Translation initiation in Saccharomyces cerevisiae ... University School of Medicine Stanford CA 94305 ... (IF2) have similar and partially overlapping functions in yeast mitochondrial

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: [email protected]

Phone: (607) 254-4835

Fax: (607) 255-6249

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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.

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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).

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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

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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+

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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

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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

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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.

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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

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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

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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.

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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

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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