From the Department of Biochemistry and the
Graduate Program in Biochemistry, Cell and Developmental
Biology, Rollins Research Center, Emory University School of Medicine,
Atlanta, Georgia 30322-3050
Received for publication, February 10, 2003, and in revised form, March 10, 2003
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ABSTRACT |
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The amino-terminal domain (ATD) of
Saccharomyces cerevisiae mitochondrial RNA polymerase has
been shown to provide a functional link between transcription and
post-transcriptional events during mitochondrial gene expression. This
connection is mediated in large part by its interactions with the
matrix protein Nam1p and, based on genetic phenotypes, the
mitochondrial membrane protein Sls1p. These observations led us to
propose previously that mtRNA polymerase, Nam1p, and Sls1p work
together to coordinate transcription and translation of mtDNA-encoded
gene products. Here we demonstrate by specific labeling of
mitochondrial gene products in vivo that Nam1p and Sls1p
indeed work together in a pathway that is required globally for
efficient mitochondrial translation. Likewise, mutations in the ATD
result in similar global reductions in mitochondrial translation
efficiency and sensitivity to the mitochondrial translation inhibitor
erythromycin. These data, coupled with the observation that the ATD is
required to co-purify Sls1p in association with mtDNA nucleoids,
suggest that efficient expression of mtDNA-encoded genes in yeast
involves a complex series of interactions that localize active
transcription complexes to the inner membrane in order to coordinate
translation with transcription.
The ~80-kb Saccharomyces cerevisiae mitochondrial
genome encodes seven oxidative phosphorylation subunits destined for
the inner mitochondrial membrane, one ribosomal protein, two rRNAs, and
a full complement of tRNAs (1). Transcription of yeast mtDNA is
initiated at multiple promoters by a dedicated mtRNA polymerase that is
encoded by the RPO41 gene (2) and homologous to the
bacteriophage T7 family of RNA polymerases (3). Most mitochondrial
transcripts are polycistronic and require extensive processing to
release the mature RNA species for proper gene expression (4). In
addition, introns are often present in certain messages that also must
be excised. In the case of the COX1 and COB
genes, this is a complex process that requires intron-encoded maturases that help catalyze splicing (5, 6). Once fully processed, mRNAs are
translated within the matrix by ribosomes associated with the
mitochondrial inner membrane (7, 8). Finally, the newly synthesized
mtDNA-encoded proteins are assembled with the corresponding
nucleus-encoded subunits to form the oxidative phosphorylation enzyme
complexes in the inner membrane. The execution and coordination of
these events are necessary for normal cellular respiration.
We previously examined the mechanism of mitochondrial gene expression
in the budding yeast S. cerevisiae and elucidated a pathway
of transcription-coupled events that involves an amino-terminal domain
(ATD)1 of mtRNA polymerase
(9-11). Over the course of these experiments, several mutations in the
yeast mtRNA polymerase ATD have been characterized, including two
deletion mutations, rpo41 Analogous in some ways to the carboxyl-terminal domain of RNA
polymerase II (13), the ATD of mtRNA polymerase is the binding site for
at least one factor involved in post-transcriptional events.
Specifically, it is the interaction point for Nam1p (10), a protein
involved in RNA processing and translation (14-16). Two additional
lines of evidence support a functional role for this interaction in
coordinating mitochondrial translation. First, nam1 Media--
Yeast were grown as described (19) in complete YPG
(glycerol) medium, YPD (dextrose) medium, or synthetic dextrose (SD) medium with the necessary nutritional supplements as indicated. The
bacto-yeast extract, dextrose, and yeast nitrogenous base (without
amino acids) were obtained from Difco. Glycerol and peptone were
obtained from Fisher.
Yeast Strains and Plasmid Construction--
The yeast strains
used in this study were derived from DBY2006 ( Radiolabeling of Mitochondrial Translation Products in
Vivo--
An overnight culture (2 ml) of each yeast strain was grown
to saturation in YPG (or YPD for petite mutants) medium and used to
inoculate a 15-ml SD culture supplemented with the appropriate amino
acids. These cultures were then incubated at 30 °C for 2 h
prior to overnight incubation at 37 °C. These cultures were then
diluted to an A600 of 0.6 with SD medium. After
incubation at 37 °C for 2.5 h, cycloheximide (Spectrum
Laboratory Products, Inc.) was added to 250 µg/ml in 10 ml of each
culture, and the incubation was continued for 5 min prior to the 15-min
incubation with 100 µCi of [35S]methionine (PerkinElmer
Life Sciences). Labeling was stopped by addition of 4 ml of 1 mM Na2SO4 and 1% casamino acids,
followed by an additional 10-min incubation. The cells were harvested
by centrifugation, and mitochondria were prepared as described (20). The procedure was identical for labeling done at 30 °C except that
cycloheximide was added to a concentration of 150 µg/ml. Mitochondrial protein concentration was determined using the Bio-Rad Protein Assay kit. Mitochondrial protein (15 µg) was separated on a
17% SDS-PAGE gel. The gel was then treated with Enlightening (PerkinElmer Life Sciences), dried, and exposed to x-ray film for
40-72 h.
