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Molecular and Cellular Biology, May 2000, p. 3677-3684, Vol. 20, No. 10
Department of Biomolecular
Chemistry1 and Institute for Enzyme
Research and the Department of Biochemistry,2
University of Wisconsin, Madison, Wisconsin 53706, and
Department of Molecular and Cellular Biology, Faculty of
Biotechnology, University of Gdansk, 80-822 Gdansk,
Poland3
Received 20 October 1999/Returned for modification 7 December
1999/Accepted 28 February 2000
The mitochondrial matrix of the yeast Saccharomyces
cerevisiae contains two molecular chaperones of the Hsp70 class,
Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In
vitro, radiolabeled Yfh1 was not imported into ssc1-3
mutant mitochondria, remaining in a protease-sensitive
precursor form. As reported earlier, the Yfh1 intermediate form was
only slowly processed to the mature form in Hsp70s are ubiquitous molecular
chaperones found in all major organelles of eukaryotes that function by
binding transiently to exposed hydrophobic sequences in unfolded or
partially folded proteins (3, 11, 36). Hsp70s' chaperone
activity is driven by their inherent ATPase activity, which regulates
binding and release of substrate polypeptides. Through this mechanism,
Hsp70s function in diverse cellular processes such as protein folding, protein translocation across membranes, and assembly and disassembly of
multimeric structures (6, 19, 21).
Ssc1, an essential heat shock protein of the 70-kDa class (Hsp70), is a
key component of the mitochondrial protein import machinery of
Saccharomyces cerevisiae (15, 23, 45).
Mitochondrial import is a multistep process necessary for the
biogenesis of most mitochondrial proteins (30, 31, 37, 39).
The vast majority of mitochondrial proteins are translated in the
cytosol with an N-terminal presequence that targets the preprotein to receptors on the outer mitochondrial membrane. After passage
through a proteinaceous channel of the outer mitochondrial membrane,
the presequence is then driven across the inner mitochondrial membrane through a protein channel and into the matrix by the membrane potential. Cooperating with cochaperones and components of the inner
mitochondrial membrane, matrix-localized Ssc1 binds the emerging
preprotein while processing proteases cleave the presequence. Ssc1,
acting in an ATP-dependent manner, is required for the further translocation of the remaining preprotein across the inner
mitochondrial membrane. Subsequent folding of the imported
mitochondrial protein within the matrix is, at least in some cases,
dependent upon Ssc1 and other molecular chaperones.
Although for many years Ssc1 was the only known Hsp70 of mitochondria,
a second matrix-localized Hsp70, Ssq1, has recently been identified
(40). Ssq1 is not essential; however, cells lacking Ssq1
grow extremely slowly at low temperatures such as 23°C. While Ssq1
function is not required for the proper import or processing of several
mitochondrial proteins including cytochrome b2
(Cytb2) and the Fe/S protein of the cytochrome
bc1 (Cytbc1) complex
(40), recent studies demonstrate a role for Ssq1 in the
maturation of Yfh1 (26), a nucleus-encoded
mitochondrial protein involved in iron homeostasis (1, 5,
32). In in vitro import assays, the Yfh1 precursor was processed
in two steps by the mitochondrial processing peptidase (MPP),
generating first an intermediate and then the functional mature form
(4). Unlike wild-type mitochondria, mitochondria
lacking Ssq1 accumulated the intermediate form of Yfh1,
which was only slowly processed to the mature form (26).
Thus far, Yfh1 is the only substrate identified whose maturation is
affected by the absence of Ssq1.
Yfh1 is the orthologue of the human protein frataxin. Mutations in the
frataxin gene are associated with the neurodegenerative disease
Friedreich's ataxia (7). Iron accumulates within the affected cells, and the activity of Fe/S-containing proteins, such as
aconitase, is reduced (27, 35). Accumulation of iron, along
with a reduction in the activity of Fe/S-containing proteins, has also
been observed for mitochondria isolated from yeast strains with
mutations in YFH1 (1, 12). It is thought that the
increased levels of iron result in increased production of oxygen
radicals within mitochondria (16). Since Fe/S centers are
particularly sensitive to oxidative damage (13), the
decrease in the activity of Fe/S-containing enzymes has been attributed
indirectly to iron accumulation. Consistent with this idea,
yfh1 cells are more sensitive to oxidative agents than are
wild-type cells (1).
The phenotypes of Yeast strains, plasmids, media, and chemicals.
