From the Department of Medicine, Division of
Hematology-Oncology, University of Pennsylvania, Philadelphia,
Pennsylvania 19104 and the ¶ Department of Physiology, University
of Pennsylvania School of Medicine,
Philadelphia, Pensylvania 19104
Received for publication, November 27, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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J-proteins are molecular chaperones with a
characteristic domain predicted to mediate interaction with Hsp70
proteins. We have previously isolated yeast mutants of the
mitochondrial Hsp70, Ssq1p, in a genetic screen for mutants with
altered iron homeostasis. Here we describe the isolation of mutants of
the J-domain protein, Jac1p, using the same screen. Mutant
jac1 alleles predicted to encode severely truncated
proteins (lacking 70 or 152 amino acids) were associated with
phenotypes strikingly similar to the phenotypes of ssq1
mutants. These phenotypes include activation of the high affinity
cellular iron uptake system and iron accumulation in mitochondria. In
contrast to iron accumulation, Fe-S proteins of mitochondria were
specifically deficient. In jac1 mutants, like in
ssq1 mutants, processing of the Yfh1p precursor protein from intermediate to mature forms was delayed. In the genetic backgrounds used in this study, jac1 null mutants were
found to be viable, permitting analysis of genetic interactions. The
Iron is required as a cofactor for critical proteins involved in
diverse biological processes including oxidation-reduction, cellular
respiration, oxygen interaction, and metabolic conversions (1).
However, iron can also be extremely toxic. The toxicity of iron results
from interaction with reactive oxygen intermediates, generating highly
toxic hydroxyl radicals that damage nearby macromolecules, including
lipids, DNA, and proteins (2). Cells cope with this dual nature of iron
in part by homeostatic regulation of cellular iron uptake. In
Saccharomyces cerevisiae and other organisms, iron uptake is
induced by iron deprivation and repressed by iron sufficiency (3). The
components of the iron uptake system of yeast include a surface
reductase encoded by FRE1 that is regulated by iron levels
through changes in its transcription (4). We used this property of the
FRE1 gene to select mutants that failed to repress
FRE1 expression in response to iron. The types of mutants selected included mutants with perturbed cellular iron uptake (5, 6),
iron sensing (7), or intracellular iron distribution (8, 9). This last
category of mutants, which included ssq1 mutants, exhibited
misregulated activation of high affinity cellular iron uptake and
accumulation of iron within mitochondria (8). The SSQ1 gene
encodes a low abundance Hsp70 chaperone of the mitochondrial matrix
(10). We now describe the isolation of jac1 mutants with similar phenotypes using the same selection scheme. Jac1p contains a
J-domain with a signature HPD (histidine, proline, aspartate) tripeptide motif (11) predicted to mediate interaction with Hsp70s
(12). Thus, Jac1p may be the functional partner of Ssq1p with activity
in controlling iron trafficking into the cell and mitochondria.
Culotta and co-workers (11) have identified jac1
and ssq1 mutants by using a different genetic screen that
selects for improved growth of a yeast strain deficient in cytosolic
superoxide dismutase (Sod1p). Mutations of jac1 or
ssq1 were associated with deficient activities of Fe-S
proteins (11). Fe-S clusters are modular units in which iron and sulfur
are coordinated in various combinations and linked to the peptide
backbone of proteins via cysteine sulfurs. Because of their ability to
donate or accept electrons with tremendously varied range of
potentials, these clusters are involved in many fundamental biological
processes, ranging from cellular respiration to metabolic conversions
(13). Although the clusters can be synthesized non-enzymatically
in vitro (14), recent genetic and biochemical evidence
suggests that their in vivo formation is catalyzed by
specific enzymes (15-18). In bacteria, several of these proteins are
associated together on the isc operon (16), and their
precise biological roles in Fe-S cluster synthesis are the subject of
ongoing work (19-22). Homologs of Ssq1p (called HscA or Hsc66) and
Jac1p (called HscB or Hsc20) have been found on the isc
operon, suggesting a role in Fe-S cluster synthesis or maintenance (16,
23). Overexpression of these proteins is required for overexpression of
Fe-S proteins in bacteria (24). In a recent study, Vickery and
co-workers (21) demonstrated a three-way interaction of the Hsp70
(Hsc66), a J-protein co-chaperone (Hsc20), and another isc
operon-encoded protein, IscU. IscU may provide a scaffold for
assembling intermediates of the Fe-S cluster assembly process (20).
Here we characterize the phenotypes of jac1 mutants with
altered iron homeostasis and their similarity to phenotypes of
ssq1 mutants. We also demonstrate genetic interactions
between JAC1 and SSQ1.
Growth Media
Rich medium consisted of 1% yeast extract, 2% peptone, 100 µg/ml adenine, and various carbon sources. In some experiments 2%
raffinose was used (YPAR), and in other experiments 2% glucose was
used (YPAD). To induce the GAL10 promoter, the carbon source used was 2% raffinose and 0.5% galactose. Expression from the GAL10 promoter was turned off by growth in identical medium
without galactose. For experiments with different concentrations of
iron, standard defined medium with 2% raffinose was modified by
omission of iron. Medium was autoclaved and iron was added back from a filter sterilized stock of 100 mM ferric ammonium sulfate.
To achieve severe iron deprivation, defined medium without added iron
was supplemented with 10 µM chelator bathophenanthrolene disulfonate (Fluka). Methods for growing yeast, crossing strains, sporulation, tetrad dissection, and yeast transformation have been
described (25).
Yeast Strains, Mutant Selection, and Crosses
Haploid strain 61q (MAT Mutant Selection--
61q was subjected to mutagenesis by UV
irradiation to produce a mortality of 40-70%. The mutagenized
population was diluted and allowed to form colonies from single cells
on YPAD agar plates. These colonies were replicated to agar plates
containing defined medium supplemented with 10 µM copper
sulfate and 20 µM ferric ammonium sulfate. After 4 days,
some of the colonies developed papillae, which were streaked for single
clones on YPAD plates and evaluated further. The mutants derived from
61q by this procedure included UV6.3 (jac1-3,
rho°) and UV6.30 (jac1-30, rho°).
The related parental strains CM3260 (MAT Interruption of AFT1--
In strains CM3260, 8A, and 2C, the
AFT1 open reading frame
(ORF)1 was interrupted by
insertion of a URA3 marker gene at a unique HindIII site in the genome (7), creating strains
CM3260 Meiotic Mapping--
Plasmid pRS406-JAC1 was inserted at the
MscI site of the JAC1 locus in CM3262, creating
strain JM1 (URA3::JAC1). JM1 was
crossed with 8A (jac1-3, ura3-52) and
sporulated, and the tetrads were scored for non-repressed ferric
reductase (mutant iron regulatory phenotype) and Ura+
growth. All Ura+ tetrads lacked the mutant iron regulatory phenotype.
Plasmid Shuffling--
A shuffle strain for JAC1 was
constructed as follows. A diploid resistant to cycloheximide, YPH501
(cyh2/cyh2), was transformed with the construct jac1KOhis.
