Mitochondrial Genes and theirExpression: YeastPiotr P. SlonimskiCentre de Genetique Moleculaire du Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France

Giovanna CarignaniUniversity of Padova, Padova, Italy

Mitochondria possess their own genome, which encodes a

small part of the proteins that make up the organelle.

The remaining proteins, i.e. the majority, are encoded by the

nuclear genome. Mitochondrial life thus depends on the

coordinated expression and interaction of nuclear genes

and genes present on the organellar genome (mtDNA)

itself. Although mtDNA encodes very few subunits of the

energy-transducing complexes of the inner mitochondrial

membrane, these subunits constitute the key elements of the

overall process: cytochrome b for the reductase activity,

cytochrome c oxidase subunit 1 for the oxidative activity, and

Atp6 for the synthetase activity. The genes coding for these

three subunits are always located in the mtDNA in all

eukaryotes, whether fungi, protists, animals, or plants. The

mtDNA is supposed to be a remnant of a prokaryotic

genome that would have originated from a symbiotic event

between different cellular species. As the majority of the

prokaryotic genes would, however, in the course of evolution,

have migrated to the nucleus of the new cell, it is surprising

that even today a mitochondrial genome exists. A plausible

reason might reside in the compulsory evolution of the

formation mechanism of the enzymatic complexes of the

inner mitochondrial membrane. This might depend on a

topological constraint of the expression and assembly of the

different subunits, some of which are very hydrophobic and

have to be transcribed, translated, inserted, and assembled

from inside the organelle. It follows then that these key genes

are present inside the mitochondrion. This would also

explain the persistence of mtDNA and its complex

machinery of expression for a billion years since the original

symbiotic event.

The knowledge of the conjoint expression of both the

mitochondrial and nuclear genes involved in energy transduc-

tion is best advanced in the study of baker’s yeast, essentially

due to the extraordinary power of molecular genetics both

in vivo and in vitro.

Yeast as a Model Organism

for the Study of Mitochondrial

Biogenesis and Function

The facultative anaerobic yeast Saccharomyces cerevi-siae has provided the main information about mito-chondrial biogenesis, from the discovery of petitemutants by Boris Ephrussi and Piotr Slonimski andtheir collaborators in the 1950s. This was followed bythe exhaustive analysis of mitochondrial genetics in the1970s in the laboratory of Gif-sur-Yvette and by theidentification of mtDNA by Gottfried Schatz, and stillremains an excellent experimental organism, owing toits capacity of surviving in the absence of competentmitochondria, to the facility of isolation or constructionof mutants (several thousand have already been charac-terized) and to the possibility of mitochondrial trans-formation. The search for nuclear genes involved inmitochondrial biogenesis and function is constantlycarried out in this organism and the results continuallylead to the isolation of functionally homologous humangenes. In particular, many mitochondrial disease geneshave been identified in this way.

Mitochondrial life depends on the coordinatedexpression and interaction of nuclear genes and genespresent on the organellar genome (mtDNA) itself. Themitochondrial genetic system is required for thesynthesis of a limited number of proteins, in particularin yeast of seven highly hydrophobic subunits of theenergy-transducing complexes of the inner membrane(Figure 1).

All the processes necessary to the expression of thesegenes (i.e., transcription, RNA processing, and trans-lation), as well as those required for mtDNA mainte-nance and integrity, take place in the same

Encyclopedia of Biological Chemistry, Volume 2. q 2004, Elsevier Inc. All Rights Reserved. 697

compartment, the mitochondrion itself, as they occur ina bacterium, the hypothetical mitochondrial ancestor.Also, the different processes are functionally linked,frequently through the activity of proteins exhibitingmultiple roles.