PJ53 is
isogenic to W303: trp1-1/trp1-1 ura3-1/ura3-1
leu2-3,112/leu2-3,112 his3-11,15/his3-11,15 ade2-1/ade2-1
can1-100/can1-100 GAL2+/GAL2+
met2-
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of the Mitochondrial Hsp70s, Ssc1 and Ssq1, in
the Maturation of Yfh1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
ssq1
mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998).
However, the intermediate form in both wild-type and
ssq1 mitochondria was entirely within the inner
membrane, as it was resistant to digestion with protease after
disruption of the outer membrane. Therefore, we conclude that Ssc1,
which is present in mitochondria in approximately a 1,000-fold excess
over Ssq1, is required for Yfh1 import into the matrix, while Ssq1
is necessary for the efficient processing of the intermediate to
the mature form in isolated mitochondria. However, the steady-state
level of mature Yfh1 in ssq1 mitochondria is
approximately 75% of that found in wild-type mitochondria, indicating
that this retardation in processing does not dramatically affect
cellular concentrations. Therefore, Ssq1 likely has roles in addition
to facilitating the processing of Yfh1. Twofold overexpression of Ssc1
partially suppresses the cold-sensitive growth phenotype of
ssq1 cells, as well as the accumulation of mitochondrial
iron and the defects in Fe/S enzyme activities normally found in
ssq1 mitochondria. ssq1 mitochondria
containing twofold-more Ssc1 efficiently converted the intermediate
form of Yfh1 to the mature form. This correlation between the observed
processing defect and suppression of in vivo phenotypes suggests that
Ssc1 is able to carry out the functions of Ssq1, but only when present
in approximately a 2,000-fold excess over normal levels of Ssq1.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
yfh1 and ssq1 cells are
very similar. Both accumulate iron, have low activity of
Fe/S-containing enzymes, and are hypersensitive to oxidative agents. To
better understand the function of Ssq1 and its relationship to Ssc1 and
Yfh1, we analyzed the translocation and maturation of Yfh1 in more
detail. Efficient maturation of Yfh1 requires the sequential action of Ssc1 and Ssq1. Ssc1 is required for translocation across the inner membrane, and subsequently Ssq1 is needed for efficient processing by
MPP. Overexpression of the abundant Ssc1 partially suppresses the
defects caused by the absence of the minor Hsp70 Ssq1, indicating that these two Hsp70s partially overlap in function.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1/met2-1 lys2-2/lys2-2 (20). PJ43B is
a wild-type haploid derivative of PJ53. The haploid ssq1
strain was made by first replacing one of the SSQ1 alleles
of PJ53 with the LYS2 gene
(ssq1::LYS2) (40) and then
sporulating the heterozygous diploid. ssq1/pSSC1 is the haploid
ssq1 strain carrying the SSC1 gene on the
centromeric plasmid, pRS316 (40, 42). PK83 carries the
ssc1-3 temperature-sensitive allele, and PK83 is its isogenic wild-type strain (15). The strain BJ3497
(pep4::HIS3 ura3-52 his200
[22]), which is defective in proteinase A, was used
for expression of the GST-Ssc1 (29) and GST-Ssq1HA (this study) fusion proteins.
426 to +754 using Pfu1 polymerase (Stratagene, La
Jolla, Calif.). YFH1 was then cloned into the pRS vectors,
pRS306 and pRS316 (42), which carry the URA3
marker. The HIS3 gene was used to replace the entire protein
coding sequence of YFH1, and the resulting pRS306YFH1::HIS3 plasmid was used to disrupt
YFH1 in the diploid PJ53 by the standard two-step disruption
procedure (34). yfh1::HIS3 derivatives were verified by PCR amplification.
Translocation of proteins into and fractionation of
mitochondria.
