From the Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, Georgia 30322
Received for publication, October 31, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Alignment of three fungal mtRNA polymerases
revealed conserved amino acid sequences in an amino-terminal region of
the Saccharomyces cerevisiae enzyme implicated previously
as harboring an important functional domain. Phenotypic analysis of
deletion and point mutations, in conjunction with a yeast two-hybrid
assay, revealed that Nam1p, a protein involved in RNA processing and
translation in mitochondria, binds specifically to this domain. The
significance of this interaction in vivo was demonstrated
by the fact that the temperature-sensitive phenotype of a deletion
mutation (rpo41 Expression of the mitochondrial genome occurs in the organelle
matrix and involves both nuclear- and mtDNA-encoded factors. In
addition to mRNAs (which usually encode protein subunits of the
enzyme complexes involved in oxidative phosphorylation), mtDNA in many
organisms also contains genes for tRNAs and ribosomal RNAs that are
necessary for mitochondrial translation. The remaining factors required
for expression and replication of the mitochondrial genome are encoded
in the nucleus and imported into the organelle. Mitochondrial
transcripts are often polycistronic and thus a large number of RNA
processing reactions is required to liberate mature RNA species (1). In
addition, RNA processing events have been implicated in the initiation
of mtDNA replication (2). Thus, a complete understanding of
mitochondrial gene expression and mtDNA replication requires broader
understanding of how these RNA processing events are accomplished
in vivo.
The mitochondrial transcription machinery in the budding yeast,
Saccharomyces cerevisiae, is well characterized (3, 4) and
involves a nucleus-encoded mtRNA polymerase (sc-mtRNA polymerase, encoded by the RPO41 gene) that is homologous to the single
subunit Escherichia coli bacteriophage RNA polymerase (5, 6)
and a transcription initiation factor sc-mtTFB (7-9). In addition, factors involved in mtRNA processing have also been identified in this
organism, including those involved in liberating tRNAs, rRNAs, and
mRNAs from polycistronic transcripts and excising introns from
certain messages (see Ref. 10 for review). One such factor, Nam1p (also
known as Mtf2p), was initially identified as a high copy
suppressor of mtDNA point mutations that affect splicing of introns
from the mitochondrial COX1 and CYTB messages
(11) and independently as a temperature-sensitive mutation affecting mitochondrial transcript levels (12). Nam1p is localized to the
mitochondrial matrix (13), and characterization of NAM1 null
mutant strains has confirmed its involvement in COX1 and CYTB intron removal and elucidated potential roles for this
protein in overall mitochondrial translation capacity and
ATP6/8 mRNA processing and/or stability (14). In
addition, crude mitochondrial transcription complexes isolated from one
NAM1 mutant strain remain competent for transcription but
exhibit an altered DNA binding activity profile (12), suggesting a
potential link between Nam1p function and the mitochondrial
transcription machinery.
We had shown previously that an amino-terminal domain of yeast mtRNA
polymerase is dispensable for transcription initiation in
vivo but nonetheless is required for stability and maintenance of
the mitochondrial genome, suggesting that additional activities may be
coupled to the transcription process in mitochondria (15). These
studies implicated amino acids 29-208 of mtRNA polymerase as a minimal
portion of the protein that harbors an independent functional domain of
the enzyme. Here we have characterized this domain further and
demonstrate that one of its functions is to provide an interaction
point for Nam1p, that may provide the means to couple factors involved
in additional aspects of RNA metabolism directly to the transcription
machinery in yeast mitochondria. In principal, such a coupling
phenomenon in mitochondria may be analogous to mechanisms of gene
expression in the nucleus, where many aspects of mRNA processing
are coupled functionally to transcription via interactions involving
the carboxyl-terminal domain of the largest subunit of RNA polymerase
II (16, 17).
Plasmid Construction--
Wild-type and mutated alleles of
RPO41 used in the plasmid shuffle assay were expressed in
yeast from the plasmid pGS348 (15), which contains a
7.2-kb,1 RPO41
gene-containing, SalI-SpeI restriction fragment
of yeast genomic DNA cloned into the shuttle vector pRS314 (18).
Construction of the rpo41 Site-directed Mutagenesis--
Specific point mutations in the
RPO41 gene were generated by a two-step, megaprimer PCR
protocol as follows. The PCR template (pBS348) consisted of a 2.1-kb
SalI-BamHI restriction fragment from pGS348,
which contains the amino-terminal extension of RPO41 and
upstream sequences, cloned into pBSII KS+ (Stratagene, Inc). Promoters
for T7 and T3 RNA polymerase flank the RPO41 insert in this
plasmid. For each mutation, a PCR was performed that utilized a
specific mutagenic oligonucleotide (synthesized by Midland Certified Reagent Co., Midland, TX) as one primer and an oligonucleotide corresponding to the T7 promoter as the second primer to generate an
~500-base pair product. This PCR product was gel-purified and used as
the source of a megaprimer (after denaturation, the 500-nucleotide strand containing the T7 primer sequence serves as the megaprimer) in a
second PCR in conjunction with a T3 primer. The 2.2-kb product of this
second PCR, which now had the desired mutation fixed in the
RPO41 gene, was digested with SalI and
BamHI and ligated into pGS348 to form a full-length mutated
RPO41 allele that can be expressed in yeast. All PCRs were
performed using Pfu Turbo DNA Polymerase (Stratagene, Inc.)
in the buffer supplied by the manufacturer and typically consisted of
25-30 amplification cycles (95 °C, 30 s; 55 °C, 1 min;
68 °C, 8 min) followed by a 12-min, 68 °C extension period at the
end of the last cycle. In all cases, the nucleotide sequence of the
portion of RPO41 open reading frame in pGS348 that was
amplified during the mutagenesis protocol was determined to ensure that
the desired site-directed mutation was the only mutation introduced
during the PCR procedure.