Expression of Mitochondrial Genes

Mitochondrial gene expression requires the coordinatedinteraction of mitochondrial and nuclear encoded

factors: the mitochondrial genome (Figure 1) participatesin the process with the genes encoding the RNA-subunitof mitochondrial RNase P (9S RNA), the 21S and 15SrRNAs, a complete set of tRNAs, and one protein of thesmall mitoribosomal subunit, Var1; in addition someintrons encode proteins necessary for RNA splicing(RNA maturases). The nuclear genome encodes allother factors necessary for mitochondrial genomemaintenance and expression, as well as all othermitochondrial proteins (,500 yeast proteins located inthe organelle were currently listed in the Yeast ProteomeDatabase, YPD, in 2001, but predictions indicate about700). These are translated in the cytoplasm, and thenimported into different compartments of mitochondriausing protein-specific import mechanisms. A mitochon-drial targeting sequence, often present at the aminoterminus of translated precursors, allows their inter-action with the mitochondrial receptor–translocatorTOM–TIM, followed by maturation of the precursorand its localization in mitochondria. Also, recent resultspoint out that nuclear-encoded mitochondrial proteins ofbacterial origin are synthesized on polysomes associatedwith the mitochondrion, while those of eukaryotic originare generally translated on free cytosolic polysomes, thuspromoting speculations about organelle origins.

General Features of Simple

and Mosaic Genes

(To describe the expression of yeast mitochondrialgenes, we distinguish between simple genes and mosaicgenes, i.e., genes containing introns and thus subject to amore complicated expression pathway.)

TRANSCRIPTION

The yeast mitochondrial genome is ,4 times larger thanmammalian mitochondrial genomes, although theycontain approximately the same number of genes. Thedifference is due to the lengths of intergenic regions andto the presence of introns in some of the yeast genes.Whereas the 16,000 base-pair mammalian mitochon-drial genome is transcribed by two opposite promoters(one for each strand), genes on the 80,000 base-pairyeast mtDNA are transcribed, singly or in clusters, fromseveral promoters (14 active promoters have beenidentified, see Figure 1). The consensus sequence ofmitochondrial promoters is a nonanucleotide50ATATAAGTA 30, whose last A is the þ1 position ofthe transcript, and termination of transcription isindicated by the dodecanucleotide consensus50AAUAAUAUUCUU30 (see Figure 2).

As in the case of bacterial transcription, all mito-chondrial genes are transcribed by the same RNA

FIGURE 1 The mitochondrial genome of Saccharomyces cerevisiae.The circular map is in agreement with the genetic results and with the

observation that mtDNA replicates by the rolling-circle mode.

Different yeast strains contain mtDNA molecules of different lengths

(comprised between 70 and 85 bp). These differences are essentiallydue to the presence/absence of introns and hypothetical open reading

frames (ORFs 1–5 in this representation). Yeast mtDNA encodes seven

highly hydrophobic subunits of the energy-transducing complexes of

the inner membrane, i.e., apocytochrome b (encoded by the cyt b, orcob-box, gene), three subunits of cytochrome c oxidase (encoded by the

cox1, cox2, and cox3 genes) and three subunits of ATP synthase

(encoded by the atp6, atp8, and atp9 genes). The cytb, cox1, and 21SrRNA genes contain introns, some of which are translated, indepen-dently or in frame with their upstream exons, to produce maturases

(e.g., the bI2 intron of the cytb gene, see Figure 3) or site-specific

endonucleases (the v intron of the 21S rRNA gene); others encodecomplex proteins with multiple functions, i.e., maturases, reverse

transcriptases, endonucleases (e.g., the aI1 intron of the cox1 gene).

Red: exons of protein-coding genes; blue: introns (aI1–aI5; bI1–bI5;

v); hatched blue: intron-encoded ORFs; green: hypothetical ORFs;yellow: 9S, 15S, and 21S RNAs; solid circles: tRNAs. Flags indicate

the transcription initiation sites and their orientation. The orientation

of all the transcription units is clock-wise, with the exception of

the tRNAthr1 gene (open circle), which is transcribed from theopposite strand.