Mitochondria were isolated from PK82, PK83, PJ43B,
ssq1, and ssq1/pSSC1 strains as previously described
(15). [35S]methionine-labeled precursor
proteins were synthesized in vitro using a coupled rabbit reticulocyte
lysate system (Promega). Methods for import experiments have been
described previously (15). Briefly, 50 µg of mitochondrial
protein for the PJ43B, ssq1, ssq1/pSSC1, PK82, or
PK83 strain was incubated for 5 min at 25°C, or 15 min at 37°C for
PK82 and PK83. Mitochondrial samples were then incubated at 25°C with
lysate containing 35S-labeled Yfh1 or
Cytb2167DHFR (15) for the indicated times before
addition of a mixture of 0.25 µM valinomycin, 4 µM antimycin A, and
10 µM oligomycin which results in dissipation of the membrane potential. Half of each sample was then treated with proteinase K at a
final concentration of 100 µg/ml for 15 min on ice. Twenty-five micrograms of mitochondrial protein was then analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
ssq1 yeast strains. After import, mitochondrial samples were divided in half and reisolated by centrifugation for 5 min at
16,000 × g at 4°C. Each pellet was resuspended in
SEM (250 mM sucrose, 1 mM EDTA, 10 mM MOPS [morpholinepropanesulfonic
acid], pH 7.2) or EM (1 mM EDTA, 10 mM MOPS, pH 7.2) buffer at a
concentration of 50 µg of mitochondrial protein/500 µl of buffer
and incubated on ice for 15 min. Mitochondria and mitoplasts, obtained
by incubating isolated mitochondria in hypotonic EM buffer, were
treated with and without 50 µg of proteinase K for 15 min on ice.
Samples were centrifuged for 15 min at 14,000 × g to
isolate the mitochondria or mitoplasts. Samples were separated by
SDS-PAGE, transferred to nitrocellulose, and subjected to
PhosphorImager analysis (Molecular Dynamics). Samples were then
analyzed by immunoblot analysis using polyclonal antibodies against
Cytb2 (this study) or Mge1 (29) using the
Renaissance detection kit from New England Nuclear Labs (Boston,
Mass.).
Respiratory enzyme assays and measurement of mitochondrial
iron.
Activities of the respiratory enzymes, succinate
dehydrogenase-cytochrome c oxidoreductase
(SDH-Cytbc1) and Cytbc1, were measured in
mitochondria isolated from PJ43B, ssq1, and ssq1/pSSC1
yeast strains grown to an optical density at 600 nm of 0.5 to 1.0 at 34°C in YP medium containing 2% galactose (15).
SDH-Cytbc1 activity was measured using succinate as the
substrate as previously described (46), except that
2,6-dichloroindophenol was used in place of cytochrome c
(41). Cytbc1 activity was measured using reduced ubiquinone as a substrate. The reduction of cytochrome c was
determined as previously described (17, 41). Cellular
aconitase activity was measured by monitoring the decrease in the
absorbance of the substrate cis-aconitate at 240 nm as
described previously (10, 41).
Purification of GST-Ssq1HA and GST-Ssc1 proteins.
BJ3497
cells harboring the GST-SSC1 (29) or
GST-SSQ1-HA (this study) overexpression plasmid were grown
to an optical density at 600 nm of 3.0 in 6 liters of synthetic
complete medium without uracil containing 2% galactose at 30°C.
Cells were harvested by centrifugation for 5 min at 2,700 × g. The cells were washed with cold water and resuspended in
90 ml of buffer P (20 mM NaPi [pH 7.3], 150 mM NaCl, 1%
[vol/vol] Triton X-100, 0.1% 
-mercaptoethanol, 1 mM
phenylmethanesulfonyl fluoride, 1 mM TPCK
[L-chloro-3{4-tosylamido}-4-phenyl-2-butanone], and 2 µg each of pepstatin A and leupeptin per ml). The cell suspension was
frozen in liquid nitrogen, thawed on ice, and lysed using a French
press at 20,000 lb/in2. The mixture was centrifuged at
100,000 × g for 60 min. The cleared extract was loaded
directly onto a 5-ml glutathione agarose column equilibrated with
buffer P containing 0.1% Triton X-100 and 10% (vol/vol) glycerol. The
column was washed with 10 volumes of buffer P containing 1 M NaCl,
0.1% Triton X-100, and 10% glycerol and subsequently with 10 volumes
of the same buffer containing 150 mM NaCl. Protein was eluted with
buffer P containing 20 mM glutathionine adjusted to pH 7.3 and 10% glycerol.
Protein quantitation using immunoblot analysis.
To determine
Yfh1 levels, mitochondrial proteins purified from wild-type (PJ43-2B)
and ssq1 strains were subjected to SDS-PAGE, transferred
to nitrocellulose (Protran; Schleicher & Schuell Co., Keene, N.H.), and
immunoblotted with antibodies to Yfh1 and Mge1 (29).
