rho
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
prepriming complex, and ClpX
functions in activation of bacteriophage Mu DNA synthesis (5). In both cases, genetic and biochemical studies have established an essential role for chaperones in the propagation of these genomes. In contrast, the physiological importance of a general chaperone function in the
propagation and maintenance of chromosomal DNA is much less clear.
Biochemical studies have shown that bacterial RNA polymerase can be
protected against thermal denaturation and inactivation by DnaK and its
cochaperones (6). In addition, a bacterial DNA initiator protein, DnaA,
can be protected and reactivated by DnaK in vitro (7, 8).
Even less is known about chaperone functions in eukaryotic DNA
replication. Mammalian homologs of DnaK and DnaJ have been shown to
protect purified DNA polymerase 
against heat inactivation (9);
however, the physiological relevance of this result remains to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
hsp78 mutant
strain (17), and their isogenic wild-type strains were described
previously. The BY4742 strain (his3, leu2,
lys2, ura3,
hsp78::KAN; kindly provided by M. Boguta, Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Warsaw, Poland) was used to test the effects of
overproduction of Mip1p.
70 °C.
20, 17-mer; New England Biolabs Inc.) was used as a template
as indicated. The reaction was started by addition of purified Mip1p,
and the samples were incubated at 30 °C for 10 min. The rate of
incorporation of radioactive dTTP into DNA was measured by
precipitating the labeled DNA with 750 µl of 10% trichloroacetic
acid and 100 mM sodium pyrophosphate. The precipitates were
collected on glass-fiber filters (Whatman GF/C) and then washed three
times with 1 ml of 1 M hydrochloric acid and 100 mM sodium pyrophosphate and once with 2 ml of ethanol. The
filters were dried, transferred to scintillation fluid, and counted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Restoration of thermally inactivated mtDNA
synthesis is mediated by the bichaperone Hsp70-Hsp78 system.
A, shown is a scheme for the isolation of mitochondria for
mtDNA synthesis measurements. Wild-type (WT),
mdj1-5, and hsp78 yeast strains were grown in
glucose-containing synthetic complete dropout medium for 12 h at
25 °C. At A600 = 2.0, 2 liters of each
culture was harvested, and mitochondria (Mitoch.) were
isolated. The rest of the culture was incubated for 30 min at 39 °C
and then for another 30 min at 46 °C. Next, the culture was divided.
One-half was harvested immediately, and mitochondria were isolated. The
other half was incubated further for 2 h at 25 °C in the
presence of cycloheximide (150 µg/ml final concentration) and then
harvested, and mitochondria were isolated. B, mtDNA
synthesis in organelle was measured as the incorporation of
[3H]dTTP into mtDNA as described under "Experimental
Procedures." Triplicate samples were analyzed for two independent
mitochondrial preparations. Bars indicate the average
values, and ranges of the results obtained are indicated.
hsp78 cells, mtDNA synthesis decreased significantly
under severe heat stress conditions (Fig. 1). But, similar to the
mdj1-5 strain, a significant restoration of mtDNA synthesis
was not observed following the recovery phase (Fig. 1). From these
results, we concluded that a bichaperone (Hsp70-Hsp78) system is
essential for reactivation of mitochondrial proteins involved in mtDNA
synthesis following severe heat shock.
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Fig. 2.
Inactivation-reactivation pattern of
mtDNA synthesis during heat shock treatment does not depend on the
overproduction of Mip1p. A and B, wild-type (WT)
cells and isogenic cells harboring plasmid pYesMIP1, respectively, were
grown in galactose-containing synthetic complete dropout medium to
induce overexpression of Mip1p polymerase. Other growth conditions,
isolation of mitochondria, and measurement of mtDNA synthesis in
organelle were as described in the legend to Fig. 1. Triplicate
samples were analyzed for two independent mitochondrial preparations.
