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Translation initiation in Saccharomyces cerevisiae mitochondria: Functional interactions
among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-
formyltransferase, and novel protein Rmd9p
Elizabeth H. Williams*1, Christine A. Butler*, Nathalie Bonnefoy† and Thomas D. Fox*
*Department of Molecular Biology and Genetics
Cornell University
Ithaca, NY 14853
USA
†Centre de Génétique Moléculaire
Laboratoire propre du CNRS associé à l’Université Pierre et Marie Curie
91198 Gif-sur-Yvette cedex
France
1 Present Address: Department of Developmental Biology
Stanford University School of Medicine
Stanford CA 94305
Genetics: Published Articles Ahead of Print, published on December 28, 2006 as 10.1534/genetics.106.064576
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Running Title: Mitochondrial translation initiation
Key Words:
Mitochondrial gene expression
Initiation factor 2
fMet-tRNAfMet
methionyl-tRNA-formyltransferase
Corresponding Author:
Thomas D. Fox
Department of Molecular Biology and Genetics
Biotech Building
Cornell University, Ithaca, NY 14853-2703
E-mail: tdf1@cornell.edu
Phone: (607) 254-4835
Fax: (607) 255-6249
3
ABSTRACT
Rsm28p is a dispensable component of the mitochondrial ribosomal small subunit in
Saccharomyces cerevisiae that is not related to known proteins found in bacteria. It was
identified as a dominant suppressor of certain mitochondrial mutations that reduced translation
of the COX2 mRNA. To explore further the function of Rsm28p, we isolated mutations in other
genes that caused a synthetic respiratory defective phenotype together with rsm28∆. These
mutations identified three nuclear genes: IFM1, which encodes the mitochondrial translation
initiation factor 2 (IF2), FMT1, which encodes the methionyl-tRNA-formyltransferase, and
RMD9, a gene of unknown function. The observed genetic interactions strongly suggest that the
ribosomal protein Rsm28p and Ifm1p (IF2) have similar and partially overlapping functions in
yeast mitochondrial translation initiation. Rmd9p, bearing a TAP-tag, was localized to
mitochondria and exhibited roughly equal distribution in soluble and membrane-bound fractions.
A small fraction of the Rmd9-TAP sedimented together with presumed monosomes, but not with
either individual ribosomal subunit. Thus, Rmd9 is not a ribosomal protein, but may be a novel
factor associated with initiating monosomes. The poorly respiring rsm28∆, rmd9-V363I double
mutant did not have a strong translation defective phenotype, suggesting that Rmd9p may
function upstream of translation initiation, perhaps at the level of localization of mitochondrially
coded mRNAs.
4
INTRODUCTION
Translation initiation appears to be a key point of regulation in the expression of
mitochondrial genes in Saccharomyces cerevisiae. Both the level and location of protein
synthesis within the organelle are strongly influenced by membrane-bound mRNA-specific
translational activators that recognize target sites in the leaders of mitochondrially coded
mRNAs (FIORI et al. 2003; GREEN-WILLMS et al. 2001; RÖDEL 1997; SANCHIRICO et al. 1998;
STEEL and BUSSOLI 1999). mRNA features necessary for the selection of translation start sites
include both the initiation codon itself and other features of the mRNA (BONNEFOY and FOX
2000; FOLLEY and FOX 1991; MULERO and FOX 1994). However, the mechanisms by which
mitochondrial translation is initiated are poorly understood, owing largely to the absence of in
vitro systems derived from the organelles. Furthermore, the extraordinary divergence of
mitochondrial genetic systems from their eubacterial ancestors and from each other (GRAY et al.
2004) limits the degree to which mechanisms can be inferred by the identification of
components homologous to those of bacteria. Therefore, genetic analysis is an important tool
for the further study of translation initiation in mitochondria.
The mitochondrially coded COX2 mRNA contains within its open reading frame
antagonistic signals that affect translation efficiency: a positive-acting sequence within the first
15 codons (BONNEFOY et al. 2001) and inhibitory sequence elements further downstream
(WILLIAMS and FOX 2003). Mutations in the positive-acting sequence strongly reduce translation
of the cox2 mRNA and produce nonrespiratory growth phenotypes due to cytochrome oxidase
deficiency. It is not known whether the mutations affect initiation, elongation, or both. These
cox2 mutations can be suppressed by compensating mutations in the COX2 reading frame,
overproduction of the COX2 mRNA-specific translational activator Pet111p, overproduction of
the large subunit mitochondrial ribosomal protein MrpL36p, and by a dominant chromosomal
mutation that alters the structure of the small subunit mitochondrial ribosomal protein Rsm28p
(BONNEFOY et al. 2001; WILLIAMS et al. 2005; WILLIAMS et al. 2004).
5
Rsm28p, which has no detectable homology to bacterial ribosomal proteins, is required
for fully efficient translation of at least the COX1, COX2, and COX3 mRNAs as judged by
expression of a reporter gene inserted into each mitochondrial locus (WILLIAMS et al. 2005).
However, it is not essential for mitochondrial translation since rsm28∆ mutants are able to grow
on nonfermentable carbon sources, albeit with reduced efficiency. The dominant suppressor
mutation, RSM28-1, is an internal in-frame deletion of 67 codons that appears to increase or
alter the activity of the protein, improving expression of the cox2 mutant mRNAs. Interestingly,
RSM28-1 also weakly suppresses both cox2 and cox3 initiation codon mutations (WILLIAMS et
al. 2005). These findings suggest that Rsm28p could have a positive role in translation initiation
that is enhanced by the internal deletion.
To examine further the function of Rsm28p we have screened for additional mutations
that enhance the translation defect caused by rsm28∆, thereby producing synthetic respiratory
defective growth phenotypes. Two of the genes identified in this screen encode mitochondrial
translation initiation factor 2 (IF2) and the mitochondrial methionyl-tRNA-formyltransferase,
strongly suggesting that Rsm28p in fact does have a role in translation initiation. The third
gene identified in this screen, RMD9, had not previously been ascribed to any defined cellular
process but is now implicated in mitochondrial gene expression.
MATERIALS AND METHODS
Yeast strains, media, and genetic methods: S. cerevisiae strains relevant to this study
are listed in Table 1. Strains used were isogenic or congenic to D273-10B (ATCC #25627),
except for YSC1178-7500474. Yeast were cultured in either complete medium (1% yeast
extract, 2% bacto-peptone, 50 mg adenine/L) or synthetic complete media (0.67% yeast
nitrogen base supplemented with appropriate amino acids) containing 2% glucose, 2%
raffinose, or 3% ethanol / 3% glycerol. Standard genetic techniques were performed as
described (Fox et al. 1991; Guthrie and Fink 1991; Sherman et al. 1974). Nonrespiring mutant
6
strains were tested for the presence of wild-type mtDNA (rho+) by mating to rho0 strains
DA1rho0 or DL2rho0 and scoring growth of the resulting diploids on a nonfermentable carbon
source.