Relative concentrations of Yfh1 were determined by densitometrically comparing Yfh1 and Mge1 signals on exposed film (X-O; Eastman Kodak
Co., Rochester, N.Y.) using Ofoto (Light Source Computer Images, Inc.)
and ScanAnalysis (Biosoft) software programs. Ssc1 and
F1 levels were determined as described above, except
that mitochondrial protein purified from the ssq1/pSSC1 yeast strain was also examined and polyclonal antibodies against Ssc1
(38) and F1 (this study) were utilized. For
quantitation of Ssq1 and Ssc1, fixed amounts of mitochondrial protein
purified from the ssq1/pRS316SSQ1-HA strain
(40) mixed with varying amounts of purified GST-Ssc1 or
GST-Ssq1HA were analyzed as described above, except that monoclonal
antibodies against hemagglutinin (HA) or polyclonal antibodies against
Ssc1 (38) were utilized. Densitometric signals from serial
dilutions of GST-purified proteins were compared to the signals from a
constant dilution of mitochondria, and the relative amounts of Ssc1 and
Ssq1 were determined by comparing the level per microgram of
mitochondrial protein. Similar results were obtained using a different
polyclonal antiserum generated from the last 14 amino acids of Ssc1
(29).
. Each fusion protein
was expressed in Escherichia coli and purified by adsorption
to glutathione-agarose beads. Eluate from the beads was used to
inoculate rabbits. Grazia Isaya (Mayo Clinic) kindly provided Yfh1 antiserum.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Ssc1 is required for import of radiolabeled Yfh1 into isolated
mitochondria.
To evaluate the contributions of Ssc1 to the
maturation of Yfh1, we tested whether functional Ssc1 was necessary for
the import of Yfh1 into mitochondria. Mitochondria were isolated from
yeast strains carrying a temperature-sensitive allele of
SSC1, ssc1-3, grown at the permissive temperature
of 25°C. Isolated mitochondria were preincubated at 37°C for 15 min
to induce the mutant phenotype (15) and then incubated at
25°C with 35S-labeled Yfh1. In wild-type mitochondria,
Yfh1 was processed to the mature form, which was resistant to
exogenously added proteinase K (Fig. 1A)
(26). In ssc1-3 mitochondria, Yfh1 was not
processed to either the intermediate or the mature form. Furthermore,
the precursor form was accessible to exogenously added proteinase K,
indicating that Yfh1 preprotein was not imported into mitochondria in
the absence of Ssc1 function (Fig. 1A). Despite the inability to import
Yfh1, the ssc1-3 mitochondria used in this experiment were translocation competent, as demonstrated by the import and processing of Cytb2167DHFR (Fig. 1B), a fusion
protein construct lacking the intact Cytb2 heme binding
domain, thus making its maturation independent of Ssc1 function
(45). We conclude that Ssc1 is required for the
translocation of the precursor form of radiolabeled Yfh1 into isolated
mitochondria, as it is for many matrix-localized mitochondrial proteins
(30).
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) were compared. To induce the mutant phenotype of
Ssc1-3, mitochondria were preincubated for 15 min at 37°C. After 5 or
15 min, further import was stopped by dissipating the membrane
potential. Samples were then divided; half were treated with proteinase
K (+PK), and half were not (Relative levels of Ssc1 and Ssq1 within mitochondria.
The
above results, coupled with previously reported data, indicate that
both Ssc1 and Ssq1 function in the maturation of Yfh1 (26).
To better understand the functional relationship between Ssc1 and Ssq1,
we determined the relative amounts of the two proteins in wild-type
mitochondria. Mitochondrial lysates were first separated on an
SDS-polyacrylamide gel and stained using Coomassie blue dye.
Densitometric analysis of the stained gel indicated that Ssc1 protein
made up approximately 2% of the mitochondrial protein profile (data
not shown), consistent with previously published values
(33). To estimate the amount of Ssc1 relative to Ssq1 in
isolated mitochondria, we performed immunoblot analysis on samples
containing fixed amounts of mitochondrial protein mixed with known
amounts of purified GST-Ssc1 or GST-Ssq1HA as described in
Materials and Methods (Fig. 2). We
currently do not have antibodies specific to Ssq1, and therefore, we
utilized an HA-tagged version of Ssq1 for our analysis which is
functional in vivo (40). Ssc1 and Ssq1 were detected by
immunoblot analysis using polyclonal antibodies specific to Ssc1 or
monoclonal antibodies specific for the HA epitope, respectively. By
comparison of the signal from isolated mitochondria with that of
purified GST fusion proteins, we calculated that Ssc1 is 1,000 to 2,000 times more abundant than Ssq1 in wild-type mitochondria. Taken
together, these data suggest that Ssc1 and Ssq1 make up
approximately 1.0 to 2.0% and 0.001 to 0.002% of mitochondrial
protein, respectively.