Phenotypic Analysis of RPO41 Mutations by Plasmid
Shuffle--
Routine growth and transformation of yeast strains, as
well as standard growth media preparation, were accomplished as
described by Sherman (20). The yeast strain GS112 ( Yeast Two-hybrid Analysis--
The yeast strain Y190 (a
gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 + URA3::GAL-lacZ + LYS2::GAL-HIS3 cyhr)
was used for all two-hybrid analyses. This strain contains two Gal4p-inducible reporter genes integrated in its genome that allow two-hybrid positives to be selected based on a His+ growth
phenotype and screened using a blue color-generating RNA Isolation and Northern Analysis of Mitochondrial
Transcripts--
A 7-ml culture of each yeast strain analyzed was
grown at 30 °C in YPG medium to stationary phase. This culture was
used to inoculate 450 ml of SD medium supplemented with leucine (100 mg/liter), adenine (20 mg/liter), and uracil (20 mg/liter) to a
starting A600 of 0.025. This culture was
subsequently grown at 37 °C on an orbital shaker (200 rpm) to a
final A600 of 0.8. Yeast cells were harvested by
centrifugation and resuspended in spheroplasting buffer (1.35 M sorbitol, 0.1 M EDTA, 0.1%
A Conserved Amino-terminal Domain in sc-mtRNA Polymerase--
The
mtRNA polymerase of S. cerevisiae is related to
bacteriophage RNA polymerases (e.g. T7, T3, and SP6) but
contains a unique amino-terminal extension (Fig.
1). In an earlier study (15), we reported
the analysis of a series of RPO41 deletion mutations that
had no effect on protein stability or localization, and ultimately revealed the existence of a functional amino-terminal domain in mtRNA
polymerase between amino acids 28 and 208 that is required for mtDNA
maintenance and can function in trans. Whereas mtRNA polymerases from most species contain significant amino acid identity in those regions involved in catalytic activity (i.e. the
bacteriophage T7 family-like domains), the amino-terminal extensions
are not well conserved between species. However, we were able to align the amino acid sequence of the amino-terminal extension of the S. cerevisiae enzyme with that from two other fungal species, Neurospora crassa and Schizosaccharomyces pombe,
and found that the most conserved region corresponded to amino acids
110-205 of the S. cerevisiae mtRNA polymerase (Fig. 1). In
particular, the region encompassing amino acids 117-155 exhibited 18%
amino acid identity (identical residues in all three species) and 38% amino acid similarity (identical or similar amino acids in all three
species). The degree of similarity in this region is emphasized further
if pairwise comparisons are made. For example, the S. cerevisiae and N. crassa proteins are 35% identical
and 56% similar in this region.
To test the hypothesis that the conserved region of the mtRNA
polymerase amino-terminal extension composes all or part of the
important functional domain (aa 28-208) we identified previously (15),
we made a series of point mutations in the S. cerevisiae RPO41 gene by site-directed mutagenesis that changed residues that
are conserved in all three of the fungal mtRNA polymerases (Fig. 1).