Screen for mutations creating a synthetic Pet- phenotype in the presence of
rsm28∆: The wild-type RSM28 gene with roughly 500 flanking base-pairs on both sides was
isolated by PCR amplification from strain NAB97. The resulting fragment was cleaved with XbaI
and inserted into XbaI-cleaved pTSV31A, a high copy 2µ ADE3 URA3 plasmid kindly provided
by J. Pringle, to generate pCB8. Strain CAB67 was transformed with pCB8, and the
transformant was subjected to mutagenesis with ethylmethane sulfonate as described
(LAWRENCE 1991). Mutagenized cells were plated for single colonies on complete
nonfermentable medium (YPEG) and incubated at 30° for five to six days. Nonsectored red
colonies were picked and restreaked to YPEG (#1). These streaks were printed to complete
fermentable medium, YPD, to allow plasmid loss, and the YPD plates were printed to medium
containing 5-fluoroorotic acid. Following growth on 5-fluoroorotic acid, the cells lacking the
plasmid pCB8 were printed to YPEG (#2). Putative mutants grew on YPEG (#1), before
plasmid loss, but not on YPEG (#2), after plasmid loss. Putative mutants lacking pCB8 were
tested for rho+ by mating to DL2rho0. rho+ putative mutants were next mated to the rsm28∆
rho0 strain NAB109rho0 to test for the presence of a recessive nuclear mutation causing a
synthetic Pet- phenotype: five putative mutants gave Pet+ diploids in this cross, suggesting they
had new recessive mutations. These diploids were sporulated, and in each case Pet+
segregated 2:2. MATa spores bearing each mutation were crossed back to each of the original
mutants, and the phenotypes of the resulting diploids scored. This complementation analysis
indicated that the mutations identified three distinct genes.
Isolation and identification of genes interacting with rsm28∆: Functional genes
corresponding to the mutations causing synthetic Pet- phenotypes were isolated by
transformation of three mutant strains with libraries of wild-type S. cerevisiae DNA. Pet+
7
transformants were screened by PCR for the absence of plasmid-borne RSM28. FMT1 was
identified in transformants of CAB74 bearing genomic fragments inserted into YCP50 (ROSE et
al. 1987). IFM1 was identified in transformants of CAB75 bearing genomic fragments inserted
into pFL44L (STETTLER et al. 1993). RMD9 was the gene isolated in transformants of CAB76
bearing a cDNA inserted into pFL61 (MINET et al. 1992). RMD9 was also isolated from a library
of genomic fragments in YEP24 (GREEN-WILLMS et al. 1998). Chromosomal mutations were
identified by PCR amplification of mutant genes from genomic DNA and sequence analysis of
the total amplification products. Sequencing of the FMT1 gene from wild-type strains of the
D273-10B (ATCC 25657) and S288C (GOFFEAU et al. 1996) genetic backgrounds revealed an
error in the original reference genomic sequence (GOFFEAU et al. 1996). A 1 nucleotide insertion
in the C-terminal coding sequence relative to the database sequence extended the predicted
polypeptide from 393 to 401 residues (GenBank accession no. AY490279).
Mitochondrial isolation, subfractionation and protein analyses. Mitochondria were
isolated and purified on Nycodenz gradients from yeast cells grown on complete medium
containing raffinose as previously described (GLICK and PON 1995). Mitochondrial ribosomes
were extracted from purified mitochondria as previously described (WILLIAMS et al. 2005) and
layered onto a 39 ml continuous 15-30% sucrose gradient containing 100mM NH4Cl, 10mM
Tris, 10mM Mg acetate pH 7.4, 7 mM beta-mercaptoethanol, 0.2% Triton X-100, 0.5 mM PMSF,
one complete protease inhibitor mini tablet without EDTA (Roche). Gradients were centrifuged,
fractionated and subjected to SDS gel electrophoresis and Western blotting as previously
described (WILLIAMS et al. 2005). Rmd9p-TAP was detected by incubation of the blots with
peroxidase-anti-peroxidase soluble complex (Sigma). Mrp7p and Mrp13p were detected using
mouse monoclonal antibodies (FEARON and MASON 1988; PARTALEDIS and MASON 1988) as
previously described (WILLIAMS et al. 2005).
In vivo pulse-labeling with 35S-methionine in the presence of cycloheximide was
performed as described (FOX et al. 1991) with the following modifications. Cells were grown to
8
saturation in liquid 1% yeast extract, 2% bacto-peptone, 2% raffinose and then transferred to
synthetic complete medium lacking Met (0.67% yeast nitrogen base, 0.08% CSM-Met (Bio 101,
Inc.), 2% raffinose). After labeling for 30 minutes the cells were chased with unlabeled 2.5 mM
methionine for 30 minutes before isolation of crude mitochondria.
RESULTS
Isolation of mutations in three genes that cause synthetic Pet- phenotypes with
rsm28∆: To explore the function of Rsm28p, we took advantage of the fact that it is dispensable
for respiratory growth by looking for mutations in other genes that would cause a synthetic Pet-
(respiratory defective) phenotype together with an rsm28∆::LEU2 mutation. Starting with an
rsm28∆, ade2, ade3, ura3 strain (CAB67) containing a multicopy plasmid bearing RSM28,
ADE3, and URA3 (pCB8), we used a modification of the sectored colony screen (BENDER and
PRINGLE 1991) to identify mutants that could not grow on nonfermentable medium if the plasmid
was lost (Materials and Methods). The screen yielded five independent recessive nuclear
mutations that caused Pet- growth phenotypes only in an rsm28∆ background. These five
mutations identified three complementation groups (Materials and Methods): two groups with
two linked mutations each, and one group with the remaining mutation.
Three rsm28∆ strains (CAB74, CAB75 and CAB76), each containing a mutation from
one of the three complementation groups, were transformed with libraries of wild-type DNA
fragments (Materials and Methods). Pet+ transformants were isolated, and those containing
plasmids bearing RSM28 were identified by PCR and discarded. Characterization of the
remaining complementing plasmids, and further analysis, revealed that this screen had
identified FMT1, IFM1 and RMD9 as genes interacting with RSM28.
The dispensable mitochondrial methionyl-tRNA-formyltransferase, Fmt1p, is
essential for respiratory growth in the absence of Rsm28p: Overlapping genomic clones
9
that complemented the respiratory defect of strain CAB74 (Materials and Methods) all contained
the gene FMT1, encoding the mitochondrial methionyl-tRNA-formyltransferase (LI et al. 2000).
We confirmed that this candidate gene was indeed the active locus by isolating a plasmid
(pEHW255) bearing only the wild-type FMT1 gene and showing that it too complemented when
transformed into CAB74. Finally, sequencing of this gene amplified from CAB74 genomic DNA
revealed a frameshift mutation truncating the normally 401 amino acid protein after residue 335
(Table 2). To generate a true null allele, we constructed an fmt1∆::URA3 complete deletion. As
previously reported (LI et al. 2000), the absence of Fmt1p had virtually no effect on respiratory
growth (Fig. 1). However an rsm28∆, fmt1∆ double mutant haploid, EHW468, failed to respire,
although the cells remained stably rho+ (Fig. 1). Since a complete block in mitochondrial
translation destabilizes mtDNA, producing rho- mutants (MYERS et al. 1985), this result indicates
that residual mitochondrial translation occurs in the absence of both Rsm28p and Fmt1p.