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The intermediate and mature forms of radiolabeled Yfh1 are located
within the matrix of wild-type and ssq1
mitochondria.
Although Ssq1 is of low abundance, it plays an
important role in the maturation of Yfh1 (26). According to
previous studies confirmed in this report, Yfh1 can be imported into
mitochondria isolated from strains carrying mutations in
SSQ1. However, it accumulates as a protease-resistant
intermediate form (Fig. 3A) (26). To better define the contribution of Ssq1 to Yfh1
maturation, we thoroughly analyzed the state of the intermediate form
of Yfh1. We determined the intramitochondrial location of the
intermediate form to ascertain whether it is completely translocated
into the matrix or whether it exists as an intermembrane space
intermediate, exposing the amino-terminal presequence to the matrix but
retaining the remainder of the protein in the intermembrane space.
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ssq1 strains
were fractionated following the import of radiolabeled Yfh1. In both wild-type and ssq1 mitochondria, the intermediate and
mature forms of Yfh1 remained protease resistant following a hypotonic treatment that ruptures the outer, but not the inner,
mitochondrial membrane, forming mitoplasts. Under these
conditions, proteins of the intermembrane space, such as
Cytb2, are released from mitochondria and any residual
protein is degraded by added protease, while matrix proteins,
such as Mge1, remain resistant to protease (Fig. 3B). The
intermediate and mature forms of Yfh1 were resistant to digestion with
proteinase K in both wild-type and ssq1 mitoplasts, as
well as intact mitochondria, indicating that the intermediate and
mature forms of Yfh1 are located within the matrix (Fig. 3A). In
several experiments, a decrease in the intensity of the radiolabeled intermediate and mature form of Yfh1 in ssq1 mitochondria
was observed. To determine if this effect was specific for Yfh1, we cotranslocated a commonly used fusion protein which localizes to the
matrix, Su9DHFR. A similar decrease in the signals of the two proteins
was observed in the mitoplast preparations (data not shown), suggesting
that ssq1 mitochondria are slightly more susceptible to
breakage during the procedure used to generate mitoplasts than are
wild-type mitochondria.
ssq1 mitochondria accumulate 10-fold more iron than
normal wild-type levels of 3 to 7 pmol of Fe/µg of mitochondrial
protein (26, 41). To determine whether the high amounts of
iron cause the observed processing defect of Yfh1 in ssq1
mitochondria, mitochondria were isolated from wild-type and
ssq1 yeast strains grown in low-iron-containing media.
Under these conditions, both ssq1 and wild-type
mitochondria have approximately 3 pmol of Fe/µg of
mitochondrial protein (Fig. 3C). The maturation of radiolabeled Yfh1 was monitored using mitochondria isolated from wild-type and
ssq1 yeast strains grown in nutrient-rich and low-iron
minimal media (Fig. 3C). Radiolabeled Yfh1 was processed efficiently in wild-type mitochondria, while the processing defect of Yfh1 persisted in ssq1 mitochondria, indicating that lack of Ssq1
function, not iron, is responsible for the Yfh1 processing defect
associated with ssq1 mitochondria.
Taken together, these results suggest that the abundant Ssc1 is
required for translocation of the Yfh1 preprotein across the inner
membrane, while the rare Ssq1 is needed for the subsequent posttranslocational processing of the intermediate to the functional mature form.
Levels of Yfh1 in ssq1 mitochondria.
In in
vitro translocation experiments, ssq1 mitochondria
inefficiently process the matrix-localized intermediate form of Yfh1 to
the mature form (Fig. 3). To determine whether this in vitro
observation translates into lowered amounts of Yfh1 in
ssq1 mitochondria in vivo, we directly determined the
relative levels of Yfh1 in mitochondria isolated from wild-type and
ssq1 mitochondria using Yfh1-specific antibodies.
Relative to the control, Mge1, Yfh1 levels in ssq1
mitochondria were about 75% of those in wild-type mitochondria (Fig.