The mitochondrial phenotype (assessed by growth on YPG medium) of each
of these mutations was determined using a standard plasmid-shuffle
strategy, and the effects of these mutations were compared with the
phenotypes of two previously characterized deletion mutations in this
region (rpo41 Nam1p Interacts with the Amino-terminal Domain of Yeast mtRNA
Polymerase--
The observed phenotypes of the amino-terminal domain
mutations suggest that this region of the protein is involved in
coupling some critical function to the transcription process in
mitochondria (15). This coupling capacity could occur directly, by
virtue of a structural or catalytic role for this domain, or indirectly through the binding of other mitochondrial regulatory factors, or
perhaps both. To test the hypothesis that other mitochondrial factors
are involved, we screened a library of yeast genomic DNA fragments for
proteins that bind to the amino-terminal portion of yeast mtRNA
polymerase using a yeast two-hybrid assay. Our strategy was to identify
proteins that interact specifically with the amino-terminal domain of
mtRNA polymerase that was implicated here and in our previous studies
(15). To accomplish this, we created two yeast two-hybrid bait
plasmids. The first, pAS-RPO, contained RPO41 sequences that
encompassed an intact amino-terminal domain (encoding aa 27-633) and
the second, pAS-RPO
In our initial two-hybrid screen, the bait plasmid used (pAS-RPO)
encoded only amino acids 27-633 of mtRNA polymerase, which primarily
contains just the amino-terminal extension of mtRNA polymerase. To
ensure that Nam1p interacts with full-length mtRNA polymerase, we
constructed a two-hybrid bait plasmid (pAS-RPO-FL) that encodes the
entire mature protein (amino acids 27-1351), and we tested it for the
interaction with Nam1p in the two-hybrid assay. We found that Nam1p was
able to interact with its amino-terminal target in the context of an
intact mtRNA polymerase as well (Table I), thus this interaction could be
analyzed using either the amino-terminal extension alone or in the
context of the full-length mtRNA polymerase. We next determined the
effect of the amino-terminal domain mutations on the ability of mtRNA
polymerase to bind Nam1p in the two-hybrid assay (Table I). The
rpo41 Nam1p Overexpression Rescues the Temperature-sensitive Phenotype of
the rpo41 Mutations in the Amino-terminal Domain of mtRNA Polymerase Result
in Mitochondrial RNA Transcript Defects Consistent with Perturbation of
Nam1p Function--
A documented phenotype of NAM1 null
mutations (14) is the specific reduction of the intron-containing
mitochondrial transcripts encoding cytochrome b (COB) and
cytochrome c oxidase subunit 1 (COX1). We had
reported previously that a deletion of the amino-terminal extension of
mtRNA polymerase (rpo41 Amino-terminal extensions present in many mtRNA polymerases
distinguish them from the related bacteriophage enzymes and provide the
mitochondrial enzymes with localization information as well as
additional function (6, 15). Previously, we demonstrated that the
amino-terminal extension of sc-mtRNA polymerase harbors a functional
domain involved in mtDNA stability, and we proposed that this domain
could function by coupling additional activities to the transcription
process (15). Because numerous RNA processing events are required for
normal mitochondrial gene expression and mtDNA replication (1, 2), we
hypothesized (15) that one process that may be coupled to transcription
may be RNA processing. Here we report the characterization of a
collection of RPO41 deletion and point mutations, the
results of which demonstrate that Nam1p, a mitochondrial matrix protein
implicated in translation and RNA processing events (11, 13, 14),
interacts physically and functionally with an amino-terminal domain of
sc-mtRNA polymerase. By using a two-hybrid protein interaction assay,
we have demonstrated that Nam1p binds specifically to the
amino-terminal extension of sc-mtRNA polymerase. The fact that the
rpo41 The physiological significance the Nam1p/mtRNA polymerase interaction
was demonstrated as follows. First, there is general correspondence
between Nam1p binding and growth phenotypes of mtRNA polymerase mutants
on YPG medium (growth on which requires mitochondrial respiration). Two
mutations that eliminate Nam1p binding altogether,
rpo41 In addition to the growth data discussed above, our analysis of mtRNA
species in RPO41 mutant strains also links amino-terminal domain function to Nam1p. All of our amino-terminal deletions and point
mutations exhibit marked reductions in the steady-state levels of
mature COX1 and CYTB mRNAs (Fig. 4). Although
the precise function of Nam1p is unknown, it is clear that
NAM1 null mutations affect a subset of mtRNA transcripts in
yeast mitochondria. In particular, Nam1p appears to be involved in the
processing and/or stability of the two intron-containing primary
transcripts that contain COX1 and CYTB and,
perhaps to a lesser degree, the 21 S rRNA (11, 14). However, Nam1p
function is not limited to effects on intron removal because strains
that contain an intronless mitochondrial genome still display a
temperature-sensitive petite phenotype and instability of the mature
ATP6 mRNA (14), which is cotranscribed with
COX1. Finally, overall translation capacity is reduced in
NAM1 null mutant strains (11), suggesting a dual role for
Nam1p in RNA processing/stability and translation, or that these two
processes are coupled in yeast mitochondria. Regardless of the precise
activity of Nam1p in mtRNA metabolism, our data demonstrate that
mutations that affect the amino-terminal domain of mtRNA polymerase
lead to mtRNA transcript defects that are entirely consistent with
disruption of Nam1p function. The simplest explanation for these data
is that at least one function of Nam1p is carried out in association
with mtRNA polymerase (i.e. Nam1p is coupled to
transcription). The fact that COX3 transcripts are not
grossly affected in our experiments is consistent with previously published data showing that mitochondrial transcription per
se is not perturbed to large degree by loss of Nam1p function (11, 14) or by the rpo41 This study also provides additional information regarding the function
of the amino-terminal domain of mtRNA polymerase. First, none of the
point mutations was rescued by overexpression of Nam1p, despite the
ability of some of these to interact with Nam1p to some degree in the
two-hybrid assay (Table I). Second, the R129D and N152A/Y154A mutations
exhibited more severe YPG growth phenotypes than the
rpo41 Based on the apparent homology between the amino-terminal domain of
sc-mtRNA polymerase and those of N. crassa and S. pombe (Fig. 1), we would predict that these organisms also contain
a Nam1p homolog that is localized to the mitochondrial matrix. However, our searches of currently available data bases have yet to identify any
obvious homologs of Nam1p in any organism. This brings into question
whether the amino-terminal extensions found in mtRNA polymerases from
other eukaryotes have similar functions to that proposed here for the
S. cerevisiae protein. To begin to address this question, we
have analyzed the sequences of the amino-terminal extensions of human
(27) and Xenopus (GenBankTM accession number
AF200705) mtRNA polymerases. As already mentioned, no obvious
similarity exists between the vertebrate and the fungal enzymes in
their amino-terminal extensions. However, like the fungal enzymes, the
vertebrate proteins are similar to each other in this region (Fig.