This heterozygous knockout
(JAC1/ Double Mutant--
The JAC1 shuffle strain 729-4B
and SSQ1 shuffle strain MK4-6 were mated, and a zygote was
picked by micromanipulation. The diploid was streaked on cycloheximide
plates to eject the pRS318 plasmids. The diploid, which became
auxotrophic for leucine, was then sporulated. Tetrads were dissected
and photographed after 5 days. The spore clones were transferred to
YPAD plates, and genotypes were analyzed for presence of
SSQ1,
Plasmids and DNA Constructions
F1 was identified by screening a yeast genomic library (27) for
correction of the slow growth and non-repressed reductase of 8A
(jac1-3). F1 contained a 5.3-kb insert in the vector YCp50. The insert consisted of bp 458483-463769 of chromosome VII and included JAC1. The JAC1 ORF with 500 bp of
flanking DNA was amplified from genomic DNA isolated from YPH501 by PCR
using Pfu polymerase and inserted into the vector
pCR2.1-TOPO (Invitrogen). Similarly, the ORFs and flanking regions were
amplified from the 8A (jac1-3) and 2C (jac1-30)
strains and inserted into the same vector. Several clones of each were
subjected to DNA sequencing. The wild-type and mutant JAC1
genomic fragments with SstI and XhoI ends were subcloned into pRS406 for integration into the genome at
StuI in the ura3-52 locus. The wild-type
JAC1 genomic fragment was also subcloned into the same sites
in pRS318 for use in plasmid shuffling. For obtaining high levels of
regulated expression, the JAC1 ORF and truncated variants
(with amino acids 1-114 or amino acids 1-32) were amplified from the
F1 plasmid with 5' BamHI and 3' XhoI sites and
cloned into pEMBlyex4-i (9). The plasmids, called GAL10-Jac1,
GAL10-jac1(1-114), and GAL10-jac1(1-32) could be linearized with a
unique StuI restriction site for integration at the
ura3-52 site. The SSQ1 ORF was inserted into the
BamHI site of the same vector and could also be linearized
with the unique StuI site. The JAC1 deletion
construct, jac1KOhis, was made by inserting a genomic fragment
containing JAC1 and flanking region between SalI
and KpnI sites into plasmid YCplac22. BamHI sites
were introduced at the start and stop of the open reading frame,
allowing for replacement of the entire ORF with a cassette containing
the HIS3 gene. The knockout construct could be released by
digestion with NotI and SstI. To delete
JAC1, diploids CM3263, YPH501 (28), and W303 (29) were
transformed with this fragment, and transformants were selected for
growth in the absence of histidine. In each case, correct integration
of the knockout was verified by PCR. For interruption/deletion of
SSQ1, a HIS3 cassette with BamHI ends
was inserted into BglII sites within a 2.2-kb
KpnI-EcoRI SSQ1 genomic fragment
carried on the vector pCRscript. For disruption of SSQ1,
digestion of this plasmid with KpnI and EcoRI was
performed, releasing a 3.5-kb fragment prior to transformation. The
pRS318-SSQ1 plasmid carried a 3461-bp EcoRI-SalI
genomic fragment containing SSQ1.
Bacterial Expression and Antibodies
For expression in bacteria, the JAC1 ORF was
amplified with 5' NdeI and 3' XhoI sites and
cloned into the corresponding sites of pET21b (Novagen), forming an
in-frame fusion with 6 histidines (His6). Expression in
BL21(DE3) was induced by 1 mM
isopropyl- Fractionation of Cells
Mitochondria were isolated as described (32). The
post-mitochondrial fraction was centrifuged at 386,000 × g for 20 min, and the supernatant was used as a cytosolic
fraction. Intact mitochondria were resuspended in 20 mM
Tris-HCl, pH 7.5, 0.6 M sorbitol. For determining the iron
content of mitochondria, wild-type or mutant yeast cells were grown to
steady state for 16 h in defined medium supplemented with varying
concentrations of iron. A small amount (100 nM) of tracer
55Fe (Amersham Pharmacia Biotech) was added during growth.
Mitochondria were isolated in the usual fashion, and an amount
equivalent to 250 µg of protein was exposed to 2% SDS in 50 µl for
10 min at 55 °C and suspended in 1 ml of scintillation mixture for
counting in a Beckman scintillation counter.
Mitochondrial Import
Radiolabeled precursors (Jac1p, Yfh1p, Put2p) were synthesized
in a cell-free translation in the presence of a mixture of [35S]methionine and [35S]cysteine (31).
Import reactions contained mitochondria (100 µg), radiolabeled
preprotein, and 4 mM ATP and 1 mM GTP (31). Following import at 20 °C for 15 min, reaction mixtures were treated with trypsin (0.1 mg/ml) for 30 min at 0 °C. The protease was inactivated, and the samples were analyzed by SDS-polyacrylamide gel
electrophoresis and exposed to film. Use of purified matrix processing protease (MPP) to test processing of mitochondrial precursor
proteins was as described (33).
Assays for Iron Uptake, Aconitase, Succinate Dehydrogenase,
and Heme Proteins
High affinity cellular iron uptake was measured as described
(5). Aconitase was assayed by measuring the formation of
cis-aconitate at 240 nm as described (34). Succinate
dehydrogenase was assayed on mitochondria lysed in 0.5% Triton X-100
by following the reduction of p-iodonitrotetrazolium violet
(INT) to INT-formazan as described (35). For detection of heme,
mitochondrial proteins (100 µg) separated by polyacrylamide gel
electrophoresis and transferred to nitrocellulose were incubated with
peroxide developer and chemiluminescent substrates (SuperSignal ECL,
Pierce) for 5 min. The covalently bound heme group of cytochrome
c (Cyc1p) and the heme group of the
bc1 complex (Cyt1p) were visualized by exposing
the blot to film (36).
Mutant Alleles of jac1 Isolated in a Screen for Mutants with
Altered Iron Homeostasis--
FRE1 encodes the major cell
surface reductase and is required for iron acquisition from ferric iron
chelates (4). The FRE1 promoter, which is repressed in
response to iron, was fused to the HIS3 coding region.
Mutants that failed to respond to iron-mediated repression were
selected for their ability to grow in the presence of iron and in the
absence of histidine (5). The most commonly selected mutants were
defective in the SSQ1 gene. Therefore, in order to avoid
selecting more of these mutants, the screen was modified by insertion
of an additional copy of SSQ1 in the haploid genome.
Two new mutants, UV6.3 and UV6.30 (Fig.