Erythromycin Sensitivity Assays--
Yeast strains were grown at
30 °C to mid-log phase in 5 ml of liquid YPG media. Cultures were
then diluted to an A600 of 0.4 and spotted onto
solid YPG medium containing the either 0, 5, 10, 20, or 50 µg/ml of
erythromycin (Sigma), using a 48-pin multiplex plating tool. The plates
were then incubated at 36 or 30 °C for 6 days prior to the
assessment of growth phenotypes. Complete lack of growth was scored as
sensitivity to the drug.
Petite Induction Assays and mtDNA Southern Analysis--
Yeast
strains were set up and grown overnight at 37 °C and diluted to an
A600 of 0.6 with SD medium as they were in the
radiolabeling experiments. The cultures were then incubated at 37 °C
for 3 h. These cultures were then diluted and plated for the
petite induction assay, as described previously (21). Statistical
analysis was applied to identify outlying data points (22). By using
this criterion, one data point for rpo41 Western Immunoblot Analysis--
Yeast cultures were grown and
mitochondria prepared as they were for the in vivo
translation assay described above, except that the cultures were not
incubated with cycloheximide or [35S]methionine.
Mitochondrial protein was separated by SDS-PAGE and transferred to
nitrocellulose membranes by standard protocols (23), and Western
analysis was performed as described in the ECL Western blotting
detection kit protocol (Amersham Biosciences), using anti-Cox2p,
anti-Cox1p, and anti-porin antibodies (Molecular Probes).
Translation in Organello and Isolation of Mitochondrial
Nucleoids--
Mitochondria competent for in organello
translation were prepared and stored as described (24). Mitochondria (2 mg of mitochondrial protein/sample) were thawed, and in
organello translation reactions were performed as described (24),
except 20 mM HEPES was substituted for Tris in the protein
synthesis medium. ATP, GTP, Sls1p and Nam1p Collaborate in a Pathway Required for Optimal
Mitochondrial Translation--
We identified previously (9)
SLS1 as a genetic suppressor of the mitochondrial petite
phenotypes of mtRNA polymerase ATD and NAM1 null
(nam1
Although sls1 Mutations in the ATD of mtRNA Polymerase Cause a Global Decrease in
Mitochondrial Translation--
To test the hypothesis that, like
sls1
The [35S]-labeling assays require that the cultures be
grown in synthetic glucose medium, a condition that allows growth of cells that have lost wild-type (rho+) mtDNA. Because mtRNA
polymerase is required for rho+ mtDNA maintenance (1, 2),
in principle, the translation defects observed in the in
vivo labeling assays could result from a decrease in the number of
rho+ genomes. To assess the degree of mtDNA instability in
the ATD mutant strains under these conditions, cultures were grown in the same manner as the [35S] incorporation assays and
quantitated for the amount of spontaneous petite formation (Table
I), which is largely a measure of
rho
To confirm that the observed translation defect results in decreased
steady-state levels of mtDNA-encoded proteins in the rpo41
ATD mutant strains, Western blot analysis was performed on
mtDNA-encoded Cox1p and Cox2p. Analysis of total mitochondrial protein
revealed that all rpo41 ATD mutants accumulated reduced steady-state levels of both proteins, whereas the amount of the nucleus-encoded mitochondrial protein porin was apparently unaffected (Fig. 3A). These data
demonstrate that mutations in the ATD perturb mitochondrial
translation, resulting in accumulation of less mtDNA-encoded protein as
expected.
Finally, to assess independently whether the rpo41 ATD
mutations affect mitochondrial translation, their sensitivity to the mitochondrial translation inhibitor erythromycin was measured. The
yeast cultures used in this assay were maintained on glycerol medium
throughout the experiment, selecting for maintenance of rho+ genomes. With the exception of the
rpo41-E119A-C121A mutation, all of the rpo41 ATD
mutants are hypersensitive to the mitochondrial translation inhibitor
erythromycin at 36 °C (Fig. 3B). Consistent with results
of the in vivo labeling experiments (Fig. 2A) and the steady-state analysis (Fig. 3A), the
rpo41-N152A/Y154A strain was by far the most
sensitive to the drug.