5), exhibiting 34% amino acid identity
and 53% similarity. Comparing these conserved amino-terminal regions of the vertebrate homologs to other known sequences in available data
bases, we found a match of potential significance to a protein, CRP1,
that is located in maize chloroplasts (28). Remarkably, the function of
CRP1 in chloroplasts is similar to that documented for Nam1p in yeast
mitochondria, that is, the processing and translation of specific
mRNAs. The conserved amino-terminal region in both the human and
Xenopus mtRNA polymerase is similar (20-25% identity, 40-45% similarity) to a block of amino acids that is repeated twice
in CRP1 (Fig. 5). Similar data base searches reported by Fisk et
al. (28), revealed similarity between CRP1 and a family of plant
proteins of unknown function that are related to a salt-inducible protein in tobacco. Additionally, they found that the region held in
common between CRP1 and this family of proteins was also related, although more distantly, to several other factors involved in post-transcriptional gene regulation in mitochondria including Pet309p,
a translational activator; Rpm2p, a protein subunit of mitochondrial
RNaseP (a tRNA processing factor); and threonyl-tRNA synthetase.
Recently, the homologous regions in these proteins have been postulated
to be composed of a tandemly repeated, 35-amino acid domain called a
pentatricopeptide repeat (PPR) motif that is structurally similar to
the well characterized tetratricopeptide repeat (TPR) motif (29). Based
on comparisons with a proposed consensus sequence for a PPR motif (29),
it appears that vertebrate mtRNA polymerases contain at least two PPR
repeats in the amino-terminal extension (Fig. 5). Thus, the human and
Xenopus mtRNA polymerases join this list of organelle
regulatory proteins involved in RNA processing and translation that
likely define a new PPR family of proteins. Although the function of
these CRP1-like sequences in vertebrate mtRNA polymerases is unknown,
it is interesting to speculate based on our results regarding the
function of the S. cerevisiae amino-terminal domain.
Perhaps, like yeast mtRNA polymerase, the CRP1-like domain in
vertebrate mtRNA polymerases may be involved in coupling RNA processing
or translation activities to transcription. Experiments are currently
in progress to determine whether, in fact, the amino-terminal extension
of human mtRNA polymerase has a role similar to that provided by the
yeast enzyme and to determine whether the amino acid similarity to CRP1
(i.e. the PPR motifs) is of functional and evolutionary
significance. Such experiments should lend new insights into the
regulation of human mitochondrial genome expression and replication and
its impact on human disease.
2), which impinges on this
amino-terminal domain, is suppressed by overproducing Nam1p. In
addition, mutations in the amino-terminal domain result specifically in
decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in
NAM1 null mutant yeast strains. Finally, one point mutation
(R129D) did not abolish Nam1p binding, yet displayed an obvious
COX1/CYTB transcript defect. This mutation exhibited the
most severe mitochondrial phenotype, suggesting that mutations in the
amino-terminal domain can perturb other critical interactions, in
addition to Nam1p binding, that contribute to the observed phenotypes.
These results implicate the amino-terminal domain of mtRNA polymerases
in coupling additional factors and activities involved in mitochondrial
gene expression directly to the transcription machinery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2- and
rpo41
3-containing versions of pGS348 has been
described elsewhere (15). All of the yeast two-hybrid bait plasmids
utilized in this study are derivatives of pAS1 (19), which express the
RPO41 reading frame as amino-terminal fusions to the Gal4p
DNA-binding domain. Two parental RPO41-bait plasmids were
used, pAS1-RPO and pAS1-RPO-FL, that contained amino acids 27-633 and
amino acids 27-1351 of sc-mtRNA polymerase, respectively (numbered
according to Ref. 6). To construct the corresponding mutated
RPO41-bait plasmids, restriction fragments containing the
mutated versions of RPO41 were isolated from PCR products (or from the corresponding pGS348 derivatives) and ligated into either
pAS1-RPO or pAS1-RPO-FL (Table I). The plasmid used to overproduce
Nam1p (pYES/GS-NAM1) was obtained from Invitrogen (Genestorm clone
yDL044cy). This plasmid contains a V5 epitope-tagged version of the
NAM1 gene under control of a galactose-inducible promoter.
his3-
200 leu2-3,-112 ura3-52 trp1-
1
ade2, rpo41
1::HIS3 +pGS347 (URA3,
RPO41)) was used to analyze RPO41 gene mutations by
plasmid shuffle (21). After transformation of GS112 with the desired
mutant RPO41 allele on a pGS348 plasmid, two rounds of
growth on synthetic dextrose (SD) medium containing 5-fluoroorotic acid
were used to select for strains that express the mutated version of the
RPO41 gene as their only source of mtRNA polymerase, as
described previously (15). To characterize the growth phenotypes of the
mutant strains in detail, serial 10-fold dilutions were plated onto
solid YPD (glucose-containing medium) and YPG (glycerol-containing
medium) using a 48-pin multiplex plating tool ("Frogger," Dankar,
Inc.) and grown at both 30 and 37 °C. Growth on YPG medium requires mitochondrial respiration, whereas growth on YPD does not; therefore, defective growth on YPG medium was scored as a mitochondrial petite phenotype.