1A), were selected and found
to exhibit small colony size, non-repressing surface ferric reductase,
and increased high affinity ferrous iron uptake. The mutant phenotypes
were found to be recessive. The mutants were backcrossed with a
wild-type parental strain, and the slow growth and iron related
phenotypes segregated in a 2+:2
The wild-type allele for the mutant gene in 8A was then cloned by
complementation of the slow growth and reductase phenotypes. The
complementing activity was further localized to a minimal construct
containing only the JAC1 ORF and 500 bp of 5'- and
3'-flanking DNA. To rule out the possibility that complementation by
the JAC1-containing genomic DNA fragment could be indirect,
the JAC1 ORF with its native promoter and a URA3
marker gene were inserted into the unique MscI site of the
chromosomal JAC1 locus in a wild-type strain. This strain
JM1 (URA3::JAC1) was crossed with 8A
(jac1-3, ura3-52) and sporulated. All
Ura Null Phenotype of jac1: Viability Depends on Genetic
Background--
The severity of the truncation of the predicted
JAC1 ORF in allele jac1-30 suggested that it
would be non-functional or null. The viability of this mutant was
surprising in view of an earlier work indicating that JAC1
was an essential gene required for vegetative growth (11). To directly
address the question of essentiality of JAC1 in the CM3263
genetic background in which the mutants were selected, the entire ORF
was deleted in this diploid, and the correctness of the deletion was
verified by genomic PCR. The heterozygous knockout was sporulated and
spore clones were dissected on rich (YPAD) medium and incubated at
30 °C. All four spores formed colonies, although the histidine
prototrophs carrying the knockout constructs formed much smaller
colonies than the wild-type spore clones (Fig.
2A). The tetrad clones
containing the jac1 deletions could be maintained in
culture, although they were often overgrown by more rapidly growing
suppressor mutants. Similarly, in another yeast genetic background,
YPH501, jac1 knockouts germinated and formed small colonies
(Fig. 2A). This slow growth phenotype of the jac1
deletion was more severe when incubated at elevated (37 °C) or lower
(23 °C) temperatures (data not shown). Thus, in both of these yeast
strains, jac1 deletion was deleterious but still allowed
germination and vegetative growth. We also examined the deletion
phenotype in W303, another commonly used parental strain. In this
background, the jac1 deletion underwent germination and
formed tiny macroscopic colonies consisting of several thousand cells
(Fig. 2A, enlarged panel). However,
these clones could not be propagated. Perhaps small amounts of Jac1p
carried over from the diploid into the deletion spore clones supported
a finite number of doublings prior to growth arrest. In summary,
JAC1 is required for vegetative growth in some yeast genetic
backgrounds and not in others.
Mutant jac1 Phenotype Reconstituted by Plasmid Shuffling--
In
genetic backgrounds in which the jac1 null strain was
viable, it was extremely slow growing and accumulated suppressor mutations. To accurately assess the phenotype of mutants and to more
easily work with strains carrying mutated forms of JAC1, we
used a plasmid shuffle strategy as is used to study essential genes
(26). The phenotype of the deletion (Fig. 2B,
line 1) uncovered after plasmid shuffle was
characterized by slow growth and increased cellular iron uptake (Fig.
2B, compare lines 1 and 2).
Jac1p expressed from a galactose-induced (GAL10) promoter completely corrected the slow growth and iron uptake phenotypes of the
mutant (Fig. 2B, line 3). Jac1p
truncations were constructed corresponding to the jac1-3
(amino acids 1-114) and jac1-30 (amino acids 1-32)
mutations, and these were placed under the control of a
GAL10 promoter in the shuffle strain. When these mutated forms of Jac1p were expressed by turning on the promoter, the growth
properties and iron uptake levels remained indistinguishable from
deletion mutants (Fig. 2B, compare lines
1, 4, and 5). Under these inducing
conditions, expression of truncated Jac1p (amino acids 1-114) from the
GAL10 promoter was 3-4-fold increased compared with the
wild-type protein expressed from the native promoter (data not shown).
The results suggest that the C-terminal domain of Jac1p (amino acids
115-184) is required for protein function.
Misregulated Cellular Iron Uptake of jac1-3 and jac1-30 Mutants:
Dependence on AFT1--
Mutant strains 8A (jac1-3) and 2C
(jac1-30) exhibited activity of the FRE1-HIS3
fusion gene under iron replete repressing conditions (Fig.
3A). Surface reductase
activity likewise was not repressed by iron addition to the medium (not
shown). The parental strain and the two mutants, 8A
(jac1-3) and 2C (jac1-30), were grown in media
with different concentrations of iron. The cells were washed and high
affinity ferrous iron uptake was measured by incubation with 1 µM 55Fe radionuclide (Fig. 3B).
Under iron starvation conditions, iron transport activities of
wild-type and mutants were similar; however, exposure to iron in the
growth media progressively repressed cellular iron uptake in wild-type
cells to a much greater extent than in the mutants (Fig.
3B).
Aft1p is a DNA-binding protein that interacts with target consensus
sequences in the promoters of iron-regulated genes. In the absence of
iron, Aft1p binds to these sequences and activates transcription. Aft1p
thereby controls iron-dependent expression of the
components of the high affinity ferrous transport system (37). We
wondered if the aberrant induction of high affinity ferrous transport
observed for the jac1 mutants was mediated via Aft1p. To
address this question, the AFT1 gene was interrupted and
high affinity ferrous uptake was measured in the
jac1 Mitochondrial Iron Accumulation in jac1 Mutants Subjected to Iron
Stress--
Wild-type and jac1 mutant strains were grown in
media containing different concentrations of iron with 55Fe
added as a tracer. After 16 h of growth, mitochondria were isolated and radiolabeled iron was determined by scintillation counting. At the lowest medium iron concentration of 0.1 µM, mitochondrial iron in the wild-type was 0.36 pmol/µg of protein versus 1.0 and 1.0 for the
jac1-3 and jac1-30 mutants. These mitochondrial
iron values for the mutants are equivalent or below levels of
mitochondrial iron in wild-type strains grown in standard defined
medium (usually 1-3 pmol/µg). With increasing amounts of iron in the
growth media, jac1-3 and jac1-30 mutants
accumulated tremendously increased amounts of iron in mitochondria
(Fig. 4). For the jac1-3
mutant grown in 1, 5, or 50 µM iron, mitochondrial iron
was increased to 26, 127, or 395 pmol/µg protein. For the
jac1-30 mutant the levels were 32, 186, and 348 pmol/µg
protein, respectively. These results indicate that the
jac1-3 and jac1-30 mutations lead to abnormal
trafficking of iron to mitochondria.
Deficient Iron-Sulfur and Heme Proteins in jac1
Mutants--
Iron-sulfur proteins were evaluated in the jac1-3
and jac1-30 mutants. Aconitase (Aco1p) is a mitochondrial protein
(38), which contains a 4Fe-4S cluster required to catalyze the
conversion of citrate to isocitrate (34). Aconitase protein level and
activity in the wild-type strain were not affected by iron content of
the growth medium (Fig. 5A).
By contrast, in the mutants, aconitase protein was so severely
decreased that it was not detected by immunoblotting. The activity
assay was more sensitive, and some residual activity was noted in the
jac1 mutants. Interestingly, this residual activity was
dependent on the iron content of the mitochondria. In mitochondria
isolated from the mutants grown in low iron media, aconitase activity
was present at ~10% of wild-type levels, whereas in the mitochondria
isolated from iron stressed cells exposed to 50 µM iron
in the medium, activity was further decreased or undetectable. In
summary, aconitase protein and activity were markedly diminished in the
jac1 mutants under all growth conditions. Iron accumulation
within mitochondria could be correlated with abrogation of the small
amount of residual aconitase activity, suggesting a toxic effect.