Sls1p Co-purifies with Mitochondrial Nucleoids in an mtRNA
Polymerase ATD-dependent Fashion--
The ability of Sls1p
to suppress nam1 Overexpression of Sls1p Increases the Steady-state Levels of
mtDNA-encoded Proteins in rpo41 ATD Mutant Strains Independently of Its
Role in Translation--
Because Sls1p overexpression suppresses the
translation defect in the nam1 A large body of evidence now indicates that mitochondrial
translation is primarily, if not exclusively, a membrane-associated process (7, 8, 26-29). In yeast, this is a complex process that
involves not only membrane-associated ribosomes but also mRNA-specific translational activators and numerous other proteins involved in processing and stability of mRNA that are directly or
indirectly associated with the inner membrane (4, 7). Our previous work
(9-11) in this area suggested that mtRNA polymerase is also
intricately involved in the efficiency of translation not only
directly, through synthesizing the requisite RNA species, but also via
functions of the ATD in coordinating transcription with
post-transcriptional events. In this report, we have elucidated a role
for Sls1p in mitochondrial translation, and we provide additional new
lines of evidence that support a model in which one function for the
ATD of mtRNA polymerase is to nucleate a series of interactions
involving Nam1p and Sls1p that are ultimately required to link mtRNA
polymerase to the inner membrane to facilitate efficient mitochondrial
protein synthesis. The data supporting these conclusions are discussed below.
A recent key observation that led to our initial proposal that the ATD
of mtRNA polymerase is involved in coupling transcription to
membrane-associated events is the ability of the membrane protein Sls1p
to rescue the petite phenotype of nam1 Next, we investigated whether the ability of Sls1p to rescue a
nam1 The elucidation that Sls1p is involved in translation in cooperation
with Nam1p (Fig. 1) suggested to us that one function of the pathway of
gene expression events involving Nam1p, Sls1p and the ATD that we have
described previously (9, 10) is to facilitate delivery of transcripts
to the translation machinery at the inner mitochondrial membrane. Two
additional lines of evidence provided by this study support this
conclusion. First, all mtRNA polymerase ATD mutations examined here
resulted in translation-related defects, including a significant
reduction in labeling of all mtDNA-encoded proteins in vivo
(Fig. 2A), reduced steady-state levels of the mtDNA-encoded
proteins Cox1p and Cox2p (Fig. 3A), and, with the exception
of the rpo41-E119A-C121A mutation (discussed later), an
increased sensitivity to the mitochondrial translation inhibitor
erythromycin (Fig. 3B). That the three ATD mutations that have translation defects in all three assays
(rpo41 Based on these data, we propose a revised model for mitochondrial gene
expression involving these factors (Fig.
6). In this model, Nam1p is predicted to
bind to the ATD of mtRNA polymerase to facilitate the interaction of a
transcriptionally active, nucleoid-associated mtRNA polymerase with
Sls1p at the inner mitochondrial membrane. Once this connection is
established between mtRNA polymerase and Sls1p (which may involve
additional factors), translation of the mRNA is accomplished in a
transcription-coupled manner. The precise functions of Nam1p and Sls1p
remain to be elucidated. However, based on the recent report by Fox and
colleagues (18), Nam1p may facilitate interactions between the nascent
mRNA and COX-specific translation
activators. If this were the case, then it is tempting to speculate
that, once a functionally coupled transcription/translation complex is
fully established, Nam1p would dissociate in order to locate another
template-bound mtRNA polymerase that has yet to be membrane-coupled. A
transient nature to the Nam1p interactions is postulated based
on the fact that Nam1p is not found as a nucleoid component
(see Ref. 25; this study, data not shown) and is localized primarily to
the matrix in mitochondrial fractionation studies (30). At present we
speculate that the most likely function for Sls1p in this regard is to
serve as part of a membrane-anchoring point for mtRNA polymerase during
active gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and rpo41
3, and
several single or double point mutations, which result in mitochondrial
petite phenotypes and defects in mitochondrial RNA processing and/or
mtDNA stability (10, 11). Specifically, the steady-state levels of
mature mRNA from the intron-containing COB and
COX1 genes are severely decreased in these strains, whereas those of an intronless gene, COX3, are minimally affected
(10). These intron-processing defects suggest that the ATD affects
translational efficiency (9) because studies using
mitochondria-specific translation inhibitors cause the same defects
in vivo (12). In addition, the minimal defects in
COX3 transcript levels in the rpo41 ATD mutants,
along with S1 nuclease protection assays of the ori5
promoter in an rpo41
3 strain (11), suggest that the
catalytic activity and initiation properties of mtRNA polymerase are
not drastically altered.
mutations result in similar, yet more severe, COX1 and
CYTB intron-processing defects as ATD mutations (10).