-galactosidase (LacZ) assay (19). Y190 was transformed with pAS1-RPO to generate an
RPO41-bait strain for the initial two-hybrid screen for
proteins that interact with an intact amino-terminal domain. The
resulting strain, Y190 (pAS-RPO), was subsequently transformed with a
library of yeast cDNA fragments fused to the Gal4p activation
domain in the LEU2 plasmid pACT (19). Leu+,
His+ colonies were selected on SD medium supplemented with
adenine (20 mg/liter) and containing 3-amino-1,2,4-triazole (Sigma) at 30-50 mM. We pooled the Leu+, His+
colonies from a screen of ~70,000 library plasmid transformants and
isolated plasmids from these strains en masse using a smash and grab protocol (22). In this manner, an enriched library of
potential positives (library plasmids that yielded a His+
phenotype) was generated. This library was used to transform the yeast
strain Y190 pAS1-RPO
4 that contains an RPO41-bait plasmid specifically missing the amino-terminal domain. Leu+
transformants from this transformation were replica-plated onto both
Leu
, His+ medium and Leu
,
His
medium to select for strains that were
Leu+ (i.e. contained a library plasmid), yet
His
due to lack of interaction with the amino-terminal
truncated bait. The 52 strains that met all the above criteria were
kept for further analysis. Next, the library plasmid from each of these strains was isolated and used to transform a fresh Y190 (pAS1-RPO) strain. A LacZ color assay (19) was used as our final screen for
library plasmids that encoded proteins that interacted specifically with the amino-terminal domain. Plasmids that conferred a blue color in
Y190 (pAS1-RPO) but not in Y190 (pAS1-RPO
4) were subjected to
nucleotide sequence analysis to determine the identity of the gene
implicated in the interaction.
-mercaptoethanol, pH 7.4). Zymolyase 20T (U. S. Biochemical Corp.)
was added to a concentration of 3 mg/g wet weight of cells and
incubated at 37 °C for 10 min. Six volumes of 1 M
sorbitol were added to the sample, and the resulting spheroplasts were
harvested by centrifugation (3000 × g for 5 min at
4 °C). The spheroplast pellet was then resuspended in 2 volumes of
MT buffer (400 mM mannitol, 2 mM EDTA, 50 mM Tris-Cl, pH 7.4). An equal volume of glass beads was
added, and the spheroplasts were lysed by vortexing (two 30-s pulses
separated by a 1-min incubation on ice). The resulting cell lysate was
removed from the glass beads and transferred to a fresh centrifuge
tube, and 10 volumes of MT buffer were added. The lysate was then
subjected to a low speed centrifugation (1900 × g for
5 min at 4 °C) to remove cellular debris and nuclei. The resulting
supernatant was transferred to a fresh centrifuge tube and subjected to
a final high speed centrifugation (10,500 × g for 10 min at 4 °C) to pellet mitochondria. RNA was extracted immediately
from this mitochondrial pellet as described (23) and purified further
using RNeasy columns (Qiagen). Five µg of column-purified
mitochondrial RNA from each sample was separated on a 1.3%
agarose-formaldehyde gel as described (24). Northern blotting was
performed essentially as described (15), except the random prime
radiolabeled probes used for these experiments were generated using PCR
products corresponding to the mitochondrial COX1,
COX3, and COB genes as templates. The PCR
products used as probe templates were generated using purified yeast mtDNA as a template and the following gene-specific primers: COX1, 5'-CCATTAATAATTGGAGCTACAG-3' and
5'-CCAAAGAATCAAAATAAATGCTCG-3'; COB,
5'-GGCATTTAGAAAATCAAATGTG-3' and 5'-CTGTCCATAAACACAACAATAACC-3'; COX3, 5'-ATGACACATTTAGAAAGAAGTAG-3' and
5'-TTAGACTCCTCATCAGTAGAAGA-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A region of the sc-mtRNA polymerase
amino-terminal extension is conserved in two other fungal species.
A linear representation of sc-mtRNA polymerase (encoded by the
RPO41 gene) is presented at the top of the
figure. The carboxyl-terminal portion of the enzyme that is homologous
to bacteriophage RNA polymerases is depicted as a gray box,
the mitochondrial targeting sequence by a black box, and the
amino-terminal extension by a white box. Expanded at the
bottom is an alignment of the most conserved portion of the
amino-terminal extensions from S. cerevisiae (S.c.), amino
acids 110-205; N. crassa (N.c.), amino acids
122-219; and S. pombe (S.p.), amino acids
34-117. Amino acid residues that are identical are indicated by
darker shading, and those that are similar are indicated by
lighter shading. The residues in sc-mtRNA polymerase that
were changed by site-directed mutagenesis are indicated by
arrows, at the ends of the arrows the amino acid
substitution is shown (as well as the RPO41 allele
designations, which are boxed). Also indicated is the end
point of two deletion mutations that were characterized previously
(15). The rpo41 2 allele deletes amino acids
27-117 (end point labeled
2), and thus partially impinges on the
conserved region. The rpo41
3 allele deleted
amino acids 27-212 (end point labeled
3) and therefore completely
removes the conserved region.