Nevertheless, the absence of formylated methionine on the mitochondrial initiator tRNA
enhances the modest translational defect caused by the lack of Rsm28p.
Absence of Rsm28p sensitizes mitochondrial translation to mutations altering
mitochondrial translation initiation factor 2. Overlapping genomic clones that complemented
the respiratory defect of strain CAB75 (Materials and Methods) all contained the gene IFM1,
encoding the mitochondrial homologue of bacterial translation initiation factor 2 (VAMBUTAS et al.
1991). We confirmed that this candidate gene was indeed the active locus by isolating a plasmid
(pCB12) bearing only the wild-type IFM1 gene, and showing that it too complemented when
transformed into CAB75. Finally, sequencing of this gene amplified from CAB75 genomic DNA
by PCR revealed the presence of a missense mutation, ifm1-Q234K (Table 2). Ifm1p
(mitochondrial translation initiation factor 2) is required for normal levels of mitochondrial
translation and for respiratory growth (TIBBETTS et al. 2003; VAMBUTAS et al. 1991). Since
strains carrying only the ifm1-Q234K mutation can grow on nonfermentable medium, this
missense mutation must alter or reduce, but not destroy, Ifm1p function.
10
Null mutants entirely lacking Ifm1p retain the ability to translate mitochondrially coded
mRNAs, albeit at greatly reduced rates, and can therefore maintain rho+ mtDNA (TIBBETTS et al.
2003). We constructed by transformation an ifm1∆::URA3 complete deletion mutant, CAB78,
and confirmed that it retained mtDNA. Furthermore, CAB78 crossed to wild-type DUL1 yielded
tetrads containing Ura-, Pet+, rho+ spores and Ura+, Pet-, rho+ spores in a 2:2 ratio, as expected.
To test the phenotype of ifm1∆::URA3, rsm28∆::LEU2 double null mutants we crossed CAB78
to the rsm28∆::LEU2 strain CAB67. In this case, every ifm1∆::URA3 spore was rho-, regardless
of whether it was RSM28 or rsm28∆::LEU2. Thus, it appears that reduced levels of Rsm28p in
the heterozygous rsm28∆/RSM28 diploid cells that formed these haploid spores prevented the
spores lacking IFM1 from maintaining rho+ mtDNA, presumably due to reduced translation. This
quantitative genetic interaction of null mutations is more severe than the original synthetic
defective phenotype observed with the ifm1-Q234K missense mutation, and confirms that
Rsm28p and Ifm1p are likely to have roles in the same process.
In bacteria, initiation factor 2 stimulates binding of initiator fMet-tRNAfMet to the ribosomal
small subunit, in a reaction partially dependent on formylation of the charged tRNA (LAURSEN et
al. 2005). We therefore expected that the absence of formylation caused by an fmt1∆ mutation
would have no synergistic effect on an ifm1∆ mutant. Indeed, fmt1∆, ifm1∆ double mutants
were Pet-, rho+, a phenotype indistinguishable from that of the ifm1∆ single mutant.
Partial suppression of the ifm1∆ respiratory growth defect by the dominant
RSM28-1 mutation. The synthetic defective interactions between the rsm28∆ null mutation
and both fmt1 and ifm1 mutations indicate that Rsm28p may play a role in yeast mitochondrial
translation initiation. To test further this hypothesis, we asked whether the dominant
hypermorphic allele, RSM28-1, originally selected as a suppressor of cox2 translational defects
(BONNEFOY et al. 2001; WILLIAMS et al. 2005), might also suppress the respiratory growth defect
caused by the lack of mitochondrial initiation factor 2, Ifm1p. An ifm1∆::URA3 strain was
11
crossed to an RSM28-1 strain, and the ability of haploid progeny in tetrads to grow on
nonfermentable carbon sources was scored (Fig. 2). Every tetrad contained two spores with the
ifm1∆::URA3 mutation, but half of these spores exhibited weak respiratory growth, indicating
suppression by the unlinked RSM28-1 mutation. The control cross of the ifm1∆::URA3 strain to
a wild-type RSM28 strain produced a normal 2:2 segregation of the ifm1∆::URA3 Pet-
phenotype, as expected (Fig. 2). These results support the notion that Rsm28p has a role in
mitochondrial translation initiation.
A missense mutation in RMD9, a gene of unknown function, causes respiratory
deficiency in strains lacking Rsm28p. The respiratory defect of strain CAB76, carrying a
mutation in the third complementation group causing synthetic respiratory deficiency with
rsm28∆, was complemented by a plasmid from a bank of yeast cDNAs inserted into the
expression vector pFL61 (MINET et al. 1992) (Materials and Methods). This plasmid contained
the reading frame corresponding to RMD9 (YGL107C), a gene first identified as 'Required for
Meiotic nuclear Division' (ENYENIHI and SAUNDERS 2003). This gene was also present on
several overlapping genomic fragments that complemented the CAB76 respiratory defect.
Sequencing of the RMD9 locus from strain CAB76 revealed the presence of a missense
substitution, rmd9-V363I (Table 2). This synthetic defective allele does not inactivate the gene
since a strain carrying only this mutation was respiratory competent, while an rmd9∆::URA3
allele we constructed caused a tight nonrespiratory phenotype. (This tight respiratory defect
explains the 'Required for Meiotic nuclear Division' since yeast cells must be respiratory
competent to sporulate.) In the D273-10B strain background used in this study, the
rmd9∆::URA3 mutation caused cells to become rho-, suggesting that Rmd9p is essential for
mtDNA maintenance, and therefore possibly overall mitochondrial translation. However, in
strains whose mtDNA lacks all known introns, the absence of Rmd9p only partially reduced the
stability of mtDNA (NOUET et al. 2006). Therefore, while essential for respiratory growth,
Rmd9p is not absolutely essential for residual mitochondrial gene expression.
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We further examined the phenotype of the rsm28∆, rmd9-V363I double mutant by
labeling mitochondrial translation products in vivo in the presence of cycloheximide (Fig. 4A).
By this assay, the double mutant had modestly reduced translation relative to wild-type and both
single mutant strains. Interestingly, labeling of the cytochrome oxidase subunits Cox1p, Cox2p
and Cox3p was reduced relative to labeling of apo-cytochrome b in the double mutant. This
somewhat specific reduction in cytochrome c oxidase was confirmed by spectral analysis of
cytochromes in whole cells: while cytochromes a+a3 were undetectable in the double mutant,
cytochrome b absorbance was still evident (Fig. 4B).
Rmd9p is a mitochondrial membrane protein at least partially associated with
mitochondrial ribosomes. A large scale study of fusion protein location (HUH et al. 2003) and
proteomic analysis of yeast mitochondria (SICKMANN et al. 2003) found Rmd9p in mitochondria.