Second, overexpression of the mitochondrial membrane protein Sls1p
suppresses the respiration-deficient petite phenotype of both
nam1
and mtRNA polymerase ATD mutations, indicating that
mtRNA polymerase, Nam1p, and Sls1p exist in an ordered genetic pathway
required for normal mitochondrial gene expression (9). Based on these
observations and on the proposed function of Sls1p in
post-transcription events (17), we hypothesized that these three
factors comprise important components of a mitochondrial RNA-handling
pathway that targets newly synthesized transcripts to the inner
membrane (9), where translation of mtDNA-encoded messages occurs (7,
8). Entirely consistent with this model is the recent identification of
interactions between Nam1p and the COX-specific
translational activator complex (18), implicating Nam1p as a potential
envoy between mtRNA polymerase and the translation machinery. In this
study we have demonstrated that Sls1p functions globally in
mitochondrial translation, and we provide multiple new lines of
evidence that the ATD of mtRNA polymerase is a focal point for critical
interactions with Nam1p and Sls1p that act in concert to coordinate
transcription and translation at the mitochondrial inner membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
his3-
200
leu2-3, -112 ura3-52 trp1-
1 ade2). With the exception of MSR107
(GS122 + [YEp352-V5SLS1]) and MSR112 (GS125 + [YEp352-V5SLS1]), all yeast strains have been described
previously (9, 10). The YEp352-V5SLS1 vector was created as
follows. The SLS1 gene was PCR-amplified using a 5'-primer
that annealed to a sequence 200 bp upstream of the SLS1
translation start site and a 3'-primer complementary to the 3'-end of
the SLS1 open reading frame, which also contained the V5
epitope and a SacI site at its 5'-end. The PCR product was
then ligated into the pGEM-T vector (Promega), from which the V5
epitope-SLS1 fusion allele was excised on a PstI-SacI fragment and ligated into YEp352 that
was digested with the same restriction enzymes.
2 was omitted.
Total cellular DNA was isolated from cultures grown in the same manner
for Southern blot analysis. The total DNA was digested with
NdeI and separated on a 0.8% agarose gel for Southern
analysis. The Southern blots were hybridized sequentially with
radiolabeled probes to mitochondrial encoded COX1 and
COX3 and nuclear encoded ACT1 as a normalization control.
-ketoglutarate, phosphoenolpyruvate, and
pyruvate kinase (all obtained from Sigma) comprise the
energy-regenerating system in the protein synthesis medium.
Mitochondria were resuspended in 1 ml of ice-cold protein synthesis
medium, and pyruvate kinase was added, and the sample was incubated at
room temperature for 10 min prior to the addition of 400 µM of the cross-linking reagent dithiobis(succinimidyl propionate) (Pierce). After incubation for 20 min, Tris-Cl (pH 7.4) was
added to a concentration of 20 mM, and mitochondria were pelleted by centrifugation (12,000 × g, 10 min) and
resuspended in ice-cold NE2.5 (0.38 M sucrose, 20 mM Tris-HCl (pH 7.6), 2 mM EDTA, 0.8 mM spermidine) on ice. Mitochondria were then lysed by the
addition of 12.5% Nonidet P-40 to a final concentration of 0.5%.
After incubation on ice for 10 min, DNase I was added where indicated
and then all samples were centrifuged (16,000 × g, 10 min). The supernatant was transferred to another tube and incubated on
ice for 25 min. The supernatants were then layered on top of a
20/40/60% sucrose step gradient and centrifuged in an SW 41 rotor
(111,000 × g for 75 min). The 40/60% interface was
collected, diluted to 1 ml in NE2.5 buffer, and pelleted through 40%
sucrose at 136,000 × g for 1 h in a TLA 100.3 rotor. The pellets were resuspended in NE2.5 buffer, diluted in 2×
Laemmli buffer (23) with 10%
-mercaptoethanol, vortexed, and
boiled for 5 min. Samples (10 µl) of each nucleoid fraction and of
each lysate (supernatant) fraction (2 µl) were separated by 10%
SDS-PAGE for silver staining. In parallel, a sample of each nucleoid
fraction (40 µl) and each lysate sample (20 µl) were separated on
an 8% SDS-PAGE gel and transferred to a membrane for Western analysis using an anti-V5 antibody (Invitrogen).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) mutations. Because nam1
and ATD
mutant strains all demonstrate a defect in intron processing of the
COX1 and COB genes (10, 15) and Sls1p has been
implicated to act in the same pathway of transcription-coupled events
as Nam1p and mtRNA polymerase (9), we investigated whether an
sls1
strain has a similar mitochondrial intron-processing
defect as nam1
and rpo41 ATD mutant strains.
Northern analysis of total mitochondrial RNA revealed that an
sls1
strain expressed nearly wild-type levels of
COX3 mRNA, a gene that does not contain introns, yet
does not accumulate the mature mRNA from the intron-containing
COX1 and COB genes (Fig.