698 MITOCHONDRIAL GENES AND THEIR EXPRESSION: YEAST

polymerase. This is composed of only two nuclearencoded subunits, a core RNA polymerase, Rpo41,which is similar to the very simple T-odd phages RNApolymerases, and the mitochondrial transcription factor,Mtf1, which is required for the recognition of thepromoter by the holoenzyme and is released whenRpo41 enters into the elongation mode, as happens forsigma factors of bacterial RNA polymerases. Mtf1 infact shares sequence similarity with regions 2 and 3 ofbacterial sigma factors, but, unexpectedly, crystalstructure of Mtf1 has revealed clear similarity to thefamily of RNA and DNA methyltransferases and one ofthe two human homologues of Mtf1 has been proved tomethylate a conserved stem-loop in bacterial 16S rRNAand human mitochondrial 12S RNA. This is one of theseveral evidences (the incessant definition of proteincrystal structures is currently revealing novel cases)where the structure/function relationship of a presentday protein indicates that it might have evolved from amultifunctional ancestor or from a protein with adifferent function.

RNA Processing: 50 and 30 Processing

Mitochondrial transcription units are unusual as theyare characterized by the presence of mRNAs inter-spersed with tRNA and rRNAs. In human mtDNAtRNAs genes “punctuate” the very long principaltranscription unit and their processing at both ends“release” the majority of other RNAs. In yeast themitochondrial transcription units are shorter but alsocontain various combinations of RNAs. tRNAs pre-cursors are processed at 50 end by mitochondrial RNAseP and at 30 end by a specific endonuclease (successivelytRNAs are matured by CCA addition and nucleotidemodifications). The processing of mRNAs at their 50

end, assisted by nuclear encoded factors (e.g., Cbp1 forthe cytochrome b transcript) consists in a cleavage togenerate mature mRNA at a position specific for eachtranscript. In some transcription units the transcript isfirst released by the 30 end processing of a pre-ceding tRNA or mRNA. Processing at 30 end ofmRNAs is done near the conserved dodecamer sequence50AAUAAUAUUCUU30, which is protected by a

FIGURE 2 Main features of the expression of a “simple” mitochondrial protein-coding gene. The open reading frame (ORF) of the gene located

in the mtDNA is transcribed into messenger RNA and translated into a polypeptide chain according to the general principles of molecular biology.

However, most of the elements of these two machines derive their information from a different cellular compartment than the one where theseprocesses take place. The genes coding for the transcriptional machinery as well as those involved in the processing or stability of mRNA are located

in the nucleus, the proteins synthesized in the cytoplasm and imported inside the mitochondrion where they recognize specific signals, i.e., mtDNA

or mtRNA sequences or structures indicating the beginning and the end of DNA transcription or RNA processing (e.g., Mtf1, Nam1). The next stepof gene expression is even more complex, since in addition to numerous nuclear genes coding for proteins involved in initiation/termination of

translation, as well as the translation process itself (more than seventy ribosomal proteins, e.g., Nam9, all translation factors, e.g., Cbp1, and all

specific mitochondrially located tRNA synthetases), a number of genes located in the mitochondrial genome participate in the process. They encode

essentially the catalytic RNAs: the two, large and small, rRNAs, the complete set of tRNAs and a subunit of RNAse P. As a result of this doubleinheritance scenario, a mutational lesion in any one of the elements (e.g., the nuclear encoded leucine tRNA synthetase by gene NAM2 or a mtDNA

encoded tRNA) will ineluctably and irreversibly abolish the expression of all the genes located in mtDNA.

MITOCHONDRIAL GENES AND THEIR EXPRESSION: YEAST 699

complex of three proteins before cleavage by a probablyspecific endonuclease. All these processes are assisted byseveral nuclear encoded proteins with a gene-specific ora more general role (see Figure 2).