2 and
rpo41
3, Fig.
2A). Two individual point
mutations were introduced to generate two mutant RPO41
alleles, arginine 129 changed to aspartic acid (denoted R129D) and
E179A. In addition, two double point mutant alleles were created,
E119A/C121A and N152A/Y154A. Both double-point mutant alleles, as well
as the R129D allele, exhibited a mitochondrial petite phenotype (slow growth on YPG medium) at both 30 and 37 °C, whereas the E179A mutations resulted in no discernible phenotype. Of the three mutant strains that had a mitochondrial phenotype, the E119A/C121A mutant was
the only one capable of sustained growth on YPG at 37 °C, although
its growth rate was still markedly reduced compared with the wild-type
strain at both 30 and 37 °C. This growth phenotype was almost
identical to that observed for the rpo41
2
mutation. The N152A/Y154A mutant strain grew slowly on YPG at 30 °C
and was dramatically impaired on YPG at 37 °C. This growth phenotype was similar to that observed for the rpo41
3
mutation. The R129D mutation was the most severe mutation, resulting in
the slowest growth rate on YPG at 30 °C and virtually no growth on
YPG at 37 °C. It is also noted that two of the point mutations
(R129D and N152A/Y154A) exhibited a more severe phenotype than the
rpo41
3 mutation, suggesting that this domain
when present and mutated perturbs mtRNA polymerase function more
severely than a mutation that deletes this region altogether. To
confirm that the observed phenotypes of the point mutations were not
due to altered protein stability, the expression level of each of the
mutant proteins was analyzed by western analysis (Fig. 2B).
All of these proteins were expressed at levels similar to that of the
wild-type protein. Again, these results are consistent with our earlier
analysis of the rpo41
3 mutation that removes
this domain completely but does not affect protein localization or
stability (15).
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Fig. 2.
Analysis of sc-mtRNA polymerase
amino-terminal domain mutations. A, growth phenotypes.
Shown to the left is the RPO41 allele expressed
in each strain after plasmid shuffle in GS112. At the top of
each column of panels, the growth medium (YPG or YPD) and growth
temperature (30 or 37 °C) are indicated. For example, the strain
that is expressing wild-type allele (RPO41) after plasmid
shuffle is indicated. Serial 10-fold dilutions are plated from
left to right within each panel and, in each row
of panels, identical cultures were plated. B, western
analysis of rpo41 point-mutant strains after plasmid
shuffle. The blot was first probed with an anti-RPO41p antibody and the
location of the 150-kDa, full-length mtRNA polymerase (Rpo41p) is
indicated. The RPO41 null and point-mutant strains analyzed
are indicated above each lane. The identical blot was
stripped and probed again with an antibody against Yrb1p (yeast
Ran-binding protein, a nuclear transport protein) to serve as a control
for the amount of protein loaded in each lane.
4, that does not encodes the amino-terminal
domain but does contain other RPO41 sequences (encoding aa
392-633). The screen involved several steps that ultimately selected
for plasmids encoding proteins that exhibited an interaction
specifically with the amino-terminal portion of sc-mtRNA polymerase
(see "Experimental Procedures"). From an initial screen of
~70,000 transformants, four library plasmids were identified that met
all selection criteria. The yeast genomic DNA fragment in each of these
was determined by sequencing both ends of the insert and using this
information to search the Saccharomyces Genome
Database. Two of these isolates contained the same insert containing
the entire NAM1 open reading frame (Fig.
3), a gene encoding a mitochondrial
matrix protein involved in mtRNA processing and/or translation (11, 12,
14). The specificity of the Nam1p interaction for the amino-terminal
extension of sc-mtRNA polymerase is shown in Fig. 3A.
The other two isolates corresponded to YTA5 (or
RPT2), an ATPase component of 26 S of the proteasome complex, and YGL037c, a yeast ORF of unknown function. We believe that
YTA5 is probably of no functional significance because
proteasome subunits are commonly found as false positives in yeast
two-hybrid screens, and our preliminary experiments with YGL037c
suggest that this gene product does not localize to mitochondria (data not shown). For these reasons, and because Nam1p exhibits
characteristics compatible with a protein that might interact with
mtRNA polymerase, we focused our attention on NAM1.
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Fig. 3.