Submitochondrial analysis of functional myc-epitope tagged Rmd9p revealed that it was
peripherally associated with the inner surface of the inner membrane (NOUET et al. 2006). We
examined the location of Rmd9p in mitochondria purified from a strain bearing a chromosomally
integrated RMD9::TAP fusion gene that placed the TAP-tag at the C-terminus (GHAEMMAGHAMI
et al. 2003). This strain exhibited normal respiratory growth indicating that Rmd9p-TAP was
functional. Analysis of soluble and membrane fractions (GLICK 1995) of these mitochondria
indicated that Rmd9p-TAP was roughly evenly distributed between them (unpublished data).
This behavior was reminiscent of mitochondrial ribosomal proteins (MCMULLIN and FOX 1993;
WILLIAMS et al. 2005).
To ask whether Rmd9p-TAP was associated with mitochondrial ribosomal subunits we
subjected detergent solubilized mitochondria to sucrose density gradient sedimentation under
standard, high salt (500 mM NH4Cl), conditions. Analysis of the gradient fractions revealed that
the bulk of the Rmd9p-TAP sedimented slowly, and showed evidence of proteolytic degradation.
While some Rmd9p-TAP sedimented into the gradient, there were no distinct peaks
(unpublished results). This result strongly indicated that Rmd9p-TAP is not a true ribosomal
13
protein, but did not rule out a weaker ribosomal association which could be detectable at lower
salt concentrations (DATTA et al. 2004). We therefore subjected solubilized mitochondria to
gradient sedimentation under low salt (100 mM NH4Cl) conditions (Fig. 3). In this case we
reproducibly observed a faint but distinct peak of rapidly sedimenting Rmd9p-TAP at a position
where both the small ribosomal subunit marker protein Mrp13p and the large subunit marker
Mrp7p co-sedimented. This result indicates that at the lower salt concentration some yeast
mitochondrial monosomes remain intact and that a small fraction of the Rmd9p-TAP is loosely
associated with those monosomes. There was no indication that Rmd9p-TAP is specifically
associated with either separated ribosomal subunit. In addition, some Rmd9p-TAP, but neither
ribosomal protein marker, sedimented to the bottom fractions of the gradient. The nature of this
species remains to be determined.
DISCUSSION
While the function of mitochondrial small ribosomal subunit protein Rsm28p is not
essential for mitochondrial translation or respiratory growth, previous work had suggested that it
plays a role in general translation initiation and/or early steps in elongation (BONNEFOY et al.
2001; WILLIAMS et al. 2005). This study of mutations that cause synthetic respiratory defects in
the absence of Rsm28p has provided strong evidence for a role in an early initiation step by
identifying interacting genes encoding mitochondrial translation initiation factor 2, and the
methionyl-tRNA-formyltransferase. We also identified a new gene product, Rmd9p, that, based
on our results and those of Nouet et al. (2006), is likely to participate mRNA maturation and
localization, as well as in translation initiation at the surface of the inner membrane. The
genetic interactions observed in this study are summarized in Table 3.
In bacteria, translation initiation factors 1, 2 and 3 (IF1, IF2 and IF3) participate in the
assembly of active initiation complexes containing the initiator fMet-tRNAfMet, mRNA, the small
ribosomal subunit and the large ribosomal subunit (reviewed in (LAURSEN et al. 2005). IF2 is a
14
GTP/GDP-binding protein that interacts directly with the ribosomal small subunit and the initiator
fMet-tRNA, and positions the tRNA in the ribosomal P site. IF1 and IF3 appear to promote
dissociation of ribosomal subunits prior to initiation, assist IF2 in proofreading the fMet-tRNAfMet-
initiation codon interaction, and transition the ribosome to the decoding mode.
The synthetic defective interaction between RSM28 and IFM1 can be rationalized if one
assumes that Rsm28p and Ifm1p (IF2) have partially overlapping, mutually reinforcing roles in
establishing productive initiation complexes and/or accurate positioning of the fMet-tRNAfMet on
the mRNA and ribosomal small subunit. The recessive missense mutation synthetically
defective with rsm28∆, ifm1-Q234K, affects a highly conserved residue in the G-domain of IF2,
corresponding to E. coli IF2 codon 478 (LAURSEN et al. 2003; LAURSEN et al. 2005). We do not
know whether this mutation affects the presumed GTPase activity of the protein. However,
ifm1-Q234K presumably reduces mitochondrial IF2 activity to the point that Rsm28p becomes
necessary to promote translation initiation at a level sustaining respiratory growth. In the
complete absence of both Rsm28p and Ifm1p, cells lose functional mtDNA (become rho-)
indicating a complete loss of mitochondrial translation (MYERS et al. 1985).
Supporting the notion that Rsm28p and Ifm1p have mutually supporting roles, we also
observed a ‘positive’ genetic interaction between RSM28 and IFM1. The dominant RSM28-1
mutation, which is an internal in-frame deletion of 67 codons that was isolated as a suppressor
that improves expression of translationally defective COX2 mRNAs, appears to have increased
Rsm28p activity (WILLIAMS et al. 2005). Interestingly, RSM28-1 partially suppressed the
respiratory growth defect of an ifm1∆ mutation, suggesting that this apparently hypermorphic
allele improved mitochondrial translation initiation in the absence of Ifm1p (IF2). Bacterial IF2
has also been reported to have an additional chaperone-like activity (CALDAS et al. 2000),
raising the possibility that Rsm28p might also play a role in protein folding.
Formylation of the charged initiator fMet-tRNAfMet strengthens its interaction with
bacterial IF2 (SUNDARI et al. 1976) and, to a lesser extent, yeast mitochondrial IF2 (GAROFALO
15
et al. 2003). E. coli mutants lacking the formyltransferase grow very poorly but are viable
(GUILLON et al. 1992), while yeast fmt1 mutants lacking the mitochondrial formyltransferase
exhibit essentially normal respiratory growth (LI et al. 2000; TIBBETTS et al. 2003). However, in
the absence of Rsm28p, formylation becomes required to support a level of mitochondrial
translation sufficient to sustain respiratory growth. Perhaps the absence of formylation in fmt1
mutants causes a reduction in effective mitochondrial IF2 activity, thereby producing a synthetic
defect with rsm28∆ that mimics the one produced by ifm1-Q234K. This notion predicts that a
double ifm1∆, fmt1∆ mutant would have a phenotype (Pet-, rho+) similar to the ifm1∆ single
mutant, a prediction that we have confirmed.
Apparent orthologs of bacterial IF2 and S. cerevisiae Ifm1p can be found in most
eukaryotes, and presumably perform critical functions in mitochondrial translation. One
interpretation of our results is that the small subunit ribosomal protein Rsm28p has some
functions in budding yeast mitochondrial initiation that are played by IF1 and/or IF3 in bacteria
(LAURSEN et al. 2005), and IF3 in mammalian mitochondria (BHARGAVA and SPREMULLI 2005).