1A). Therefore, the
sls1
strain has the same intron-processing defects as
nam1
and ATD mutant strains (10). Because mitochondrial
intron-processing defects correlate with translation perturbation,
these data suggested that Sls1p impacts mitochondrial translation
efficiency. To test the hypothesis directly, mtDNA-encoded proteins in
the sls1
strain were specifically labeled in
vivo using [35S]methionine in the presence of the
cytoplasmic translation inhibitor cycloheximide. Accordingly, the
sls1
strain was severely deficient in labeling of all
mtDNA-encoded products (Fig. 1B), indicating that Sls1p is,
in fact, required globally for efficient translation in yeast
mitochondria.
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Fig. 1.
Analysis of mitochondrial translation in
nam1 and sls1 null mutant
strains. A, Northern analysis of mitochondrial
transcripts. Mitochondrial RNA was extracted and prepared for Northern
analysis as described (10). The strains analyzed have been described
previously (9) and are indicated above the figure: CMW2
(wt) and CMW1 (sls1 ). Signals for the mature
mitochondrial RNA transcripts COB, COX1, and
COX3 are indicated on the left. B, specific
labeling of mitochondrial translation products in vivo.
Shown is an autoradiogram of mtDNA-encoded proteins labeled with
[35S]methionine at 30 °C in the presence of
cycloheximide as described under "Experimental Procedures." The
strains analyzed are the same as those described in A. C, overexpression of Sls1p restores in vivo
labeling of mitochondrial proteins in an nam1
strain.
Shown is an autoradiogram of mtDNA-encoded translation products labeled
in cultures at 30 °C. The strains analyzed have been described
previously (9) and are indicated at the top of each lane:
GS140 (wt), GS141 (nam1
), and ACB1
(nam1
+ pRS316-SLS1). The identity of each
labeled mtDNA-encoded protein is indicated on the left of
the figure in both B and C.
strains exhibit a virtual absence of
mitochondrial translation (Fig. 1B), nam1
mutants have been shown by others (14) to exhibit a moderate global
decrease in mitochondrial translation and an apparent lack of labeling
of Cox1p. Because we have previously placed Nam1p and Sls1p in the same
genetic pathway (9), we analyzed the effect of deleting the
NAM1 gene on mitochondrial translation in our strain
background. Similar to the results obtained with the sls1
(Fig. 1B), in vivo labeling of mtDNA-encoded
proteins revealed an extremely low if not complete absence of
translation of all mtDNA-encoded proteins in this nam1
strain (Fig. 1C). We also demonstrated previously that
overexpression of Sls1p suppresses the petite phenotype of the
nam1
mutation in this genetic background (9). Therefore,
we also examined whether Sls1p can suppress the observed translation
defect in the nam1
strain when overexpressed. Labeling of
translation products demonstrated that overexpression of Sls1p restored
mitochondrial translation in our nam1
strain to close to
wild-type levels at 30 °C (Fig. 1C), with the exception
of Cox1p, which was only moderately increased. Similar results were
obtained when in vivo labeling was performed at 37 °C
(data not shown). The similar translation phenotypes of the
nam1
and sls1
strains as well as the
ability of increased levels of Sls1p to greatly restore translation in the nam1
strain provide strong evidence that these
proteins act in the same pathway to determine the efficiency of
mitochondrial translation.
and nam1
strains, the documented
intron-processing defects observed in rpo41 ATD mutant
strains (10) result from impaired mitochondrial translation, labeling
experiments were performed in these strains. All ATD mutations tested
resulted in decreased incorporation of radiolabel into mtDNA-encoded
proteins, indicative of a global decrease in translation efficiency
(Fig. 2A). The
rpo41-N152A/Y154A mutation resulted in the most
severe global translation defect (Fig. 2A), requiring
extended exposures to reveal the extremely low amount of labeling (data
not shown). Additional experiments revealed that labeling of
translation products did not diminish when the chase time was increased
to 1 h in any of the rpo41 ATD mutants, demonstrating
that the defect is not a result of a decrease in stability of the
translation products during the labeling period (data not shown).
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Fig. 2.
Mitochondrial RNA polymerase ATD mutations
result in global mitochondrial translation defects. A,
specific labeling of mitochondrial translation products in
vivo. Shown is an autoradiogram of mtDNA-encoded proteins labeled
with [35S]methionine in the presence of cycloheximide at
37 °C as described under "Experimental Procedures." The ATD
mutation in the RPO41 gene (encoding mtRNA polymerase)
harbored by each strain tested is indicated above the figure
as follows: RPO41 (wt); rpo41 2,
deletion of amino acids 27-117 (
2);
rpo41
3, deletion of amino acids 27-212
(
3); rpo41-E119A-C121A (119-121);
and rpo41-N152A-Y154A (152-154). The identity of
each labeled protein is indicated to the left of the figure.