Succinate dehydrogenase (SDH), a protein complex of the inner membrane
containing Fe-S and heme cofactors, was also evaluated (39). Like
aconitase, SDH activity was markedly decreased in the mutants under all
conditions, and the small amount of residual activity (6% or less)
appeared to be further compromised by growth in high iron media (Fig.
5B).
Iron is inserted into heme in mitochondria (40), and many heme proteins
are found in mitochondria. Therefore, the status of several heme
proteins was evaluated in the jac1 mutants. Cytochrome c (Cyc1p) and cytochrome bc1 (Cyt1p)
are nuclear-encoded mitochondrial proteins that contain covalently
bound heme. The heme in these proteins can be visualized using enhanced
chemiluminescence substrates by virtue of its intrinsic peroxidase
activity (36). Cyt1p and Cyc1p heme levels in mitochondria were
markedly decreased in the jac1-3 and jac1-30
mutants (Fig. 6), even at the lowest
medium iron concentration tested. The levels were further reduced in mitochondria with increased iron content. Likewise, mitochondrially encoded cytochrome oxidase subunit 3 protein (Cox3p) was also reduced
in the jac1-3 and jac1-30 mutants (Fig. 6).
This decrease could reflect lack of heme or destruction of heme in the
cytochrome oxidase complex, because the proteins of the complex are
destabilized in the absence of heme cofactor (data not shown).
Alternatively, this decrease of Cox3p in jac1 mutants could
result from mitochondrial DNA damage.
Cellular Depletion of Jac1p--
Jac1p was placed under the
control of the inducible GAL10 promoter, and the wild-type
allele was replaced with this regulated construct using the shuffle
strategy. As expected, under inducing conditions for the
GAL10 promoter, Jac1p was highly expressed (Fig.
7A). This strain grew well and
was phenotypically indistinguishable from the wild-type. When the
inducer was removed, Jac1p was no longer expressed and was depleted
from the cells (Fig. 7A), and yet surprisingly, even after
prolonged incubations or addition of glucose, which is known to repress
this promoter, no slowing of growth was noted (data not shown). When we
further evaluated this strain grown in the absence of inducer,
phenotypes noted for the jac1 mutants were found to be
present to variable degrees. High affinity iron uptake was increased
3-fold compared with wild-type (Fig. 7A). Aconitase and SDH
activities were decreased to 56% and 49% of wild-type levels,
respectively (Fig. 7B). In contrast with the jac1
mutants in which aconitase protein and activity were coordinately
decreased, in these Jac1p depletion experiments, aconitase activity was
decreased but Aco1p protein level was unaltered. The implication is
that inactive Aco1p was being generated. The inactive protein might
contain a 3Fe-4S cluster as has been described for oxidatively damaged
aconitase (13). Alternatively, it may contain no cluster at all
(apoprotein) due to loss of the cluster or failure to insert the
cluster into newly synthesized protein. Furthermore, in the Jac1p
depletion experiments, heme in Cyt1p and Cyc1p was not affected, as
distinguished from the jac1 mutant phenotype (Fig.
7C). Thus, the phenotypes resulting from Jac1p depletion
were more mild than the phenotypes of the jac1 mutants, perhaps because some minimal level of Jac1p was expressed from the
GAL10 promoter even under non-inducing conditions.
Mutant (jac1-3) Is Not Generally Defective in Precursor Protein
Translocation but Is Defective in Yfh1p Processing--
The J-proteins
of mitochondria, Mdj1p and Mdj2p, cooperate with the Hsp70, Ssc1p, in
mediating import and folding of precursor proteins (41, 42). To
determine if jac1 mutants were generally defective in
protein import, we compared translocation of Put2p precursor into
mitochondria isolated from wild-type and jac1-3 strains.
Put2p is a mitochondrial matrix protein involved in proline biosynthesis. The Put2p precursor was synthesized in reticulocyte lysate in the presence of [35S]methionine (Fig.
8A, lane
1). When incubated with mitochondria, a more rapidly
migrating mature form was generated due to removal of the mitochondrial
signal sequence (lane 2), and this mature form
was protected from externally added protease (lane
3). The import, processing, and generation of the mature
protected Put2p were unaffected in ssq1-4 mutant
mitochondria (lanes 4 and 5) or in
jac1-3 mutant mitochondria (lanes 6 and 7). In summary, a general import defect was not
observed, and consistent with the import results, the
steady state levels of Put2p were not diminished in jac1-3
mitochondria (see Fig. 5A).
An unusual defect in the processing of Yfh1p precursor was observed in
ssq1 mutant mitochondria (8, 43). Yfh1p, the yeast frataxin
homolog, is required for iron homeostasis (44). The precursor is
nuclear encoded, imported into mitochondria, and proteolytically
processed at the amino terminus in two steps by the matrix processing
peptidase (45, 46). In ssq1 mutants, the second processing
cleavage is impaired. Although the functional significance of this
observation is not yet clear, we reasoned that if Ssq1p functions with
Jac1p, the jac1 mutant might exhibit a similar defect in
Yfh1p processing. Therefore, Yfh1p precursor was radiolabeled (Fig.
8B, lane 1) and imported into
wild-type and mutant mitochondria. In the wild-type mitochondria, the
precursor was rapidly processed to the mature form (m) and
protected from external protease (Fig. 8B, lanes
2 and 3). The intermediate form (i)
generated by the first processing cleavage was not observed. In
ssq1 mitochondria, most of the precursor remained in this
intermediate, partially processed form (i), and this was
protected from external protease (Fig. 8B, lanes
4 and 5). In the jac1-3 mitochondria, the intermediate was again observed, reflecting delay in the second processing step of Yfh1p, although the severity of the defect was not
as great as in the ssq1 mutant (Fig. 8B,
lanes 6 and 7). In summary, both
ssq1 and jac1 mutant mitochondria do not manifest general import defects but exhibit similar defects in Yfh1p maturation.
Jac1p Localized to Mitochondria in the Wild-type and Undetected in
the Mutants--
The initial description of Jac1p noted a potential
mitochondrial leader sequence at the amino terminus of the predicted
ORF (11). To experimentally address the question of whether the JAC1 ORF includes a mitochondrial signal sequence, the Jac1p
precursor protein was synthesized and radiolabeled in reticulocyte
lysate (Fig. 9A,
lane 1). When the Jac1p precursor was incubated
with purified MPP, the protein was processed to a more rapidly
migrating form (lane 2). Incubation with
mitochondria also generated a species with identical mobility
(lane 3). The generation of the mature product
was dependent on the presence of a membrane potential and was
completely inhibited by the addition of valinomycin (lanes 4 and 6). The mature processed species formed
after import was protected from externally added protease indicating
that this is the translocated, processed form (lane
5). Thus, Jac1p preprotein is imported into mitochondria and
proteolytically processed to a mature form by MPP. Comparison of
the precursor and mature forms would be consistent with the removal of
a 13-amino acid mitochondrial signal sequence at the amino terminus of
Jac1p.