Analysis of homologous genes in the genomes of other fungal species is largely consistent with
this possibility. No genes encoding potential mitochondrial proteins homologous to bacterial IF1
have been identified in eukaryotes (KOC and SPREMULLI 2002), although genes coding divergent
forms of this short protein could go undetected. Divergent homologs of bacterial IF3 are present
in mammals and in the fission yeast Schizosaccharomyces pombe (BHARGAVA and SPREMULLI
2005). No clear homologs are present in budding yeasts, although a possible candidate of
questionable significance has been reported to exist in the S. cerevisiae genome (KOC and
SPREMULLI 2002). In contrast, genes encoding proteins homologous to Rsm28p can be
identified in genomes of species closely related to S. cerevisiae, such as Kluyveromyces lactis,
Candida glabrata, and Ashbya gossypii, but appear to be absent in S. pombe. However, neither
RSM28 homologs nor genes encoding IF3 homologs are detectable in the genomes of Candida
albicans or Neurospora crassa.
16
The third gene identified in our synthetic defective screen with rsm28∆ was RMD9.
Rmd9p is conserved among budding yeasts, but N. crassa and S. pombe appear to lack it. It is
not closely related to any proteins of known function. In the D273-10B (ATCC 25657) strain
background employed in this study, deletion of RMD9 caused cells to lose rho+ mtDNA.
RMD9 has also been isolated independently in a screen for high-copy suppressors of a
temperature sensitive oxa1 mutation (NOUET et al. 2006). Oxa1p is a highly conserved integral
inner membrane protein that participates in the membrane insertion of mitochondrially encoded
proteins (BAUER et al. 1994; BONNEFOY et al. 1994; HE and FOX 1997; HELL et al. 1997;
KERMORGANT et al. 1997). The homologous bacterial protein, YidC, has similar functions with
respect to the plasma membrane (LUIRINK et al. 2005). The Oxa1p C-terminal domain is
exposed on the matrix side of the membrane and interacts with mitochondrial ribosomes,
apparently facilitating co-translational insertion of newly synthesized proteins (JIA et al. 2003;
OTT et al. 2006; SZYRACH et al. 2003).
Despite the genetic interactions of RMD9 with both the ribosomal protein gene RSM28
and OXA1, the roles of Rmd9p in mitochondrial gene expression remain poorly defined. Nouet
et al. (2006) localized epitope-tagged Rmd9p to the inner surface of the inner membrane, where
it could participate in localized translation initiation, possibly in conjunction with Oxa1p and
mRNA-specific translational activators (NAITHANI et al. 2003; SANCHIRICO et al. 1998). They
found that Rmd9p is not essential for all mitochondrial translation since rmd9∆ mutants
containing mtDNA that lacked introns were respiratory deficient but able to remain rho+.
Interestingly, these rmd9∆, rho+ strains exhibited profoundly lowered levels of mitochondrially
encoded mRNAs for respiratory complex subunits, consistent with a role for Rmd9p prior to
translation initiation, perhaps in mRNA processing, localization and/or stabilization. Similar
roles have been ascribed to the mitochondrial proteins Nam1p (GROUDINSKY et al. 1993; WALLIS
et al. 1994) and Sls1p (BRYAN et al. 2002; RODEHEFFER and SHADEL 2003).
17
Our isolation of the recessive rmd9-V363I missense mutation as an enhancer of the
rsm28∆ phenotype suggests that one function of Rmd9p could be participation in mitochondrial
translation initiation, an hypothesis supported by the fact that a fraction of Rmd9p was
associated with assembled mitochondrial monosomes during sedimentation of solubilized
organelles in low salt. However, Rmd9p is not a ribosomal protein. Furthermore, analysis of
mitochondrial translation products in the rsm28∆, rmd9-V363I double mutant revealed only a
modest decrease in protein synthesis, and this effect was more pronounced for the three
cytochrome c oxidase subunits than for cytochrome b. These data are consistent with the
possibility that the mutant protein Rmd9p-V363I causes a modest defect in delivery of
mitochondrially coded mRNAs to their proper location on the inner membrane, and reduces the
efficiency of translation initiation, an effect that is exacerbated by loss of RSM28 and the
resulting dependence of mitochondrial translation initiation on IF2 alone.
In view of the known functions of the other genes isolated in our screen, IFM1 and
FMT1, we propose that the budding yeast-specific protein Rsm28p functions in mitochondria in
the assembly of active translation initiation complexes capable of directing nascent chains into
the inner membrane. Rmd9p is likely to function in delivering mRNAs to initiation complexes and
in the initiation process itself. The participation of these unique budding yeast proteins in this
otherwise highly conserved process (LOMAKIN et al. 2006) is apparently a reflection of the high
degree of evolutionary divergence observed among mitochondrial genetic systems (GRAY et al.
2004).
Acknowledgements: We thank F. Lacroute for the generous gift of genomic and cDNA
libraries. E.H. Williams was a Howard Hughes Medical Institute Predoctoral Fellow. This work
was supported by grants from by the Association Française contre les Myopathies (to N.B.) and
the U.S. National Institutes of Health (Grant GM29362 to T.D.F.).
18
LITERATURE CITED
BAUER, M., M. BEHRENS, K. ESSER, G. MICHAELIS and E. PRATJE, 1994 PET1402, a
nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2
in yeast. Mol. Gen. Genet. 245: 272-278.
BENDER, A., and J. R. PRINGLE, 1991 Use of a screen for synthetic lethal and multicopy
suppressee mutants to identify two new genes involved in morphogenesis in
Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 1295-1305.
BHARGAVA, K., and L. L. SPREMULLI, 2005 Role of the N- and C-terminal extensions on
the activity of mammalian mitochondrial translational initiation factor 3. Nucleic
Acids Res. 33: 7011-7018.
BONNEFOY, N., N. BSAT and T. D. FOX, 2001 Mitochondrial translation of Saccharomyces
cerevisiae COX2 mRNA is controlled by the nucleotide sequence specifying the
pre-Cox2p leader peptide. Mol. Cell. Biol. 21: 2359-2372.
BONNEFOY, N., F. CHALVET, P. HAMEL, P. P. SLONIMSKI and G. DUJARDIN, 1994 OXA1, a
Saccharomyces cerevisiae nuclear gene whose sequence is conserved from
prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J. Mol. Biol.
239: 201-212.
BONNEFOY, N., and T. D. FOX, 2000 In vivo analysis of mutated initiation codons in the
mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter
gene ARG8m reveals lack of downstream reinitiation. Mol. Gen. Genet. 262:
1036-1046.
BRYAN, A. C., M. S. RODEHEFFER, C. M. WEARN and G. S. SHADEL, 2002 Sls1p is a
membrane-bound regulator of transcription-coupled processes involved in
Saccharomyces cerevisiae mitochondrial gene expression. Genetics 160: 75-82.
CALDAS, T., S. LAALAMI and G. RICHARME, 2000 Chaperone properties of bacterial
elongation factor EF-G and initiation factor IF2. J. Biol. Chem. 275: 855-860.