B, Southern analysis of mtDNA isolated from wild-type
(wt) and rpo41-N152A-Y154A (152-154)
mutant strains after growth under the conditions used for in
vivo labeling in A. Radiolabeled probes were hybridized
to mtDNA (COX1 and COX3 genes) as well as nuclear
DNA (ACT1 gene) as indicated to the left of the
figure.
genome production (1). The deletion mutants,
rpo41
2 and rpo41
3, produce spontaneous
petite mutants at a rate similar to the wild-type strain, indicating
that their mtDNA is as stable as in the wild-type strain under these
conditions. The rpo41-E119A/C121A mutation produced more spontaneous petite mutants than either of the deletion mutants (Table I), yet consistently incorporated more radiolabel in the
mitochondrial translation assay (Fig. 2A), indicating that the translation defect observed in the deletion mutants is likely not
the result of increased rho
genome production. The strain
that exhibited the most severe translation defect,
rpo41-N152A/Y154A (Fig. 2A), was also
the strain that exhibited the largest amount of spontaneous petite formation (Table I). However, the ~30% instability observed
presumably cannot account for the very small percentage of total
translation activity that was observed in the in vivo
labeling experiments (Fig. 2A). Furthermore, Southern
analysis of mtDNA from the rpo41-N152A/Y154A strain using two probes that hybridize to mtDNA sequences physically separated on the genome (COX1 and COX3) also
revealed that mtDNA is largely intact in this strain under these
conditions (Fig. 2B). Altogether, these data indicate that
the translation defects measured in all of the rpo41 ATD
mutant strains using the in vivo labeling approach, with the
possible exception of the rpo41-E119A/C121A strain (see "Discussion"), are not the result of mtDNA instability at the time of the assay.
Petite induction in ATD mutant strains
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Fig. 3.
ATD mutations result in reduced steady-state
accumulation of mtDNA-encoded proteins and hypersensitivity to the
mitochondrial translation inhibitor erythromycin. A, Western
analysis of mitochondrial Cox1p and Cox2p. Cultures and mitochondria
were prepared in the same manner as described for the in
vivo labeling experiments except that cycloheximide was omitted,
and the cells were not labeled with [35S]methionine.
Blots of total mitochondrial protein (40 µg/lane) were sequentially
probed using antibodies against mtDNA-encoded Cox2p and Cox1p and
porin, a nucleus-encoded mitochondrial outer membrane protein. The
mutation in RPO41 harbored by each strain tested is
indicated as described in Fig. 2A. B, erythromycin
sensitivity assays. A small volume of a liquid culture containing the
same number of cells of the indicated yeast strains were spotted onto
YPG medium without (0 µg/ml) and with the indicated amounts (5, 10, 20, and 50 µg/ml) of erythromycin after growth as described under
"Experimental Procedures." The plates were then incubated at
36 °C for 6 days. Complete lack of growth was scored as sensitivity
to the drug at the given concentration. The yeast strains analyzed are
indicated at the top of the figure in the same manner as in
Fig. 2A.
and ATD mutations (9) (Fig.
1C) led us to investigate the possibility that Sls1p interacts in some manner with mtRNA polymerase. We hypothesized that if
Sls1p interacts with the ATD, either directly or as a complex with
other proteins, it may do so in the context of the mitochondrial
nucleoid, where mtRNA polymerase is predicted to reside. To test this
possibility, nucleoids in actively translating mitochondria were
isolated from wild-type and rpo41
3 mutant strains expressing a functional V5-tagged version of Sls1p. Western analysis of
nucleoids isolated from wild-type strains confirmed that mtRNA polymerase is present in the nucleoid fraction in a
DNA-dependent manner (data not shown), demonstrating that
mitochondrial nucleoids are being isolated. Western analysis of
mitochondrial lysates using an anti-V5 antibody revealed that
expression of the tagged Sls1p was similar in both strains (Fig.
4). Analysis of the corresponding nucleoid fraction from the wild-type strain revealed the presence of
Sls1p in the nucleoid that was dependent on the presence of mtDNA (Fig.
4), indicating that Sls1p is not simply a contaminant in the fraction.
In addition, its nucleoid association was dependent on the presence of
an energy-regenerating system that is needed for optimal in
organello translation under these conditions (24). Furthermore, in
the rpo41
3 strain, a mutant in which the ATD is deleted
and does not interact with Nam1p (10), Sls1p no longer associated with
the nucleoid fraction (Fig. 4). These data indicate that Sls1p
interacts with the ATD, either directly or in a complex with other
proteins, and this interaction is necessary for the localization of
Sls1p to the nucleoid. Because Sls1p is an integral membrane protein,
these data also suggest that mtDNA is associated with the inner
membrane during active gene expression. It is noted that Nam1p was not
detected in the nucleoid fraction under these conditions (data not
shown), consistent with the results of Kaufman et al.