Processing of Mosaic Genes isMore Complex: RNA Splicing

A typical feature of yeast mtDNA, which is not sharedby human mtDNA (but is common to many mtDNAfrom plants, fungi, and protists), is that some of its genesare split by introns. In S. cerevisiae these are the 21SrRNA, cytb (cob-box) and cox1 genes. Splicing of theseintrons is catalyzed by the intron itself (catalytic RNA),and depends on the formation of a conserved RNAthree-dimensional structure, which enables them toundergo self-splicing in vitro, albeit inefficiently andunder non-physiological conditions. In vivo, however,splicing requires the presence of specific proteins thatfunction to promote a stable and active RNA confor-mation. Furthermore, some yeast mitochondrial intronsbehave as mobile genetic elements, as they can beinserted into an intronless version of the gene at the sameposition it already occupies in the intron-containingversion (intron homing) or to a novel location (introntransposition). Mitochondrial introns belong to twoclasses, group I and II, based on the secondary andtertiary structure of the intron transcript and on themechanism of splicing. The mechanism of splicing ofgroup II introns has suggested that they might beprogenitors of nuclear pre-messenger introns and ofthe spliceosome-catalyzed splicing process, with group IIintron domains having evolved into small nuclear RNAs(snRNAs).

Several features of mitochondrial introns have beenuncovered in the laboratory at Gif-sur-Yvette in the late1970s, among these some encode proteins, i.e., RNAmaturases, which assist the splicing of the intron itselfand sometimes also of a different intron (see Figure 3),and also promote intron mobility. Intron encodedproteins also belong to two groups. Group I proteinsare members of the LAGLI-DADG family of DNAendonucleases and, besides the endonuclease activitiyrequired for intron mobility via DNA (as happens forprokaryotic transposons), some of them have high-affinity RNA-binding properties necessary for their rolein splicing. Mobile group II introns (e.g., intron aI1 ofthe cox1 gene) encode multifunctional proteins,endowed with maturase and DNA endonucleaseactivity, but also encode reverse transcriptases thatbind specifically to the intron RNAs to promote bothintron mobility and RNA splicing. In the case of group IIintrons, transposition occurs via RNA, the procedureadopted by eukaryotic nuclear retrotransposons andretroviruses. Some of the mitochondrial intron encodedproteins are translated in-frame with the upstream exon

and subsequently processed to mature proteins byvarious proteases. This process shows how the differentevents of mitochondrial gene expression (in this casesplicing and translation) are strictly interconnected andalso enlightens a peculiarity of mitochondrial trans-lation, that it starts immediately after transcription, as inprokaryotes, however before RNA processing, anoccurrence unique to this system.

In vivo self-splicing of yeast mitochondrial introns isthus assisted by proteins that, in general, have the role topromote the folding of the catalytic core of the intron,inducing the formation of nucleotide interactionsrequired for catalytic activity. These proteins are thematurases encoded by the intron itself and otherproteins, encoded in the nuclear genome, that concurin assisting the splicing event. Numerous of theseproteins have been identified as suppressors of splicing-defective mitochondrial mutations and most of them areproteins that, besides their role in mitochondrialsplicing, have also other functions. An example is themitochondrial enzyme leucyl-tRNA synthetase, Nam2,which is also necessary for the splicing of the group Iintron bI4. Actually, recent interesting results indicatethat the tyrosyl-tRNA synthetase of Neurospora crassainteracts with group I introns by recognizing conservedtRNA-like structural features of the intron RNA, thussuggesting that this might be the case for other tRNAsynthetases as well. Also the Mrs2 protein, which assistssplicing of group II introns, seems to be implicated in amore general function, as a mitochondrial Mg2þ

transporter. A particularly interesting protein is Nam1,that is involved in the removal of introns from the cytband cox1 transcripts and in overall translationcapacity, and is also responsible of coupling theseprocesses to transcription, through direct interactionwith the amino terminus of mitochondrial RNA poly-merase, Rpo41.