19
CHIRON, S., A. SULEAU and N. BONNEFOY, 2005 Mitochondrial translation: elongation
factor tu is essential in fission yeast and depends on an exchange factor
conserved in humans but not in budding yeast. Genetics 169: 1891-1901.
DATTA, K., J. L. FUENTES and J. R. MADDOCK, 2004 The yeast GTPase Mtg2p is required
for mitochondrial translation and partially suppresses an rRNA methyltransferase
mutant, mrm2. Mol. Biol. Cell 16: 954-963.
ENYENIHI, A. H., and W. S. SAUNDERS, 2003 Large-scale functional genomic analysis of
sporulation and meiosis in Saccharomyces cerevisiae. Genetics 163: 47-54.
FEARON, K., and T. L. MASON, 1988 Structure and regulation of a nuclear gene in
Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of
the mitochondrial ribosome. Mol. Cell. Biol. 8: 3636-3646.
FIORI, A., T. L. MASON and T. D. FOX, 2003 Evidence that synthesis of the
Saccharomyces cerevisiae mitochondrially-encoded ribosomal protein Var1p
may be membrane localized. Eukaryot. Cell 2: 651-653.
FOLLEY, L. S., and T. D. FOX, 1991 Site-directed mutagenesis of a Saccharomyces
cerevisiae mitochondrial translation initiation codon. Genetics 129: 659-668.
FOX, T. D., L. S. FOLLEY, J. J. MULERO, T. W. MCMULLIN, P. E. THORSNESS et al., 1991
Analysis and manipulation of yeast mitochondrial genes. Meth. Enzymol. 194:
149-165.
GAROFALO, C., R. TRINKO, G. KRAMER, D. R. APPLING and B. HARDESTY, 2003
Purification and characterization of yeast mitochondrial initiation factor 2. Arch.
Biochem. Biophys. 413: 243-252.
GHAEMMAGHAMI, S., W. K. HUH, K. BOWER, R. W. HOWSON, A. BELLE et al., 2003 Global
analysis of protein expression in yeast. Nature 425: 737-741.
GLICK, B. S., 1995 Pathways and energetics of mitochondrial protein import in
Saccharomyces cerevisiae. Meth. Enzymol. 260: 224-231.
20
GLICK, B. S., and L. A. PON, 1995 Isolation of highly purified mitochondria from
Saccharomyces cerevisiae. Meth. Enzymol. 260: 213-223.
GOFFEAU, A., B. G. BARRELL, H. BUSSEY, R. W. DAVIS, B. DUJON et al., 1996 Life with
6000 genes. Science 274: 546, 563-547.
GRAY, M. W., B. F. LANG and G. BURGER, 2004 Mitochondria of protists. Annu. Rev.
Genet. 38: 477-524.
GREEN-WILLMS, N. S., C. A. BUTLER, H. M. DUNSTAN and T. D. FOX, 2001 Pet111p, an
inner membrane-bound translational activator that limits expression of the
Saccharomyces cerevisiae mitochondrial gene COX2. J. Biol. Chem. 276: 6392-
6397.
GREEN-WILLMS, N. S., T. D. FOX and M. C. COSTANZO, 1998 Functional interactions
between yeast mitochondrial ribosomes and mRNA 5'-untranslated leaders. Mol.
Cell. Biol. 18: 1826-1834.
GROUDINSKY, O., I. BOUSQUET, M. G. WALLIS, P. P. SLONIMSKI and G. DUJARDIN, 1993
The NAM1/MTF2 nuclear gene product is selectively required for the stability
and/or processing of mitochondrial transcripts of the ATP6 and of the mosaic
COX1 and CYTB genes in Saccharomyces cerevisiae. Mol. Gen. Genet. 240:
419-427.
GUILLON, J. M., Y. MECHULAM, J. M. SCHMITTER, S. BLANQUET and G. FAYAT, 1992
Disruption of the gene for Met-tRNA(fMet) formyltransferase severely impairs
growth of Escherichia coli. J. Bacteriol. 174: 4294-4301.
GUTHRIE, C., and G. R. FINK (Editors), 1991 Guide to Yeast Genetics and Molecular
Biology. Academic Press, San Diego.
HE, S., and T. D. FOX, 1997 Membrane translocation of mitochondrially coded Cox2p:
distinct requirements for export of amino- and carboxy-termini, and dependence
on the conserved protein Oxa1p. Mol. Biol. Cell 8: 1449-1460.
21
HELL, K., J. HERRMANN, E. PRATJE, W. NEUPERT and R. A. STUART, 1997 Oxa1p
mediates the export of the N- and C-termini of pCoxII from the mitochondrial
matrix to the intermembrane space. FEBS Lett 418: 367-370.
HUH, W. K., J. V. FALVO, L. C. GERKE, A. S. CARROLL, R. W. HOWSON et al., 2003 Global
analysis of protein localization in budding yeast. Nature 425: 686-691.
JIA, L., M. DIENHART, M. SCHRAMP, M. MCCAULEY, K. HELL et al., 2003 Yeast Oxa1
interacts with mitochondrial ribosomes: the importance of the C-terminal region of
Oxa1. EMBO J. 22: 6438-6447.
KERMORGANT, M., N. BONNEFOY and G. DUJARDIN, 1997 Oxa1p, which is required for
cytochrome c oxidase and ATP synthase complex formation, is embedded in the
mitochondrial inner membrane. Curr. Genet. 31: 302-307.
KOC, E. C., and L. L. SPREMULLI, 2002 Identification of mammalian mitochondrial
translational initiation factor 3 and examination of its role in initiation complex
formation with natural mRNAs. J. Biol. Chem. 277: 35541-35549.
LAURSEN, B. S., I. SIWANOWICZ, G. LARIGAUDERIE, J. HEDEGAARD, K. ITO et al., 2003
Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-
sensitive growth of Escherichia coli. J. Mol. Biol. 326: 543-351.
LAURSEN, B. S., H. P. SORENSEN, K. K. MORTENSEN and H. U. SPERLING-PETERSEN,
2005 Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev. 69: 101-
123.
LAWRENCE, C. W., 1991 Classical mutagenesis techniques. Meth. Enzymol. 194: 273-
281.
LI, Y., W. B. HOLMES, D. R. APPLING and U. L. RAJBHANDARY, 2000 Initiation of protein
synthesis in Saccharomyces cerevisiae mitochondria without formylation of the
initiator tRNA. J. Bacteriol. 182: 2886-2892.
22
LOMAKIN, I. B., N. E. SHIROKIKH, M. M. YUSUPOV, C. U. HELLEN and T. V. PESTOVA, 2006
The fidelity of translation initiation: reciprocal activities of eIF1, IF3 and YciH.
EMBO J. 25: 196-210.
LUIRINK, J., G. VON HEIJNE, E. HOUBEN and J. W. DE GIER, 2005 Biogenesis of inner
membrane proteins in Escherichia coli. Annu. Rev. Microbiol. 59: 329-355.