(25).
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Fig. 4.
Sls1p is a component of the mitochondrial
nucleoid. Mitochondrial lysates and nucleoid fractions were
prepared as described under "Experimental Procedures." V5-tagged
Sls1p was detected by Western analysis using an anti-V5 antibody
(top panel). A separate gel was run and silver-stained to
visualize the amount of protein loaded (bottom panel).
Lanes 1-4 are the mitochondrial lysates prior to
fractionation, and lanes 5-8 are the purified nucleoid
fractions. The sample analyzed is indicated at the top of
each lane: MSR107 (wt); MSR107 treated with DNase I
(wt +DNase); MSR110 ( 3) and MSR107 without the
energy regenerating system (wt
energy).
strain (Fig.
1C) and partially rescues the petite phenotypes of all the
ATD mutant strains (9), we next investigated whether the same
conditions would restore translation in the rpo41 ATD
mutants. Perhaps unexpectedly, overexpression of Sls1p did not result
in increased labeling of mtDNA-encoded proteins in any of the
rpo41 ATD mutant strains (data not shown). However, Western
analysis of Cox1p and Cox2p revealed that overexpression of Sls1p did
result in a moderate increase in steady-state levels of both
mtDNA-encoded proteins in the ATD mutant strains (Fig. 5). We note that the effect on Cox1p was
not as dramatic as Cox2p; nonetheless, this difference was
reproducible. These data indicate that Sls1p suppresses the
rpo41 ATD mutants by increasing the steady-state level of
mtDNA-encoded proteins presumably in a manner largely independent of
its effects on translation.
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Fig. 5.
Overexpression of Sls1p increases the
steady-state levels of mtDNA-encoded proteins in ATD mutant
strains. Western analysis of ATD mutants with and without a
plasmid that overexpresses Sls1p. Blots of total mitochondrial protein
(40 µg/lane) were sequentially probed with antibodies to
mtDNA-encoded Cox2p, Cox1p, and the nucleus-encoded porin. The mutation
in the RPO41 gene harbored by each strain tested is
indicated at the top of the figure in the same manner as in
Fig. 2A. The 1st to 5th
lanes are the strains without the SLS1 plasmid as
a control, and the 6th to the 10th
lanes (bracketed) are the strains expressing
SLS1 from pRMS5-6 (9), indicated as
(+SLS1).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and ATD mutations when overexpressed (9). Because Sls1p is required for proper assembly
of the respiratory chain, but not for transcription or mtDNA
maintenance per se, it has been postulated to be involved in
post-transcriptional steps of mitochondrial gene expression (17). Our
results (Fig. 1), which clearly demonstrate that our sls1
strain has the same intron-processing defect we reported previously for
nam1
and ATD mutations (10) and are globally deficient in
labeling of mtDNA-encoded proteins in vivo, establish a
function for Sls1p in mitochondrial protein synthesis.
phenotype (9) was linked to its role in
mitochondrial translation identified here. Nam1p was shown by others
(14) to be involved in overall mitochondrial translation efficiency, accompanied by a severe defect in translation of Cox1p. We confirmed a
role for Nam1p in overall mitochondrial translation (Fig.
1C) and went on to show that overexpression of Sls1p
re-established a nearly wild-type mitochondrial labeling pattern in an
nam1
strain (Fig. 1C), supporting a critical
role for Sls1p in mitochondrial translational efficiency.
Interestingly, overexpression of Sls1p in our nam1
strain
resulted in a Cox1p-specific translation defect (Fig. 1C)
that is virtually identical to that originally reported by Asher
et al. (14), suggesting that the differences observed between these two strains may involve strain-dependent
differences in endogenous expression of SLS1.
2, rpo41
3, and
rpo41-N152A/Y154A) are those that were shown
previously to negatively impact Nam1p binding (10) strongly suggests
that the observed defects result from disruption of the proposed
RNA-handeling pathway. Second, Sls1p is found associated with nucleoids
in an ATD-dependent and energy-dependent manner
(Fig. 4), strongly supporting the notion that efficient mitochondrial
translation is occurring when Sls1p is found in a complex with those
mtRNA polymerase molecules that are bound to mtDNA and presumably
actively engaged in transcription of the mitochondrial genome.
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Fig. 6.