The localization of Jac1p in cells was examined. Immunoblotting of
purified mitochondria from the wild-type strain CM3260 revealed a
specific signal migrating at 23 kDa representing mature Jac1p (Fig.
9B). The abundance of Jac1p was unchanged in mitochondria isolated from wild-type yeast grown in widely different concentrations of iron, from 0.1 to 50 µM, indicating lack of iron
dependent regulation of protein levels (Fig. 9B). In the
mutants, jac1-3 and jac1-30, Jac1p was not
detected (Fig. 9B), even with overexposure of the immunoblot
(data not shown). Thus, the jac1-3 and jac1-30 mutants appeared to be null mutants. A cytoplasmic fraction was examined by immunoblotting. Jac1p and Mir1p, a mitochondrial marker protein, were not detected in this fraction, whereas 3-phosphoglycerate kinase (Pgk1p), a cytoplasmic marker protein, was present (Fig. 9C). The data indicate that, within the detection limits of
our antibody, Jac1p was found in mitochondria but not in cytoplasm.
Genetic Interaction of JAC1 and SSQ1--
The viability of
ssq1 and jac1 individual deletions provided an
opportunity to perform epistatic analysis of these two genes. Shuffle
strains carrying ssq1 and jac1 deletions were
mated. After counterselection against the covering plasmids, the double
heterozygote was sporulated and tetrads were analyzed. Many tetrads
contained four viable spore clones, and parental ditypes, non-parental
ditypes, and tetratypes were identified (Fig.
10A, columns
1, 2, and 3-5, respectively). The
Additional support for this assertion is provided by overexpression
experiments. Overexpression of Jac1p in Mutant jac1 allele mutants were isolated by their
ability to activate the FRE1-HIS3 gene fusion, an indicator
of abnormal iron homeostasis. Mutant jac1 cells failed to
repress cellular iron uptake appropriately in response to iron
exposure, and the excess iron accumulated within mitochondria. The iron
regulatory phenotype was tightly coupled with Fe-S protein deficiency.
The most straightforward explanation for the findings is that Jac1p is
involved in the biogenesis of one or more iron-sulfur protein regulators (47). Loss of Jac1p function, then, might result in failure
to properly form these regulatory Fe-S cluster proteins, which in turn
leads to failure to turn off iron uptake to the cell and iron
accumulation in mitochondria. The identity of putative Fe-S cluster
protein regulators of iron trafficking remains to be determined. Aft1p,
an iron-sensor regulator that works by controlling transcription of
target genes (7), is required for the induced cellular iron uptake of
jac1 mutants. Aft1p might itself contain an Fe-S cluster or
interact with another protein that contains a cluster. However,
mitochondrial iron accumulation does not occur with activated
AFT1 alleles, and so accumulation of mitochondrial iron may
be due to effects of mutated jac1 on an unidentified regulatory protein. IRP1 in mammals is an Fe-S protein with regulatory effects on iron trafficking (48), although an orthologous protein in
yeast has not been described.
Jac1p belongs to the class of J-domain chaperones predicted to interact
with Hsp70 proteins. We have now provided evidence to suggest that
Ssq1p is the Hsp70 partner for Jac1p. The mutant phenotypes resulting
from loss-of-function mutations in either jac1 or
ssq1 were extremely similar. Mutant alleles of both genes were selected in screens for abnormal iron homeostasis or
Hsp70s are structurally similar proteins that contain an ATP-binding
domain and a peptide-binding domain evolved to interact with unfolded
proteins (50). Family members exist in various cellular compartments
and function by binding unfolded proteins, thereby preventing
aggregation or assisting translocation (50). A chief function of
J-proteins is to enhance delivery of chaperone substrates to the
partner Hsp70. The J-protein may also enhance the ATPase activity of
the Hsp70 (51). Subsequently, after ATP hydrolysis, the peptide
substrate binds more tightly to the chaperone, allowing it time to fold
(50). However, J-proteins and Hsp70s may interact with different
unfolded protein substrates and may even exhibit independent chaperone
activities (50). The data on the synthetic effects of the mutants and
the cross suppression data are consistent with the existence of both
independent and cooperating functions for Hsp70 and J-proteins.
A key question relates to the identity of the substrates for this
specialized chaperone system. In view of the specific impairment in
various Fe-S cluster proteins in the jac1 and
ssq1 mutants, Fe-S cluster proteins might constitute
chaperone substrates. Most mitochondrial proteins, including Fe-S
proteins, are synthesized on cytoplasmic ribosomes and translocated
into mitochondria as unfolded intermediates (52). At this stage these
apoproteins probably lack Fe-S cofactors and may be prone to
aggregation. Specifically dedicated chaperones could protect the
apoprotein while an Fe-S cluster is inserted and then assist folding or
assembly of the active conformation. Alternatively, specialized
proteins that mediate Fe-S cluster synthesis might be the chaperone
substrates. Elegant work suggests that several proteins associated on
the isc operon of E. coli mediate in
vivo synthesis or maintenance of Fe-S clusters (16, 20), and these
might require special chaperones for their assembly or activation.
Recent data from Vickery and co-workers (21) show that the IscU
protein, proposed to be a scaffold on which Fe-S cluster assembly
occurs, interacts with high affinity with the HscA (homologous to
Ssq1p) and HscB (homologous to Jac1p) chaperones. This strongly
suggests that, in bacteria, IscU is a physiologic substrate, although
perhaps not the only one. In yeast, the IscU homologs, Isu1p and Isu2p, are mitochondrial proteins that are important for iron homeostasis (49). Another protein that could be a substrate for the Ssq1p and Jac1p
chaperones is Yfh1p. Yfh1p, the yeast frataxin homolog, is clearly
involved in cellular and mitochondrial iron homeostasis (44), although
the prokaryotic homolog is not located on the isc operon
(16).
Many questions remain, and an interesting unresolved question relates
to the evolutionary conservation of these chaperones. IscS and IscU,
proteins involved in Fe-S cluster formation (20), are conserved from
bacteria (isc operon) to yeast to humans (53, 54). Ssq1p and
Jac1p are related to orthologous genes on bacterial isc
operons implicated in Fe-S cluster synthesis or maintenance (16, 23).
Therefore, it is possible that Jac1p and Ssq1p chaperones will also be
found in higher eukaryotes. If human homologs of Jac1p and Ssq1p exist,
they might be important in human diseases of iron trafficking, such as
some neurodegenerative diseases (55) and sideroblastic anemias
(56-58).
jac1
ssq1 double mutant was
more severely compromised for growth than either single mutant,
suggesting a synthetic or additive effect of these mutations.
Overexpression of Jac1p partially suppressed ssq1 slow
growth and vice versa. Similar mitochondrial localization and similar
mutant phenotypes suggest that Ssq1p and Jac1p are functional partners
in iron homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, trp1-63,
leu2-3,112, gcn4-101, his3-609,
ura3-52, FRE1-HIS3::URA3,
SSQ1::LEU2) was derived from strain 61 by insertion of an additional copy of SSQ1 carried on plasmid pRS405 and integrated at the BstEII site of the
leu2-3,112 locus.