4). Thus, there is only a minor reduction
in the steady-state level of mature Yfh1 in ssq1
mitochondria. Therefore, the inefficient processing of Yfh1 observed in
the in vitro import assay is likely revealing a kinetic effect that is
not detected in an analysis of steady-state levels.
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ssq1
cells suppressed the growth defects caused by the lack of Ssq1
function. ssq1 cells carrying YFH1 on either a
low-copy centromeric or a high-copy-number 2µm plasmid, which
increases the amount of mature Yfh1, grew no better than
ssq1 cells carrying only control vectors (data not
shown). Together, these results suggest that the inefficiency of Yfh1
processing is not responsible for ssq1 phenotypes and is,
therefore, not the sole defect of ssq1 cells.
Increased levels of Ssc1 overcome the Yfh1 processing defect in
ssq1 mitochondria.
Previously, we reported that an
additional copy of SSC1 partially suppressed the
cold-temperature growth phenotype of ssq1 yeast strains
(40). Therefore, we tested whether overexpression of Ssc1
could also overcome the processing defect of Yfh1 in ssq1 mitochondria. Radiolabeled Yfh1 was imported into mitochondria isolated
from wild-type and ssq1 yeast strains, as well as
ssq1/pSSC1, a yeast strain with SSQ1 deleted carrying an
additional copy of SSC1 on a centromeric plasmid
(40). Unlike ssq1 mitochondria, ssq1/pSSC1
mitochondria were able to process the intermediate form of Yfh1
to the mature form with nearly the same efficiency as that of wild-type
mitochondria (Fig. 5A). Therefore,
increased levels of Ssc1 restored the processing of Yfh1 to
near-wild-type efficiencies even in the absence of Ssq1, suggesting
that, when in excess, Ssc1 can compensate for Ssq1 function.
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ssq1, and ssq1/pSSC1 yeast strains. A series of
twofold dilutions of mitochondrial proteins isolated from the
three yeast strains were tested by immunoblot analysis using polyclonal
antibodies specific to Ssc1. Densitometric analysis of the immunoblots
using F1 as a control revealed that the amount of
Ssc1 was approximately twofold higher in ssq1/pSSC1 mitochondria
than in wild-type and ssq1 mitochondria (Fig. 5B). This
twofold increase in Ssc1 levels indicates that an approximately
2,000-fold excess of Ssc1 over normal levels of Ssq1
suppresses the processing defect of the Yfh1 precursor in
ssq1 mitochondria.
Increased levels of Ssc1 suppress the iron accumulation in
ssq1 mitochondria.
Previous observations
demonstrated that ssq1 mitochondria accumulate iron
(26, 41). Wild-type mitochondria contain approximately 5.0 pmol of Fe/µg of mitochondrial protein, while ssq1
mitochondria accumulated approximately 10-fold more iron than did
wild-type mitochondria (Table 1).
Therefore, we tested whether increasing the level of Ssc1 also
suppressed the accumulation of iron in ssq1 mitochondria.
Increasing the level of Ssc1 significantly reduced the amount of iron
that accumulated in the absence of Ssq1 function. However, iron levels
remained 2.5-fold higher than wild-type levels (Table 1).
|
ssq1, and ssq1/pSSC1 strains to
H2O2 and observed that ssq1
strains were extremely sensitive to H2O2
compared to wild-type cells (Fig. 6),
consistent with previously reported data (41). The
additional copy of SSC1 partially overcame the
hypersensitivity of ssq1 cells to
H2O2 but did not restore growth to wild-type
levels (Fig. 6). This increased sensitivity of ssq1/pSSC1 strains to
H2O2 compared to that of the wild type
correlates with the higher-than-normal levels of iron observed in this
strain, suggesting that, even at increased levels, Ssc1 does not
completely replace Ssq1 function.
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Increased levels of Ssc1 partially restore activity of
Fe/S-containing mitochondrial respiratory enzymes in
ssq1 mitochondria.