Bars indicate the average values, and ranges of the results
obtained are indicated. C, shown are the steady-state levels
of overexpressed Mip1p. Mitochondrial extracts prepared from cells
harboring plasmid pYesMIP1, which were treated as described for
B, were separated by SDS-PAGE and subjected to immunoblot
analysis using polyclonal antibodies specific to Mip1p. D,
cycloheximide inhibited protein synthesis in a strain overproducing
Mip1p. Cells harboring plasmid pYesMIP1 were grown in 10 ml of
galactose-containing synthetic complete dropout medium lacking
methionine at 25 °C to A600 = 3.0, and then
the culture was heat-treated as described in the legend to Fig.
1A. Following the heat treatment, two 1-ml aliquots were
shifted to 25 °C; cycloheximide (150 µg/ml) was added to one
sample; and then 15 µl of [35S]methionine (10.5 mCi/ml)
was added to both cultures. Labeling of translation products was
stopped after 2 h by addition of unlabeled methionine (4 mM), and extracts were prepared as described previously
(41). Samples were analyzed by SDS-PAGE and phosphorimaging analysis.
E, cycloheximide inhibited synthesis of overproduced Mip1p.
Cells harboring plasmid pYesMIP1 were treated as described for
D, except that, after electrophoresis, proteins were
transferred to nitrocellulose membrane, and Mip1p was detected by
immunoblotting using polyclonal antibodies specific to Mip1p and by
colorimetric assay. Bands representing Mip1p were excised from the
membrane, and their radioactivity was counted in a liquid scintillation
counter.
View larger version (22K):
[in a new window]
Fig. 3.
Contributions of reactivation and import of
Mip1p to recovery of mtDNA synthesis after heat treatment. Yeast
strains were grown as described in the legend to Fig. 2, except that,
after heat shock, each culture was divided, and at the beginning of the
low temperature recovery phase, cycloheximide (150 µg/ml) was added
to one-half (reactivation) and absent in the other half
(reactivation + import). No cell division occurred during
the recovery phase in either the presence or absence of cycloheximide.
mtDNA synthesis in organelle was measured as described under
"Experimental Procedures." Triplicate samples were analyzed for two
independent mitochondrial preparations. Bars represent
restoration of mtDNA synthesis calculated by subtracting the values
measured directly after heat shock from the results obtained following
the recovery phase. Ranges of obtained results are indicated. The
values of mtDNA synthesis measured in mitochondria isolated from cells
grown at 25 °C before the heat shock treatment were set as
100%.
hsp78 strain that recovered after heat shock in either the presence or absence of the protein synthesis inhibitor. In the
presence of cycloheximide, the restoration of mtDNA synthesis in the
hsp78 strain was at the very low level of 5% of the
value measured for mitochondria isolated from the same strain grown at
25 °C. Moreover, only a very limited 11% increase in mtDNA synthesis was observed in the absence of the protein synthesis inhibitor (Fig. 3). This result further supported our conclusion that
import of newly synthesized Mip1p polymerase was very ineffective in
restoring mtDNA synthesis, even when reactivation of endogenous Mip1p
was inhibited by lack of Hsp78p. In contrast, a high level of mtDNA
synthesis (47% of the control value) was measured following the
recovery phase in the absence of cycloheximide in the
hsp78 strain overproducing Mip1p (Fig. 3). From these
results, we concluded that, during the recovery from severe heat shock,
the reactivation of Mip1p polymerase mediated by the mitochondrial
bichaperone system is faster and more efficient than import of newly
synthesized proteins, underscoring the physiological importance for the
protein reactivation process.
View larger version (21K):
[in a new window]
Fig. 4.
Thermal inactivation of purified Mip1p
polymerase and protection of its activity by chaperone proteins.
Purified Mip1p (0.04 µM) was either added directly to the
reaction mixture (No heat treatment) or incubated for 10 min
at the indicated temperatures in 25 µl of buffer B (see
"Experimental Procedures") in the presence of the indicated
factors: 2.7 µg of primed M13 single-stranded DNA, 4.8 µg of bovine
serum albumin (BSA), Triton X-100 at a 0.01% (v/v) final
concentration, and purified chaperone proteins (Ssc1p, 0.25 µM; Mdj1p, 0.05 µM; Mge1p, 0.025 µM; and Hsp78p, 0.1 µM). Next, samples were
transferred to 30 °C for 10 min and supplemented with a radioactive
dNTP mixture and primed M13 single-stranded DNA template (if not
present during the heat treatment). DNA synthesis was measured as
described under "Experimental Procedures."