,
trp1-63, leu2-3,112, gcn4-101, his3-609, ura3-52) and CM3262
(MATa, ino1-13,
leu2-3,112, gcn4-101, his3-609,
ura3-52) were used in crosses (5). UV6.3 and UV6.30 were
backcrossed with CM3262, and spore clones were evaluated. Backcross of
UV6.3 yielded spore clones 634-3C
(FRE1-HIS3::URA3, jac1-3,
rho+) and 8A (ura3-52, jac1-3,
rho+). Backcross of UV6.30 yielded spore clones 725-3D
(FRE1-HIS3::URA3, jac1-30,
rho+) and 2C (ura3-52, jac1-30,
rho+). In prior work, mutant selection using strain 61 yielded the mutant 191-33C
(FRE1-HIS3::URA3, ssq1-4). This strain carries a mutant allele of ssq1, which is
truncated at amino acid 220 of the coding region and was previously
called ssc2-1 (8).
aft1, 8A
aft1, and 2C
aft1.
jac1::HIS3, cyh2/cyh2) was transformed with plasmid pRS318-JAC1,
carrying the JAC1 ORF, 500 bp of 5'- and 3'-flanking region
on a centromere-based plasmid with LEU2 and CYH2
genes. A tetrad clone with Leu+ and His+
growth, 729-4B, was identified and designated as a shuffle strain (MATa, ura3-52,
lys2-801(amber), ade2-101(ochre),
trp1-
63, his3-
200,
leu2-
1,
jac1::HIS3
(pRS318-JAC1)). A shuffle strain for SSQ1 was constructed in
analogous fashion. The SSQ1 gene was interrupted with
HIS3 in the YPH501 (cyh2/cyh2) diploid.
This diploid was transformed with plasmid pRS318-SSQ1 sporulated, and a
tetrad clone with Leu+ and His+ growth, MK4-6,
was identified and used as a shuffle strain (MAT
, ura3-52, lys2-801(amber),
ade2-101(ochre), trp1-
63,
his3-
200, leu2-
1,
jac1::HIS3
(pRS318-SSQ1)). For counterselection against the presence of the
plasmid pRS318, cells were exposed to 10 µg/ml cycloheximide added to
agar plates containing growth media (26).
ssq1::HIS3,
JAC1, and
jac1::HIS3 alleles using
genomic PCR. The primers for this analysis were as follows. For
JAC1, primer 011700B (5'-GACGCAAGAACAGACCTC-3') and primer
041100A (5'-CGACCACAAGGAATTTGGAAC-3') produced a product of 802 bp. For
jac1, primer 101499E
(5'-CGCTCACCAAGCTCTTAAAACG-3') and primer 041100A (see above) produced
a 539-bp product. For SSQ1, primer 021198C
(5'-CCTGAAACAAACTTCAG-3') and 013098B (5'-CACCCGCTTGCCCATCAACA-3') produced a 928-bp product. For
ssq1, primer
101499E (see above) and 013098B (see above) produced a 275-bp product.
-D-1-thiogalactopyranoside for 3 h at
37 °C. The overexpressed Jac1p-His6 fusion protein was
isolated from inclusion bodies, purified on a nickel-nitrilotriacetic acid column (Qiagen), and eluted with 50 mM Tris-HCl, pH
8.0, 8 M urea, 0.4 M imidazole, and 5 mM
-mercaptoethanol. The eluate was analyzed by
SDS-polyacrylamide gel electrophoresis and found to contain a major
band by staining with Coomassie Blue. The band was excised from the gel
and used to inject rabbits for generation of polyclonal antibodies. The
carboxyl-terminal 109 amino acids of the SSQ1 ORF was
expressed in pET21b with His6 tag, purified from the
soluble fraction of the bacterial lysate using the
nickel-nitrilotriacetic acid column as above, and used to immunize
rabbits. Antibodies directed against Aco1p (30), Mir1p, and Por1p (31)
have been described previously. Commercially available antibodies used
in some experiments included mouse monoclonal antibodies directed against Cox3p or Pgk1p (3-phosphoglycerate kinase) (Molecular Probes).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pattern in the tetrads, consistent
with involvement of a single nuclear locus. Crosses revealed that the
two independent isolates contained allelic mutations.
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Fig. 1.
Panel A, strain nomenclature.
The original mutant isolates obtained from the selection scheme were
named UV 6.3 and UV6.30. After backcrossing to the wild-type, tetrad
clones 8A and 2C were identified containing mutant alleles, called
jac1-3 and jac1-30, respectively. The alleles
were sequenced and the mutation in jac1-3 was found to
alter nucleotide 344 of the coding from T to G, generating a stop codon
at position 115. The mutation in jac1-30 was found to alter
nucleotide 97 from C to T, generating a stop codon at position 33. Panel B, domain structure of Jac1p. The predicted domain
structure of Jac1p includes a mitochondrial signal sequence
(S), J-domain (J), and carboxyl-terminal domain
of unknown function. The J-domain includes the characteristic HPD
(histidine, proline, aspartate) at residues 48-50. The locations of
jac1-3 and jac1-30 mutations are depicted by
upward arrows. The domain structure of the
bacterial J-protein Hsc20 is shown. Identity refers to the percentage
of amino acid identity between Hsc20 and Jac1p in the J domain or
C-terminal domain.
tetrad clones exhibited slow growth and
non-repressing reductase (mutant phenotype), indicating absence of
recombination between the marked wild-type JAC1 and the 8A
mutant. Hence, meiotic mapping also supported that the mutation in 8A
responsible for the mutant phenotypes was in JAC1 or closely
linked to this locus. The mutant alleles were rescued from strains 8A
(jac1-3) and 2C (jac1-30) by PCR of the
JAC1 ORF and flanking regions. DNA sequence analysis revealed single nucleotide changes in each case. For the
jac1-3 allele, nucleotide 344 was changed from T to G,
altering codon 115 from TTA (leucine) to TGA (Stop). The
jac1-3 mutant allele codes for a truncated protein lacking
its carboxyl one-third. For the jac1-30 allele, nucleotide
97 was changed from C to T, altering codon 33 from CAA (glutamine) to
TAA (Stop), and is thereby predicted to code for a severely truncated
protein (Fig. 1).
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Fig. 2.