Nam1p interacts specifically with the
amino-terminal extension of sc-mtRNA polymerase and suppresses a
mutation in this region. A, -galactosidase activity
(dark color in lower panel) indicates a
two-hybrid interaction between Nam1p and sc-mtRNA polymerase in the
lacZ-reporter strain Y190, which that contains a plasmid
(pAS1-RPO) encoding an intact amino-terminal domain. The same strain
containing a plasmid (pAS-RPO
4) that is missing the amino-terminal
domain, but contains other sc-mtRNA polymerase sequences, produces no
-galactosidase activity (upper panel, no dark
color). B, overproduction of Nam1p suppresses the YPG
growth defects of a rpo41
2 strain but not a
rpo41
3 strain. Five strains that were streaked
onto a YPG plate and grown at 36 °C are shown. The yeast strain
GS124 contains a plasmid encoding the rpo41
2
allele as its only source of sc-mtRNA polymerase; the yeast strain
GS125 contains a plasmid encoding the rpo41
3
allele as its only source of sc-mtRNA polymerase; and GS122 is the
isogenic wild-type strain that has the wild-type RPO41 gene
provided on a plasmid as its only source of sc-mtRNA polymerase (15).
GS124 and GS125 transformed with an empty URA3 plasmid
(YEp352) or with a URA3 plasmid that overexpresses Nam1p
(pYES/GS-NAM1) are indicated.
2 mutant, which retains a largely intact
amino-terminal domain (Fig. 1), exhibited a reduced, yet significant,
ability to interact with Nam1p, whereas the
rpo41
3 mutant, which is deleted for the
amino-terminal domain altogether, did not interact in this assay (Table
I). Of the point mutated proteins, the N152A/Y154A mutant was the only
one that did not interact with Nam1p to some degree in this assay. All
of the mutated RPO41 fusion proteins shown in Table I were expressed at least as well as the wild-type fusion protein (data not
shown); thus those mutations reported to disrupt the Nam1p interaction
did not dramatically affect expression or stability of the two-hybrid
bait protein.
Results of a yeast two-hybrid analysis of the interaction of Nam1p with
the amino-terminal domain of sc-mtRNA polymerase
2 Mutation--
To determine whether the Nam1p interaction
with the amino-terminal domain of mtRNA polymerase that we identified
by two-hybrid analysis is of physiological significance, we next tested
whether overexpression of Nam1p could rescue the phenotype of mutations in the amino-terminal domain. To accomplish this we utilized a high
copy plasmid (pYES/GS-NAM1) that expresses an epitope-tagged version of
the NAM1 gene from a galactose-inducible promoter (see "Experimental Procedures"). Expression levels of the tagged version of Nam1p from this promoter, even without galactose induction, are
capable of complementing a chromosomal NAM1 disruption,
confirming that the tagged Nam1p is functional in vivo (data
not shown). Introduction of this Nam1p-overproducing plasmid resulted
in significant rescue of the temperature-sensitive phenotype of the
rpo41
2 mutation but not that of the
rpo41
3 mutation (Fig. 3B). These
results are consistent with the fact that the
rpo41
2-encoded mtRNA polymerase is still
capable of interacting with Nam1p to some extent in the two-hybrid
assay, whereas that encoded by rpo41
3 is not
(Table I). None of the amino-terminal domain point mutations was
suppressed by overproducing Nam1p under these conditions (data not
shown). In the case of the N152A/Y154A protein, this result is
consistent with an inability to interact with Nam1p in the two-hybrid
assay. However, the E119A/C121A and R129D proteins still interact with Nam1p to some degree in the two-hybrid assay, yet their mitochondrial defects cannot be rescued by overexpression of Nam1p, suggesting that
there are other defects that are contributing to the observed phenotypes of these mutations (see "Discussion").
3) did not affect
transcription initiation from the mitochondrial ori5
promoter in vivo (15); however, we did not attempt to
analyze other mitochondrial transcripts at that time. To address
whether mutations in mtRNA polymerase amino-terminal domain exhibit a
nam1-like phenotype, we analyzed mitochondrial transcripts
in these strains by northern analysis (Fig.
4). Total mtRNA was analyzed from each
strain after growth at 37 °C for five generations and compared with
a NAM1 null strain grown at 30 °C, which exhibits the
diagnostic COX1 and CYTB transcript defects. As
observed in the NAM1 null strain, all of the mtRNA polymerase mutant strains exhibited a marked reduction in the steady-state levels of both CYTB and COX1
messages. In contrast, the steady-state level of COX3, a
mitochondrial transcript that is largely unaffected by Nam1p function
(14), was not dramatically altered under these conditions and served as
an indicator that mitochondrial transcription per se was not
globally affected by these mutations. This conclusion is supported by
the additional observation that the overall mitochondrial transcript
profile in all of the strains, as judged by ethidium bromide staining (data not shown), was virtually identical.
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Fig. 4.
Northern analysis of mitochondrial
transcripts from amino-terminal domain mutant yeast strains. Five
micrograms of mtRNA was analyzed from the yeast strains indicated at
the top of each lane. RNA was isolated from the
RPO41 mutant ( 2,
3, E119A/C121A, N152A/Y154A, and
R129D) and the corresponding isogenic wild-type strain
(RPO41) after growing in SD medium for five generations at
37 °C. RNA was isolated from the NAM1 null mutant strain
(nam1) and its corresponding isogenic wild-type strain
(NAM1) after growing in SD medium at 30 °C. The resulting
RNA blot was successively hybridized with COB,
COX1, and COX3 exon probes (as indicated to the
left of the figure). The size of each transcript detected is
indicated in kb to the right of the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 mutation did not abolish Nam1p binding
in this assay, whereas the rpo41
3 mutation did, suggests that the primary binding site being assayed here lies
between the end points of these two deletions (amino acids 118-208).