MCMULLIN, T. W., and T. D. FOX, 1993 COX3 mRNA-specific translational activator
proteins are associated with the inner mitochondrial membrane in
Saccharomyces cerevisiae. J. Biol. Chem. 268: 11737-11741.
MINET, M., M.-E. DUFOUR and F. LACROUTE, 1992 Complementation of Saccharomyces
cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs. Plant J. 2: 417-
422.
MULERO, J. J., and T. D. FOX, 1994 Reduced but accurate translation from a mutant
AUA initiation codon in the mitochondrial COX2 mRNA of Saccharomyces
cerevisiae. Mol. Gen. Genet. 242: 383-390.
MYERS, A. M., L. K. PAPE and A. TZAGOLOFF, 1985 Mitochondrial protein synthesis is
required for maintenance of intact mitochondrial genomes in Saccharomyces
cerevisiae. EMBO J. 4: 2087-2092.
NAITHANI, S., S. A. SARACCO, C. A. BUTLER and T. D. FOX, 2003 Interactions among
COX1, COX2 and COX3 mRNA-specific translational activator proteins on the
inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae.
Mol. Biol. Cell 14: 324-333.
NOUET, C., M. BOURENS, O. HLAVACEK, S. MARSY, C. LEMAIRE et al., 2006 Rmd9p
controls the processing/stability of mitochondrial mRNAs and its overexpression
compensates for a partial deficiency of Oxa1p in Saccharomyces cerevisiae.
Submitted for publication.
23
OTT, M., M. PRESTELE, H. BAUERSCHMITT, S. FUNES, N. BONNEFOY et al., 2006 Mba1, a
membrane-associated ribosome receptor in mitochondria. EMBO J. 25: 1603-
1610.
PARTALEDIS, J. A., and T. L. MASON, 1988 Structure and regulation of a nuclear gene in
Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit
of the mitochondrial ribosome. Mol. Cell. Biol. 8: 3647-3660.
PEREZ-MARTINEZ, X., S. A. BROADLEY and T. D. FOX, 2003 Mss51p promotes
mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p.
EMBO J. 22: 5951-5961.
RODEHEFFER, M. S., and G. S. SHADEL, 2003 Multiple interactions involving the amino-
terminal domain of yeast mtRNA polymerase determine the efficiency of
mitochondrial protein synthesis. J. Biol. Chem. 278: 18695-18701.
RÖDEL, G., 1997 Translational activator proteins required for cytochrome b synthesis in
Saccharomyces cerevisiae. Curr. Genet. 31: 375-379.
ROSE, M. D., P. NOVICK, J. H. THOMAS, D. BOTSTEIN and G. R. FINK, 1987 A
Saccharomyces cerevisiae genomic plasmid bank based on a centromere-
containing shuttle vector. Gene 60: 237-243.
SANCHIRICO, M. E., T. D. FOX and T. L. MASON, 1998 Accumulation of mitochondrially
synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting
information in untranslated portions of their mRNAs. EMBO J. 17: 5796-5804.
SHERMAN, F., G. R. FINK and C. W. LAWRENCE, 1974 Methods in yeast genetics. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
SICKMANN, A., J. REINDERS, Y. WAGNER, C. JOPPICH, R. ZAHEDI et al., 2003 The
proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci.
USA.
24
STEEL, K. P., and T. J. BUSSOLI, 1999 Deafness genes: expressions of surprise [In
Process Citation]. Trends Genet 15: 207-211.
STETTLER, S., N. CHIANNILKULCHAI, S. HERMANN-LE DENMAT, D. LALO, F. LACROUTE et
al., 1993 A general suppressor of RNA polymerase I, II and III mutations in
Saccharomyces cerevisiae. Mol. Gen. Genet. 239: 169-176.
SUNDARI, R. M., E. A. STRINGER, L. H. SCHULMAN and U. MAITRA, 1976 Interaction of
bacterial initiation factor 2 with initiator tRNA. J. Biol. Chem. 251: 3338-3345.
SZYRACH, G., M. OTT, N. BONNEFOY, W. NEUPERT and J. M. HERRMANN, 2003 Ribosome
binding to the Oxa1 complex facilitates co-translational protein insertion in
mitochondria. EMBO J. 22: 6448-6457.
TIBBETTS, A. S., L. OESTERLIN, S. Y. CHAN, G. KRAMER, B. HARDESTY et al., 2003
Mammalian mitochondrial initiation factor 2 supports yeast mitochondrial
translation without formylated initiator tRNA. J. Biol. Chem.
VAMBUTAS, A., S. J. ACKERMAN and A. TZAGOLOFF, 1991 Mitochondrial translational-
initiation and elongation factors in Saccharomyces cerevisiae. Eur. J. Biochem.
201: 643-652.
WALLIS, M. G., O. GROUDINSKY, P. P. SLONIMSKI and G. DUJARDIN, 1994 The NAM1
protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript
processing/stabilisation, is located in the yeast mitochondrial matrix. Eur. J.
Biochem. 222: 27-32.
WILLIAMS, E. H., N. BSAT, N. BONNEFOY, C. A. BUTLER and T. D. FOX, 2005 Alteration of
a novel dispensable mitochondrial ribosomal small subunit protein, Rsm28p,
allows translation of defective COX2 mRNAs. Eukaryot. Cell 4: 337-354.
WILLIAMS, E. H., and T. D. FOX, 2003 Antagonistic signals within the COX2 mRNA
coding sequence control its translation in Saccharomyces cerevisiae
mitochondria. RNA 9: 419-431.
25
WILLIAMS, E. H., X. PEREZ-MARTINEZ and T. D. FOX, 2004 MrpL36p, a highly diverged
L31 ribosomal protein homolog with additional functional domains in
Saccharomyces cerevisiae mitochondria. Genetics 167: 65-75.