TRANSLATION

Translation of mitochondrial mRNAs resembles moreclosely its prokaryotic than eukaryotic counterpart withrespect to the spectrum of antibiotics inhibiting mito-chondrial translation. Also, mitochondrial mRNAs areuncapped and lack the poly(A) tail, as prokaryoticmRNAs. However mitochondrial translation has uniquefeatures, e.g., the use of an alternative genetic code andthe composition of mitochondrial ribosomes. These aremade up of nuclearly encoded proteins (with theexception of Var1), the majority of which have norecognizable homology to known proteins, i.e., areunique. It has been suggested that these proteins havemore specialized functions connected with the couplingof the translation process to the inner mitochondrialmembrane, where the seven hydrophobic mtDNA-encoded proteins have to be assembled.

700 MITOCHONDRIAL GENES AND THEIR EXPRESSION: YEAST

The 50-untranslated leader sequences (UTLs) ofmitochondrial mRNAs contain sequence or structuralsignals that indicate the translation initiation site and thatare recognized by mRNA-specific activator proteins (e.g.,Cbs1, Cbs2, and Cbp1 for cytb, Pet309 for cox1, Pet 111for cox2 mRNA). Some of the translational activators,that mediate mRNA interactions with mitochondrialribosomes, are organized on the surface of the innermitochondrial membrane in a way that facilitatesassembly of the inner membrane complexes. Assemblyof these complexes necessitates also other proteins, e.g.,Oxa1 is necessary for the correct assembly of cytochromec oxidase and the ATP synthase complex.

The recent observed interaction of some membrane-bound specific activators with two proteins, Nam1 andSls1, that, besides being involved in posttranscriptionalevents and in translation, are able to bind the amino-terminal domain of mitochondrial RNA polymerase,suggests that expression of mtDNA-encoded genesinvolves a complex series of interactions that localizeactive transcription complexes to the inner membrane,in order to coordinate translation with transcription.

In this way synthesis of the mitochondrially encodedproteins is coordinated with their assembly into multi-meric respiratory complexes. On the other hand, theposttranslational targeting mechanism of nuclearencoded subunits seems to be preceded by mRNAlocalization in cytoplasmic ribosomes tightly associatedwith the organelles, thus facilitating the localization ofproteins. This suggests a topological coordinationbetween translation of the mitochondrial and nuclear-encoded subunits and their assembly into higher ordercomplexes.

SEE ALSO THE FOLLOWING ARTICLES

Adrenergic Receptors † Mitochondrial DNA † Mito-chondrial Inheritance † mRNA Polyadenylation inEukaryotes † mRNA Processing and Degradationin Bacteria † Pre-tRNA and Pre-rRNA Processingin Bacteria † Pre-tRNA and Pre-rRNA Processing inEukaryotes

FIGURE 3 Specific features of the expression of a “mosaic” mitochondrial protein-coding gene. Several yeast mitochondrial genes containintrons and some of these introns contain ORFs (see Figure 1). The expression of such “mosaic” genes occurs in two successive steps supplementary

to the general features of expression of “simple” genes (see Figure 2). The primary, unspliced RNA transcript (pre-mRNA) for apocytochome b acts

as a messenger for a protein translated from the upstream exon ORF of cytochrome b and the ORF of the successive intron. This fusion protein,mRNA maturase (in the example shown referred to as the bI4 RNA maturase) promotes catalytically the excision of the intervening sequence from

the pre-mRNA and ligation of cytochrome b exonic RNA leading to the formation of cytochrome b mRNA. The maturase recognizes specific

structures of the intervening sequence, helps the productive folding and facilitates the activity of the ribozyme. At the same time the maturase

activity destroys the RNA which codes for itself and thus exerts a negative feedback for its own biosynthesis. In consequence the amount ofmaturase in the organelle is very low, and the protein can be detected only when its splicing function is impaired. Generally, the maturases act

selectively on the introns which encode them. A notable exception is the maturase shown in the example, i.e., the maturase encoded by the fourth

intron of the cytochrome b gene (bI4): its activity is essential also for the splicing of the fourth intron (aI4) of a different gene, cox1, which encodes

the subunit one of cytochrome c oxidase. In this manner the expression of the key catalysts of the electron transfer cascade may be coordinated.