Panel A, null phenotype of
jac1 depends on genetic background. Heterozygous deletions
of JAC1 were constructed in diploid strains: YPH501, CM3263,
and W303. These strains were sporulated; tetrads were dissected,
arrayed in vertical columns on rich medium agar plates, and incubated
at 30 °C for 4 days. In the YPH501 panel,
column 5 was not a true tetrad. In the
W303 panel, column 4 (tetrad 4) was photographed at higher magnification and this is shown
in the right-hand panel. Panel B,
JAC1 alleles examined by plasmid shuffling. JAC1
shuffle strain, 729-4B
( jac1::HIS3,
(pRS318-JAC1)), was transformed with various constructs
targeted to the ura3-52 locus. These constructs were:
1, pRS406, empty vector; 2, pRS406-JAC1,
wild-type JAC1 allele including the native promoter;
3, GAL10-Jac1, JAC1 ORF under the control of the
GAL10 promoter; 4, GAL10-jac1-(1-114), truncated
Jac1p of 114 amino acids under the control of the GAL10
promoter; 5, GAL10-jac1-(1-96), truncated Jac1p of 96 amino
acids under the control of the GAL10 promoter. The
transformants were analyzed by plating 5-fold dilutions of 1 × 105, cells in a 5-µl volume on agar plates containing
YPAR supplemented with 0.5% galactose to induce the GAL10
promoter and 10 µg/ml cycloheximide to counterselect against the
covering plasmid. Plates were incubated at 30 °C for 4 days before
photography. For measurement of high affinity ferrous iron uptake,
cells were taken from the photographed plates and grown under inducing
conditions (YPAR with 0.5% galactose) prior to assay of high affinity
ferrous iron uptake using 55Fe radionuclide.
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Fig. 3.
Panel A, misregulation of
FRE1 in jac1-3 and jac1-30 mutants.
Strains carrying the FRE1-HIS3 construct integrated into the
genome of a his3 strain were evaluated for
their ability to grow on standard defined medium (1 µM
iron) in the absence or presence of histidine. Strains 61q
(JAC1), 634-3C (jac1-3), and 725-3D
(jac1-30), all of which contained the FRE1-HIS3
construct integrated into the genome, were evaluated for their ability
to grow on standard defined medium in the absence or presence of
histidine. Growth of jac1-3 and jac1-30 mutants
on the histidine-free plate reflects activity of the
FRE1-HIS3 fusion under iron replete conditions. Panel
B, misregulated cellular iron uptake in jac1 mutants.
Yeast strains CM3260 (JAC1), 8A (jac1-3), and 2C
(jac1-30) were grown in modified defined medium. This
medium contained glucose as a carbon source and iron chelator
bathophenanthrolene disulfonate (10 µM) or different
concentrations of iron (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 µM) added as ferric ammonium sulfate. The cells were
grown to stationary phase, diluted 10-fold into media of the same
composition, and allowed to enter logarithmic growth prior to assay of
high affinity ferrous iron uptake using 55Fe radionuclide.
Panel C, increased high affinity ferrous iron uptake in the
jac1 mutants is AFT1-dependent.
Strains CM3260 (JAC1), 8A (jac1-3), and 2C
(jac1-30) were each transformed with a construct to
interrupt the AFT1 gene. Strains were grown in YPAD, and
iron uptake was measured.
aft1 double mutants. The results showed
equivalent low levels of uptake for all strains lacking
AFT1, regardless of the absence or presence of
jac1 mutations (Fig. 3C). Therefore, the
increased cellular iron uptake due to jac1 mutations was
completely dependent on an intact copy of AFT1.
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Fig. 4.
Accumulation of mitochondrial iron in
jac1 mutants. Yeast strains CM3260
(JAC1), 8A (jac1-3), and 2C
(jac1-30) were grown in defined medium containing 2%
raffinose and varying concentrations of iron (0.1, 1.0, 5.0, and 50 µM). 55Fe radionuclide was added as a tracer,
and, after 16 h of growth, mitochondria were isolated and the iron
content determined by scintillation counting of radionuclide in the
samples. The iron content is displayed as picomoles of iron per
microgram of mitochondrial protein.
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Fig. 5.
Panel A, deficiency of
aconitase protein and activity in jac1 mutants. Aconitase
activity was assayed in mitochondria isolated from yeast strains CM3260
(JAC1), 8A (jac1-3), and 2C
(jac1-30) grown in defined medium with different iron
concentrations (0.1, 1.0, 5.0, and 50 µM). The units of
activity are nanomoles of cis-aconitate/mg of mitochondrial
protein/min. The aconitase protein level (Aco1p, 85 kDa) was assayed by
immunoblotting with a monospecific antibody.
-1-Pyrroline-5-carboxylate dehydrogenase (Put2p, 61 kDa), a
mitochondrial matrix protein, was evaluated as a control. Panel
B, deficiency of SDH activity in jac1 mutants. SDH was
assayed in the various mitochondria described in panel A.
The units of activity are nanomoles of INT-formazan/mg of mitochondrial
protein/min. Mean and standard deviations of three replicate assays for
aconitase and SDH activity are depicted.
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Fig. 6.
Deficiency of heme proteins in
jac1 mutants. Mitochondria were isolated from
yeast strains CM3260 (JAC1), 8A (jac1-3), and 2C
(jac1-30) grown in defined medium and exposed to varying
concentrations of iron (0.1, 1.0, 5.0, and 50 µM). Porin
(Por1p, 31 kDa), a mitochondrial outer membrane protein, was evaluated
as a control. Heme groups in cytochrome c (Cyc1p heme, 12 kDa) and the bc1 heme complex of cytochrome
c1 (Cyt1p heme, 31 kDa) were evaluated by heme
blotting. Cytochrome oxidase subunit 3 (Cox3p, 24 kDa) was assayed by
immunoblotting with a monoclonal antibody.
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Fig. 7.
Depletion of Jac1p. The JAC1
ORF was placed under the control of the GAL10 promoter and
the wild-type allele in YPH499 was replaced with this regulated
construct, GAL10-Jac1. The wild-type strain, YPH499, and the GAL10-Jac1
strain were grown in non-inducing medium (2% raffinose, no galactose)
for 96 h. During this period, cells were maintained in logarithmic
growth by dilution once each day. Panel A, Jac1p was
evaluated by immunoblotting on purified mitochondria, and iron uptake
was measured on whole cells. Panel B, aconitase protein
(Aco1p) was assayed by immunoblotting; aconitase activity and SDH
activity were assayed on purified mitochondria. Panel C,
heme cofactor in cytochrome c (Cyc1p heme) and cytochrome
c1 of the bc1 complex
(Cyt1p heme) were evaluated by heme blotting.
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Fig. 8.
Panel A, import of Put2p into
isolated mitochondria. The Put2p precursor
( -1-pyrroline-5-carboxylate dehydrogenase) was synthesized in
reticulocyte lysate in the presence of [35S]methionine.
The precursor (lane 1) was incubated with 100 µg of isolated mitochondria (Mito) from strains 61 (WT, lanes 2 and 3),
191-33C (ssq1-4, lanes 4 and
5), or 8A (jac1-3, lanes 6 and 7). After 15 min, samples were exposed to 0.1 mg/ml
trypsin (Trypsin, lanes 3,
5, and 7), and the protease was inactivated. The
mitochondria were retrieved by pelleting at 12,000 × g
and analyzed by SDS-polyacrylamide electrophoresis. The Put2p precursor
(p) and mature forms (m) are indicated.