Consistent with this proposal, alignment of the amino-terminal extensions of S. cerevisiae, N. crassa, and
S. pombe revealed that, although not highly conserved
overall, the region exhibiting the highest degree of
identity/similarity corresponds to amino acids 117-155 of the S. cerevisiae protein (Fig. 1).
3 and N152A/Y154A, exhibit a similar reduction of growth rate on YPG medium at 30 °C and a severe growth defect on YPG at 37 °C (Fig. 2A). Although less severe,
mutations that retain some level of Nam1p binding,
rpo41
2 and E119A/C121A, also exhibit
significant YPG growth defects. In the rpo41
2
case, defective Nam1p binding appears to make a significant
contribution to the phenotype because the YPG growth defect of this
strain can be rescued by overproducing Nam1p (Fig. 3B). This
is consistent with the fact that the end point of the
rpo41
2 deletion impinges on, but does not
eliminate, the most conserved residues identified in the fungal mtRNA
polymerase alignment (Fig. 1). Thus, it is possible that this mutation
reduces the affinity of the interaction but retains the major binding
determinants for Nam1p. Also consistent with a defect in Nam1p binding
is the fact that the rpo41
3 deletion completely removes the conserved domain and cannot be rescued by
overexpression of Nam1p. Thus, suppression of YPG growth defects by
Nam1p overexpression requires the presence of an intact amino-terminal domain, an observation most easily explained by a direct interaction between these two factors.
3 mutation (15).
3 mutation (Fig. 2A), which is
completely devoid of this region (Fig. 1). In fact, the R129D mutation
was still capable of interacting with Nam1p, but exhibited the most
severe mitochondrial phenotype (capable only of slow growth on YPG at 30 °C, Fig. 1). All of these data suggest that point mutations in
the amino-terminal domain can be more deleterious than mutations that
remove this domain altogether. Several explanations can potentially account for these observations. One possibility is that the
amino-terminal domain is involved not only in Nam1p binding, but also
in some other function. Although speculative at this point, it is
tempting to consider that the amino-terminal domain of sc-mtRNA
polymerase may be involved in binding the primary RNA transcript during
transcription, perhaps to facilitate threading of the RNA into a
coupled RNA-processing machinery during transcription. In this regard,
it is noteworthy that the amino-terminal domain of T7 RNA polymerase,
the closest relative to mtRNA polymerases, has been implicated in
nascent RNA binding (25). If this is the case, then mutations in the amino-terminal domain could affect Nam1p binding, RNA binding, or both,
resulting in the more severe phenotypes observed with particular point
mutations. An alternative explanation is that a higher order complex is
involved that contains not only mtRNA polymerase and Nam1p but also
other factors involved in RNA processing/stability or translation. If
this were the case, one could envision how point mutations result in
the observed phenotypes by disrupting the binding of Nam1p, the binding
of other factors, or both. Perhaps consistent with this idea is the
observation that splicing of the group I intron (bI5) in the
mitochondrial CYTB gene is facilitated by the Cbp2 protein
in a transcription-dependent manner in vitro (26), suggesting that other factors involved in mtRNA processing events
may indeed function in a transcription-coupled manner in vivo. Determination of the precise composition of the Nam1p-mtRNA polymerase complex and assignment of additional functions to the amino-terminal domain remain important goals.
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Fig. 5.
The amino-terminal extensions of vertebrate
mtRNA polymerases have a repetitive amino acid motif similar to that
found in CRP1, a protein involved in RNA-processing and translation in
chloroplasts. The human mtRNA polymerase is diagrammed at the
top in the same manner as the yeast enzyme in Fig. 1. A
linear representation of the Zea mays CRP1 protein is
also presented. The region of amino acid sequence similarity that is
common to vertebrate (human and Xenopus) mtRNA polymerases
and CRP1 is depicted as a hatched box. Shown at the bottom
is a ClustalW (30) alignment of two regions in CRP1 (CRP-box1 and
CRP-box2) with the analogous region of the human and Xenopus
mtRNA polymerases. Black-boxed letters denote amino acid
identity, and gray-boxed letters indicate amino acid
similarity. Recent evidence suggests that these regions of CRP1 are
composed of a tandemly repeated, 35-amino acid domain called a PPR
motif that appears to define a new family of proteins involved in RNA
processing and translation in organelles (29). Based on a proposed
consensus sequence for a PPR motif (29), it appears that the vertebrate
mtRNA polymerases contain at least two PPR repeats in the
amino-terminal extension (indicated at the bottom of the
figure).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Yuan Wang for early contributions to this work, Melissa McKay for suggesting the PCR mutagenesis protocol, and Dr. Bonnie Seidel-Rogol for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by NHLBI Grant HL-59655 from the National Institutes of Health (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.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, Rollins Research Center, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-3798; Fax: 404-727-3954; E-mail: gshadel@emory.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M009901200
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase pair; PCR, polymerase chain reaction; aa, amino acids.
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