The highly reactive hydroxyl
radicals generated from excess free iron can damage the Fe/S centers of
Fe-containing mitochondrial proteins, resulting in a decrease in their
activity (13). ssq1 mitochondria show a
decrease in the enzymatic activities of several respiratory components
containing Fe/S clusters: aconitase, Cytbc1, and SDH
(41, 43). Therefore, we tested whether the
overproduction of Ssc1 restored the activity of these proteins. The
activity of aconitase is reduced to 7% in ssq1 cells,
but increasing the level of Ssc1 restored the activity of aconitase in
ssq1/pSSC1 cells to 58% of wild-type levels (Table 1). Using
isolated mitochondria, we measured the activity of certain complexes of
the electron transport chain that contain proteins with Fe/S centers,
the Cytbc1 complex and the SDH complex. Assaying
Cytbc1 activity directly by supplying the reaction
with reduced ubiquinone, we determined that ssq1
and ssq1/pSSC1 mitochondria maintained 33 and 90% of
wild-type Cytbc1 activity, respectively (Table 1). To
measure the activity of the SDH complex, we assayed
SDH-Cytbc1 activity. The rate-limiting step of
ubiquinone-mediated mitochondrial electron transport is diffusion
of the ubiquinones within the inner membranes (8, 28).
Therefore, the measured activity of the SDH-Cytbc1 assay is
indicative of the activity of the SDH complex itself. We found that the
ssq1 mitochondria possess only 8% of wild-type SDH-Cytbc1 activity, suggesting a strong defect in SDH
activity itself, consistent with previously reported data (41,
43). The ssq1/pSSC1 mitochondria had 54% of the
SDH-Cytbc1 activity of wild-type mitochondria. We conclude
that increasing Ssc1 levels approximately twofold can partially restore
the activity of Fe/S-containing proteins. This suppression may well be
the indirect result of lowering the free iron level within
ssq1 mitochondria.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The in vitro data reported here support a sequential mode of
action of the two mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1; first, import across the mitochondrial inner membrane requires Ssc1 function, and second, processing of the intermediate to the mature form of the protein within the matrix is
facilitated by Ssq1. It is likely that Ssc1 drives import of Yfh1 in a
manner similar to the way it functions in the import of many
polypeptides (30). The effect of Ssq1 on Yfh1 maturation is
likely direct, as reducing the amount of iron accumulating in
ssq1 mitochondria did not alleviate the processing
defect. However, the mechanism of a specific role of Ssq1 in
facilitating the second cleavage of Yfh1 by MPP is unresolved
(4). Perhaps it binds Yfh1 at a specific site(s),
maintaining the intermediate form in a conformation that allows the
second cleavage site to become accessible to MPP. In the absence of
Ssq1, the Yfh1 intermediate may fold into a conformation that masks
this cleavage site.
However, Ssq1 is not uniquely required for the processing of the intermediate form of Yfh1, as increasing the levels of Ssc1 twofold suppresses this maturation defect. Since Ssc1 is normally 1,000 to 2,000 times more abundant than Ssq1, we were surprised that increasing the level of the very abundant Ssc1 twofold was able to significantly suppress the processing defect of Yfh1 in vitro. Normally, Ssc1 is likely occupied in carrying out chaperone functions in the mitochondrial matrix. Therefore, little Ssc1 might be free and available for interaction with Yfh1. Even though the additional copy of SSC1 results in only a twofold increase in the total amount of Ssc1 protein, doubling the protein concentration might dramatically increase the level of "free" Ssc1. For example, if 5% of Ssc1 is normally unbound to substrates and free for interaction, a twofold increase in total Ssc1 concentration would be equivalent to a 20-fold increase in available Ssc1.
Based on the assumption that the free pool of Ssc1 is normally relatively small, we propose two possible explanations for the suppression. (i) Ssq1 and Ssc1 could differ in the specificity of their interactions with peptides, with Ssq1 having a very high affinity for a critical sequence in Yfh1. According to this scenario, Ssc1 would be capable of interacting with this Yfh1 sequence, but with a much lower affinity than Ssq1. Thus, a higher concentration of Ssc1 would be required to attain effective interactions with Yfh1. (ii) On the other hand, sequences within Ssq1, outside the peptide binding cleft, may serve to target Ssq1 to a particular site within the mitochondrial matrix, which facilitates interaction with the incoming precursor Yfh1 polypeptides. For example, factors may exist which interact with both Ssq1 and the intermediate form of Yfh1. By this model, Ssq1 might not have an intrinsic higher affinity for Yfh1 but rather have a very high effective concentration near Yfh1 precursors. Such factors may associate with the mitochondrial inner membrane, since matrix fractions alone were unable to process Yfh1 efficiently in vitro (4). If this model is correct, very high levels of free Ssc1 in the matrix would be required to obtain the same effective concentration as Ssq1.