26
TABLE 1. Strains used in this study
Strain Genotypea Source
DA1rho0 MATα ade2 [rho0] (FOLLEY and FOX 1991)
DL2rho0 MATa lys2 [rho0] (FOLLEY and FOX 1991)
DUL1 MATα lys2 ura3∆ [rho+] (FOLLEY and FOX
1991)
CAB67 MATα ade2-101 ade3-24 rsm28∆::LEU2
leu2-3,112 ura3-52 [rho+]
This study
CAB74 MATα ade2-101 ade3-24 rsm28∆::LEU2
leu2-3,112 ura3-52 fmt1-1 [rho+]
This study
CAB75 MATα ade2-101 ade3-24 rsm28∆::LEU2 leu2-
3,112 ura3-52 ifm1-Q234K [rho+]
This study
CAB76 MATα ade2-101 ade3-24 rsm28∆::LEU2
leu2-3,112 ura3-52 rmd9-V363I [rho+]
This study
CAB78 MATa arg8::hisG his3∆HindIII leu2-3,112 lys2
ura3-52 ifm1∆::URA3 [rho+]
This study
CAB104 MATa arg8::hisG his3∆HindIII leu2-3,112 lys2
ura3-52 rmd9-V363I (ade2-101 and/or ade3-24)
[rho+]
This study
EHW227 MATα arg8::hisG his3∆HindIII leu2-3,112 lys2
ura3 RSM28-1 [rho+]
This study
EHW467 MAT� arg8::hisG his3∆HindIII leu2-3,112 lys2
ura3-52 fmt1∆::URA3 [rho+]
This study
EHW468 MATα ade2-101 ade3-24 leu2-3,112 ura3-52
+
This study
27
fmt1∆::URA3 rsm28∆::LEU2 [rho+]
EHW469 MATα ade2-101 ade3-24 leu2-3,112 ura3-52
fmt1∆::URA3 rsm28∆::LEU2 [rho+]
This study
NAB97 MATa arg8::hisG RSM28-HA his3∆HinDIII
leu2-3,112 lys2 ura3-52 [rho+, cox2-22]
(WILLIAMS et al. 2005)
NAB109rho0 MATa arg8∆::hisG rsm28∆::URA3 his3∆HinDIII
leu2-3,112 lys2 ura3-52 [rho0]
This study
NB40-36a MATα lys2, leu2-3,112, arg8::hisG, ura3-52
[rho+]
(PEREZ-MARTINEZ et
al. 2003)
NB80 MATa arg8::hisG his3∆HindIII leu2-3,112 lys2
ura3-52 [rho+]
(BONNEFOY and FOX
2000)
PJD1 MATα ade2-101 ade3-24 leu2-3,112 ura3-52
[rho+]
This study
YSC1178-7500474 MATa his3-delta1 leu2-delta0 ura3-delta0
met15-delta0 RMD9-TAP [rho+]
(GHAEMMAGHAMI et al.
2003)
a Mitochondrial genotypes are in brackets.
28
TABLE 2. Substitutions that caused synthetic respiratory defects in the absence of Rsm28p.
Gene ORF DNA sequence Protein sequence
FMT1 Deletion of C at base 1006 Truncation after residue 335
IFM1 C at base 700 to A Q at position 234 to K
RMD9 G at base 1087 to A V at position 363 to I
29
Table 3. Phenotypes of single and double mutants.
Genotype Respiratory
growth rho+ mtDNA
RSM28 IFM1 FMT1 RMD9 + +
rsm28∆ IFM1 FMT1 RMD9 + +
RSM28 IFM1 fmt1∆ RMD9 + +
rsm28∆ IFM1 fmt1∆ RMD9 - +
RSM28 ifm1∆ fmt1∆ RMD9 - +
RSM28 ifm1-Q234K FMT1 RMD9 + +
rsm28∆ ifm1-Q234K FMT1 RMD9 - +
RSM28 ifm1∆ FMT1 RMD9 - +
rsm28∆ ifm1∆ FMT1 RMD9 - -
RSM28-1 IFM1 FMT1 RMD9 + +
RSM28-1 ifm1∆ FMT1 RMD9 +/- +
RSM28 IFM1 FMT1 rmd9-V363I + +
rsm28∆ IFM1 FMT1 rmd9-V363I - +
RSM28 IFM1 FMT1 rmd9∆ - -a
rms28∆ IFM1 FMT1 rmd9∆ - -
aDeletion of RMD9 causes cells to become rho- in the D273-10B strain background, where
mtDNA contains introns. In strains whose mtDNA lacks introns, rmd9∆ does not prevent
maintenance of mtDNA (NOUET et al. 2006).
30
Figures Legends
Figure 1. Mutations in FMT1 cause a synthetic respiratory defect with rsm28∆. Strains with the
relevant genotypes indicated in the sector diagram were streaked to complete glucose medium
and then printed to complete nonfermentable (Ethanol-Glycerol) and fermentable (Glucose)
media, followed by incubation at 30˚ for 6 and 1 days, respectively. The same streaks were also
mated to a rho˚ tester strain (DL2rho0) and then printed to complete nonfermentable medium to
reveal mtDNA maintenance (Crossed to rho° Ethanol-Glycerol). Strains were, clockwise from
upper left: NB80, EHW467, CAB67, EHW468, CAB74, EHW469 (Table 1).
Figure 2. The dominant mutation RSM28-1 partially suppresses the ifm1∆::URA3 mutation.
The ifm1∆::URA3 strain CAB78 was crossed with the RSM28-1 strain EHW227 (ifm1∆ X
RSM28-1), and the wild-type RSM28 strain NB40-36a (ifm1∆ X RSM28). The diploids were
induced to sporulate and twenty tetrads were dissected from each cross on complete glucose
medium. After growth of the spore clones the plates were replicated to complete
nonfermentable medium and incubated at 30° for four days. The figure shows five tetrads
representative of the twenty analyzed from each cross.
Figure 3. A small fraction of Rmd9p-TAP co-sediments with mitochondrial ribosomes
(monosomes). Ribosomes were extracted from purified mitochondria of strain YSC1178-
7500474 (expressing Rmd9p-TAP) and sedimented through a continuous 15-30% sucrose
gradient containing 0.1 M NH4Cl. Gradient fractions were precipitated, and analyzed by Western
blotting to detect Rmd9p-TAP, the large ribosomal subunit protein Mrp7p (FEARON and MASON
1988) and the small subunit protein Mrp13p (PARTALEDIS and MASON 1988). Top and Bottom
denote the orientation of the gradient. The arrow indicates the peak fraction of intact
monosomes.
31
Figure 4. Mitochondrial protein synthesis and cytochrome spectra of the rsm28∆, rmd9-V363I
double mutant. (A) Mitochondrial translation products were labeled with [35S]methionine in the
presence of cycloheximide, and crude mitochondria were isolated (Materials and Methods). The
strains were RSM28, RMD9 (PJD1); rsm28∆, RMD9 (CAB67); rsm28∆, rmd9-V363I (CAB76);
and RSM28, rmd9-V363I (CAB104), as indicated. Samples were applied to a 15%
polyacrylamide-SDS gel, which was dried and autoradiographed. The major mitochondrial
translation products are indicated. (B) Low temperature cytochrome spectra were recorded
after addition of dithionite to whole cells grown on complete galactose medium at 28°, as
described (CHIRON et al. 2005). Absorption maxima for cytochromes a+a3, b, c1, and c are 602,
558, 552 and 546 nm, respectively. Strains were the same as in (A).
GlucoseEthanol-Glycerol Crossed to rho°Ethanol-Glycerol
RSM28FMT1
RSM28fmt1∆
rsm28∆FMT1
rsm28∆fmt1∆
rsm28∆fmt1-1
rsm28∆fmt1∆
Figure 1
ifm1∆ XRSM28-1
Figure 2
ifm1∆ XRSM28
Figure 3
Top Bottom
Rmd9p-TAP
Mrp7p
Mrp13p
Figure 4
Cox1p
Cyt b
Cox2p
Cox3p
RSM28
, RM
D9
rsm
28∆, R
MD9
rsm
28∆, r
md9
-V36
3I
RSM28
, rm
d9-V
363I
A
B
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