MITOCHONDRIAL GENES AND THEIR EXPRESSION: YEAST 701

GLOSSARY

self-splicing The intrinsic ability of some introns to remove them-

selves and link together the two adjacent RNA exons, by

transesterification reactions.

spliceosome The large RNA–protein body upon which splicing of

nuclear mRNA precursors occurs.

suppressor gene A gene which, by mutation, is able to eliminate the

effects of a mutation in another gene.

TOM–TIM Proteins forming receptor complexes that are needed fortanslocation of proteins across the mitochondrial outer (TOM) or

inner (TIM) membrane.

FURTHER READING

Barrientos, A. (2003). Yeast models of human mitochondrial diseases.

IUBMB Life 55, 83–95.

Belfort, M. (2003). Two for the price of one: A bifunctional intron-encoded DNA endonuclease–RNA maturase. Genes Dev. 17,

2860–2863.

Bolduc, J. M., Spiegel, P. C., Chatterjee, P., Brady, K. L.,

Downing, M. E., Caprara, M. G., Waring, R. B., and Stoddard,B. L. (2003). Structural and biochemical analyzes of DNA and

RNA binding by a bifunctional homing endonuclease and group

I intron splicing factor. Genes Dev. 17, 2875–2888.

Chen, X. J., and Clark-Walker, G. D. (2000). The petite mutation inyeasts: 50 years on. Int. Rev. Cytol. 194, 197–238.

Contamine, V., and Picard, M. (2000). Maintenance and integrity of

the mitochondrial genome: A pletora of nuclear genes in thebudding yeast. Microbiol. Mol. Biol. Rev. 64, 281–315.

Dieckmann, C. L., and Staples, R. R. (1994). Regulation of

mitochondrial gene expression in Saccharomyces cerevisiae. Int.Rev. Cytol. 152, 145–181.

Dujon, B., Colleaux, L., Jacquier, A., Michel, F., and Monteilhet, C.

(1986). Mitochondrial introns as mobile genetic elements: The role

of intron-encoded proteins. Basic Life Sci. 40, 5–27.

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chondrial gene expression. Crit. Rev. Biochem. Mol. Biol. 30,

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and flanking exons in wild-type and box3 mutants of cytochrome b

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introns. Annu. Rev. Biochem. 64, 435–461.

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mitochondria. Nat. Rev. Mol. Cell. Biol. 3, 555–565.

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Long mRNAs coding for yeast mitochondrial proteins of prokar-

yotic origin preferentially localize to the vicinity of mitochondria.

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BIOGRAPHY

Giovanna Carignani obtained her degree in pharmacy in 1960 at the

University of Trieste (Italy). She is an Assistant Professor of

Molecular Biology of the Faculty of Medicine of the University of

Padova (Italy).

Piotr P. Slonimski obtained his M.D. degree in 1947 at the University of

Cracow (Poland) and soon after he joined the Centre National de la

Recherche Scientifique (CNRS), Laboratory of Boris Ephrussi, in Paris.

He became Director of the CNRS Laboratory of Physiological Genetics

in 1960, then Director of the CNRS Center of Molecular Genetics of

Gif-sur-Yvette until 1992. He was Professor of Molecular Genetics at

the Sorbonne, at the University Pierre et Marie Curie (Paris VI) and

Guest Professor at several American and European universities. He was

in charge of the first French government Genome Project (Groupement

de Recherches et d’Etudes sur les Genomes, GREG) and is active now,

as Emeritus Professor, in the field of genome bioinformatics.

702 MITOCHONDRIAL GENES AND THEIR EXPRESSION: YEAST