Overexpression of Ssc1 not only suppressed the in vitro Yfh1 processing
defect but also partially suppressed the in vivo phenotypes of iron
accumulation, cold sensitivity, and reduced activity of Fe/S-containing
proteins. This cosuppression lends credence to the idea that the
similarity of ssq1 and yfh1 mutant phenotypes is
at least partially due to the role of Ssq1 in Yfh1 maturation. However,
the steady-state level of Yfh1 in mitochondria lacking Ssq1 is 75% of
that found in wild-type mitochondria, raising the question of whether
this small reduction in Yfh1 levels is sufficient to cause the observed
phenotypes of ssq1 mutants. Since increasing the level of
mature Yfh1 did not suppress the growth phenotypes of ssq1
mutant cells, the cause of the multiple ssq1 mutant
phenotypes must be more complicated than simply the reduction in the
efficiency of processing of the intermediate form of Yfh1. One can
envision two possible explanations for this result. (i) Ssq1
facilitates the proper folding of mature Yfh1. Therefore, the mature
form of Yfh1 is not folded properly in ssq1 mitochondria.
To test this possibility, we assessed aggregation and altered protease sensitivity of Yfh1 in ssq1 mitochondria. However, no
differences were observed in these experiments between wild-type and
ssq1 mitochondria (data not shown). Therefore, at this
time, there are no experimental data to support this model. (ii) Ssq1
is involved in functions in addition to the maturation of Yfh1. These
functions could involve the maturation of other proteins, which have
yet to be identified, or other roles of Ssq1 in the matrix as discussed below.
The phenotypes of ssq1 mutant strains are partially
suppressed by a 2,000-fold excess of Ssc1 over normal levels of
Ssq1, suggesting that, even at increased levels, Ssc1 is not
completely replacing Ssq1 function. Perhaps Ssc1 levels must be
increased more than twofold to achieve full suppression. However, we
found that ssq1 cells harboring a high-copy 2µm plasmid
containing SSC1 did not enhance suppression over that seen
with a centromeric plasmid. In fact, expression of Ssc1 from this
SSC1::2µm plasmid inhibited growth of wild-type
cells (data not shown). Possibly, excess Ssc1 inappropriately binds to
folded proteins, causing deleterious effects on the cell.
On the other hand, the partial suppression of ssq1 defects
by Ssc1 suggests that Ssq1 has a specific function in mitochondria that
cannot be replaced by Ssc1. For example, Ssq1 could play a unique role
in the maintenance and/or assembly of Fe/S proteins (41,
43), which would explain not only the partial suppression of
ssq1 defects but also the inability of near-wild-type
levels of mature Yfh1 to suppress ssq1 phenotypes as
well. Genetic data support a connection between Ssq1 and other
proteins, Nfu1, Isu1 and Isu2, and Nfs1, believed to be involved in the
assembly and/or maintenance of Fe/S centers (25, 41, 43). In
nitrogen-fixing bacteria, orthologues of Isu1, Isu2, Nfu1, and Nfs1 are
required for the synthesis of the Fe/S cluster of nitrogenase (9,
14, 48). Interestingly, orthologues of these genes along with
genes encoding an hsp70 and a DnaJ-like protein reside within the same operon in Azotobacter vinelandii and non-nitrogen-fixing
bacteria, suggesting the existence of a general macromolecular system
for the biogenesis of Fe/S proteins (47). Therefore, it is
possible that Ssq1 plays a chaperone-type role in maturation of
proteins containing Fe/S clusters. For example, Ssq1 might assist in
the assembly of a multimeric complex, composed of the proteins
mentioned above, which functions in the assembly or repair of Fe/S
proteins. However, at this time, a direct role for Ssq1 or any of these gene products in the assembly or repair of Fe/S proteins remains to be
clearly separated from the indirect effects that may be caused by an
alteration in iron homeostasis. Clearly, more work needs to be done to
resolve the connections between the complex phenotypes described
for ssq1 strains.
| |
ACKNOWLEDGMENTS |
|---|
We thank Grazia Isaya for generously providing the Yfh1 antibodies and A. Dancis for helpful suggestions.
This work was supported by the National Institutes of Health (GM27870) to E.A.C. and the Biotechnology Training Program (5T32GM08349) and the Cremer Basic Sciences Fellowship Fund (C.V.). The work of J.M. was partially supported by the Polish State Committee for Scientific Research Project 6P04A06017.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Phone: (608) 262-1358. Fax: (608) 262-5253. E-mail: ecraig{at}facstaff.wisc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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