View larger version (17K):
[in a new window]
Fig. 5.
Mitochondrial chaperone proteins prevent
aggregation of Mip1p polymerase. Purified Mip1p (0.2 µM) was incubated for 10 min at 42 °C in 25 µl of
buffer B in the absence or presence of purified chaperone proteins (as
described in the legend to Fig. 4), and then monomeric and aggregated
forms were separated by centrifugation (as described under
"Experimental Procedures"). A, SDS-PAGE of soluble (top
(T)) and aggregated (bottom (B)) fractions. Mip1p
was stained by Coomassie Brilliant Blue. B, DNA synthesis
activity of the top fractions. 5-µl aliquots were added to the
reaction mixture containing activated double-stranded DNA as a
substrate, and DNA synthesis was measured as described under
"Experimental Procedures."
View larger version (13K):
[in a new window]
Fig. 6.
Thermally inactivated mtDNA polymerase is
reactivated by the mitochondrial bichaperone Hsp70-Hsp78 system.
Purified Mip1p (0.04 µM) was inactivated by incubation
for 10 min at 42 °C in 25 µl of buffer B; purified chaperone
proteins were added (as described in the legend to Fig. 4); and samples
were incubated for 20 min at 30 °C. Next, reaction mixtures were
supplemented with a radioactive dNTP mixture and primed M13
single-stranded DNA template, and DNA synthesis was measured as
described under "Experimental Procedures." DNA synthesis activity
of untreated Mip1p added directly to the reaction mixture was set as
100%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
hsp78 mutations
indicated that the bichaperone Hsp70-Hsp78 system is essential for the
restoration of mtDNA synthesis in vivo. Results of in
vitro experiments using purified proteins confirmed the ability of
the mitochondrial bichaperone system to reactivate aggregated Mip1p
polymerase. Specifically, both the Hsp70 and Hsp78p components of the
bichaperone system are required for the efficient reactivation of
Mip1p. However, a distinction between protection and reactivation of
Mip1p polymerase is probably artificial, as one can imagine that, under
changing environmental conditions, both protection and reactivation
take place at the same time. This is supported by the fact that the bichaperone Hsp70-Hsp78 system was involved in both functions.
hsp78 strain, we have demonstrated that the
reactivation of endogenous Mip1p is more efficient in its contribution
to the restoration of mtDNA synthesis than to import of newly
synthesized Mip1p when Mip1p is produced at physiological levels. The
contribution of imported Mip1p is comparable to that provided by the
reactivation of endogenous Mip1p only when Mip1p is overproduced. To
our knowledge, these are the first experiments to directly estimate the
relative contributions of the reactivation of a protein
versus protein synthesis to the restoration of a physiologically important process following heat shock. It was shown
previously that other cellular processes, e.g. mRNA
splicing (38) and mitochondrial translation (18, 39), are disrupted upon severe heat shock conditions and are reactivated in
chaperone-mediated processes. However, in these cases, it was
impossible to determine the protein substrates that were reactivated by
chaperones and thus difficult to estimate the physiological importance
of protein reactivation to the restoration of splicing and translation.
) (10). The molecular
mechanism of this phenomenon remains to be elucidated. We would like to
stress, however, that anybody who studies the specific role of
molecular chaperones in mtDNA metabolism should not forget about their
general chaperone function and should keep in mind that, because of the
thermal sensitivity of mtDNA synthesis, the use of
temperature-sensitive mutants would be significantly limit.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. E. A. Craig, C. Pfund, B. Schilke, and R. Lightowlers for discussions and critical reading of the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by Polish State Committee for Scientific Research Project 6P04A06017.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.
To whom correspondence should be addressed. Tel.:
48-58-301-2241 (ext. 313); Fax: 48-58-301-9222; E-mail:
marszalek@biotech.univ.gda.pl.
Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.M201756200
1 A. Germaniuk and J. Marszalek, unpublished data.
| |
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES
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