Panel B, the Yfh1p precursor radiolabeled by synthesis in
reticulocyte lysate (lane 1) consisted of the
full-length product (p) and a truncated form arising from
internal initiation (*). The mitochondrial import assay was performed
as described in panel A. Yfh1p precursor was processed in
two steps, and in ssq1-4 and jac1-3 mutant
mitochondria, both the intermediate form (i) and mature
(m) forms were observed.
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Fig. 9.
Panel A, import and
processing of Jac1p precursor into isolated mitochondria. The
radiolabeled Jac1p precursor was treated with MPP (lane
2) or incubated with 100 µg of wild-type mitochondria
(lanes 3-6). In some cases, 5 µg/ml
valinomycin (Val, lanes 4 and
6) was added to the mitochondria prior to the addition of
the precursor. After 15 min, samples were exposed to 0.1 mg/ml trypsin
(Trypsin, lanes 5 and 6),
and the protease was inactivated. The mitochondria were retrieved by
pelleting at 12,000 × g and analyzed by
SDS-polyacrylamide electrophoresis. The Jac1p precursor (p)
and mature forms (m) are indicated. Panel B,
Jac1p unregulated by iron in the wild-type and undetectable in the
jac1 mutants. Mitochondria were isolated from strains CM3260
(JAC1), 8A (jac1-3), and 2C
(jac1-30) grown in 0.1, 1, 5, or 50 µM iron
added to modified defined medium. The mitochondria were probed with
antibodies to Jac1p or Put2p ( -1-pyrroline-5-carboxylate
dehydrogenase). Jac1p migrated at 23 kDa, and Put2p migrated at 61 kDa.
Panel C, localization of Jac1p in cells. A cytoplasmic
fraction (C) was compared with a mitochondrial fraction
(M) isolated from wild-type cells (YPH499). These fractions
were evaluated by immunoblotting with antibodies against Jac1p (23 kDa), Mir1p (32 kDa), a protein of the mitochondrial inner membrane,
and Pgk1p (45 kDa), 3-phosphoglycerate kinase, a cytoplasmic
protein.
ssq1 spore clones formed small colonies
(
s),
jac1 spores
formed smaller colonies (
j), and the double
mutants formed tiny, albeit viable colonies (
). In
many cases, the double mutant spore clones did not grow after
inoculation of liquid or solid media cultures. Thus, the double mutants
were clearly more compromised than either of the single mutants in
terms of viability and growth. The data suggest that the two genes may
serve some overlapping and some non-overlapping functions.
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Fig. 10.
Genetic interactions of SSQ1
and JAC1. Panel A, synthetic
effects of combining jac1 and
ssq1 mutations. Shuffle strains for
JAC1 and SSQ1 were mated, and the diploid was
treated with cycloheximide to counterselect against the covering
plasmids for both genes. The diploid was sporulated, and PCR was used
to analyze the tetrads for segregation of JAC1,
jac1::HIS3,
SSQ1, and
ssq1::HIS3 alleles.
Typical tetrads are shown: 1 (parental ditype), 2 (non-parental ditype), and 3-5 (tetratypes) The
corresponding mutant genotypes are shown in the table below:
j
(
jac1::HIS3),
s
(
ssq1::HIS3),
(
jac1::HIS3,
ssq1::HIS3). Panel
B, overexpression effects. The SSQ1 shuffle strain
(MK4-6) was transformed with integrating plasmids linearized at
StuI and targeted to the ura3-52 locus: pRS406
(1), pRS406-SSQ1 (2), GAL10-Ssq1 (3),
or GAL10-Jac1 (4). The JAC1 shuffle strain
(729-4B) was similarly transformed with the integrating plasmids:
pRS406 (5), pRS406-JAC1 (6), GAL10-Ssq1
(7), and GAL10-Jac1 (8). The transformants were
suspended in water, and 5-fold dilutions of 105 cells were
plated on agar plates containing 2% raffinose, 0.5% galactose, and 10 µg/ml cycloheximide to counterselect against the covering plasmids.
Panel C, strains YPH499 (1) or YPH499 transformed
with GAL10-Ssq1 (2) or GAL10-Jac1 (3) were grown
in medium containing 2% raffinose and 0.5% galactose. Strains 61 (4), 191-33C (ssq1-4) (5), and 8A
(jac1-3) (6) were grown in medium containing 2%
raffinose. Mitochondria were isolated and analyzed by immunoblotting
with antibodies to Ssq1p, Jac1p, and Put2p.
ssq1 mutants clearly suppressed the slow growth phenotype (Fig. 10B,
compare 1 versus 4). Overexpression of
Ssq1p in
jac1 mutants also enhanced growth,
but the effect was not as marked (Fig. 10B, compare
7 to 5). Approximately 3-fold overexpression of
Jac1p was achieved without overexpression of Ssq1p, and vice
versa overexpression of Ssq1p was achieved without
overexpression of Jac1p (Fig. 10C). In the mutant 33C
(ssq1-4), Ssq1p was undetectable without alteration of the
Jac1p level, and in strain 8A (jac1-3), Jac1p was
undetectable without alteration of the Ssq1p level (Fig.
10C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sod1 suppression (11). The iron trafficking
abnormalities were similar for both mutants, characterized by induction
of high affinity cellular iron uptake and accumulation in mitochondria.
Mutants of jac1 and ssq1 (11, 49) exhibited
multiple Fe-S protein deficiencies. Protein translocation into
jac1 or ssq1 (10) mutant mitochondria was not
generally impaired. However, a delay in the second processing cleavage
of translocated Yfh1p was observed for both mutant mitochondria in
in vitro import assays. Finally, genetic evidence for the
interaction exists. The double mutant (
ssq1
jac1) was even more slow growing than either single
mutant, indicating an additive or synthetic effect. The overexpression of Jac1p was able to significantly compensate for the slow growth of
the
ssq1 mutant, and reciprocally the
overexpression of Ssq1p was able to slightly compensate for the slow
growth of the
jac1 mutant. Finally, both Ssq1p
(10) and Jac1p have been localized to mitochondria and thus reside in
the same cellular compartment, as would be required for them to interact.
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FOOTNOTES |
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* This work was supported by Hematology Clinical Research Grant T32 HL 07439-21 (to R. K.), National Institutes of Health Grant DK53953 (to A. D.), National Institutes of Health Grant GM57067 and American Heart Association Grant 9951300U (both to D. P.), and National Research Service Award Fellowship NS11166 (to D. M. G.).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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
215-573-6275; Fax: 215-573-7049; E-mail:
adancis@mail.med.upenn.edu.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M010695200
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ABBREVIATIONS |
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The abbreviations used are: ORF, open reading frame; PCR, polymerase chain reaction; SDH, succinate dehydrogenase; MPP, matrix processing peptidase; bp, base pair(s); kb, kilobase pair(s); INT, p-iodonitrotetrazolium violet.
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1. | Brittenham, G. (2000) in Hematology: Basic Principles and Practice (Hoffman, R. , Benz, E. , Shattil, S. , Furie, B. , Cohen, H. , Silberstein, L. , and McGlave, P., eds) , pp. 397-428, Churchill Livingstone, Philadelphia |
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