Model describing critical interactions
required to coordinate transcription and translation at the inner
mitochondrial membrane. The mitochondrial inner membrane is shown
with associated gene-expression components (e.g. RNA
processing factors, ribosomes, and other translation components)
indicated as large shaded rectangles. Nam1p is shown binding
to the ATD of mtRNA polymerase (10) in order to facilitate the
interaction of mtDNA-bound (i.e. transcription-competent)
mtRNA polymerase with Sls1p at the inner membrane. Based on its
ATD-dependent localization to mtDNA nucleoids (Fig. 4),
Sls1p is shown in association with the ATD of mtRNA polymerase that is
engaged in transcription. However, it is noted that whether such an
interaction is direct or mediated by other proteins is currently not
known. In this model, Sls1p is postulated to function as part of an
interaction point for the ATD, perhaps to localize mtRNA polymerase and
its associated nascent transcript to the inner membrane to facilitate
RNA interactions with the translational activators. The nascent
transcript emerging from mtRNA polymerase represents one of the
cytochrome oxidase subunit mRNAs (e.g. COX1,
COX2, or COX3) and is shown interacting with the
membrane-associated COX translational-activator complex.
Based on the recent identification of the interactions between Nam1p
and members of this complex (18), we show Nam1p binding here as well,
perhaps to help establish binding of the 5'-untranslated region of the
mRNA with its cognate translational activator. After a
membrane-associated mtRNA polymerase complex is established, Nam1p is
postulated to dissociate, perhaps to locate the next mtRNA polymerase
to be coupled to the membrane.
Whereas the vast majority of data presented herein are consistent with
this model, it is clear that mutations in the ATD can cause multiple
defects in mitochondrial gene expression that are not as easily
explained. First, although we were successful at eliminating mtDNA
instability as an explanation for the observed overall translation
defects seen in most of ATD mutant strains tested (Table I), the
rpo41-E119A-C121A is a notable exception. This mutation
resulted in a relatively large defect in steady-state accumulation of
Cox1p and Cox2p (Fig. 3A). In fact, its steady-state defect
was comparable with that of the rpo41-N152A/Y154A
strain (Fig. 3A), despite the fact that its in
vivo labeling capacity was substantially greater (Fig.
2A), and it was not hypersensitive to erthyromycin (Fig.
3B). This mutation also does not appear to disrupt Nam1p
binding (10). Altogether, these results suggest that the
rpo41-E119A-C121A defect is not due to disruption of the
proposed translation-coordination function of the ATD but rather its
mtDNA instability phenotype (Table I). Second, overexpression of Sls1p
is unable to rescue the in vivo labeling defect in the rpo41 ATD mutant strains (data not shown) but does
moderately increase the steady-state level of Cox1p and Cox2p in the
ATD mutants (Fig. 5), suggesting that Sls1p has a role in assembly of
the oxidative phosphorylation complexes as originally postulated by
others (17). These data indicate the ability to Sls1p to partially
rescue the ATD mutant phenotypes (9) is most likely through this second
function and not it ability to reestablish normal membrane coupling of
mtRNA polymerase through the ATD. One possibility is that
overexpression of Sls1p may allow increased numbers of membrane
complexes to form in the ATD mutant strains and, even though they are
not "wild-type" complexes, facilitates assembly (and hence
stability) of the proteins that do manage to get translated, thus
partially rescuing the mutant phenotype. In the case of
nam1 strains, a similar scenario is envisioned where the
ability of Sls1p to increase the number of membrane sites would, in
principle, increase the probability that normally coupled complexes
would form because the ATD is intact in these strains, thus providing
an explanation for the nearly full rescue of the nam1
phenotype by Sls1p overexpression (9). This interpretation is entirely
consistent with the proposal that Nam1p functions to facilitate
formation of the membrane-coupled mtRNA polymerase complex involving
Sls1p (Fig. 6), but itself is not an active member of the complex once
it is formed. Finally, the rpo41-R129D mutation results in
only a moderate reduction in mitochondrial translation (data not
shown), yet causes the most severe glycerol growth phenotype (10),
suggesting that this mutation in the ATD of mtRNA polymerase can
compromise additional cellular functions.
In conclusion, we have elucidated important new aspects of how
mitochondrial gene expression is accomplished in yeast. Our results
indicate that the primary mechanism involves a complex series of
interactions that ensure coordination of transcription and translation
of mitochondrial transcripts at the inner mitochondrial membrane that
is mediated through the ATD of mtRNA polymerase. It will be of great
general importance to determine whether a similar mechanism is
operating in human cells, where defects in mitochondrial gene
expression can cause and exacerbate disease states and impact the
aging process.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-59655 (to G. S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 404-727-3798; Fax: 404-727-3954; E-mail: gshadel@emory.edu.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M301399200
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ABBREVIATIONS |
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The abbreviation used is: ATD, amino-terminal domain.
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