Artigo 1 - Mitochondrial Evolution

2012; doi: 10.1101/cshperspect.a011403Cold Spring Harb Perspect Biol Michael W. Gray Mitochondrial Evolution

Subject Collection Mitochondria

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EncephalopathyS) Metabolism in Ethylmalonic2Altered Sulfide (H

Valeria Tiranti and Massimo Zeviani

Mitochondrial Membrane during ApoptosisPermeabilization of the Outer−−Where Killers Meet

Tom Bender and Jean-Claude Martinou

Mitochondrial Metabolism, Sirtuins, and AgingMichael N. Sack and Toren Finkel

and Parkin: Links to ParkinsonismMitochondrial Quality Control Mediated by PINK1

Derek Narendra, John E. Walker and Richard Youle

Mechanisms of Protein Sorting in Mitochondria

Pfanner, et al.Diana Stojanovski, Maria Bohnert, Nikolaus

Mitochondrial EvolutionMichael W. Gray

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Mitochondrial Evolution

Michael W. Gray

Centre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry andMolecular Biology, Dalhousie University, Halifax, Nova Scotia B3M 4R2, Canada

Correspondence: [email protected]

Viewed through the lens of the genome it contains, the mitochondrion is of unquestionedbacterial ancestry, originating from within the bacterial phylum a-Proteobacteria(Alphaproteobacteria). Accordingly, the endosymbiont hypothesis—the idea that the mito-chondrion evolved from a bacterial progenitor via symbiosis within an essentially eukaryotichost cell—has assumed the status of a theory. Yet mitochondrial genome evolution has takenradically different pathways in diverse eukaryotic lineages, and the organelle itself is increas-ingly viewed as a genetic and functional mosaic, with the bulk of the mitochondrial prote-ome having an evolutionary origin outside Alphaproteobacteria. New data continue toreshape our views regarding mitochondrial evolution, particularly raising the question ofwhether the mitochondrion originated after the eukaryotic cell arose, as assumed in theclassical endosymbiont hypothesis, or whether this organelle had its beginning at thesame time as the cell containing it.

In 1970, Lynn Margulis published Origin ofEukaryotic Cells, an influential book that ef-

fectively revived the long-standing but mostlymoribund idea that mitochondria and plastids(chloroplasts) evolved from free-living bacte-ria via symbiosis within a eukaryotic host cell(Margulis 1970). The discovery in the 1960s ofDNA within these organelles together with therecognition that they contain a translation sys-tem distinct from that of the cytosol were two ofthe observations that Margulis marshaled insupport of the endosymbiont hypothesis of or-ganelle origins. Indeed, throughout her career,Margulis forcefully argued that symbiosis is apotent but largely unrecognized and unappre-ciated force in evolution (Margulis 1981). Tech-nological developments in DNA cloning andsequencing in the 1970s and 1980s opened the

way to the detailed characterization of mito-chondrial genomes and genes, and the gen-eration of key molecular data that were instru-mental in affirming a bacterial origin of themitochondrial and plastid genomes, allowingresearchers to pinpoint the extant bacterial phy-la to which these two organelles are most closelyrelated. Over the past several decades, numerousreviews have documented in detail the bio-chemical and molecular and cell biologicaldata bearing on the endosymbiont hypothesisof organelle origins (Gray 1982, 1983, 1989a,b,1992, 1993, 1999; Gray and Doolittle 1982; Wal-lace 1982; Cavalier-Smith 1987b, 1992; Gray andSpencer 1996; Andersson and Kurland 1999;Gray et al. 1999, 2001, 2004; Lang et al. 1999;Andersson et al. 2003; Burger et al. 2003a; Bul-lerwell and Gray 2004). Various endosymbiotic

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models proposed over the years have been com-prehensively critiqued (Martin et al. 2001),while the debates surrounding the endosymbi-ont hypothesis have been recounted in an en-gaging perspective that traces the developmentof ideas regarding organelle origins (Sapp1994). Within a historical context, the presentarticle emphasizes more recent data and in-sights that are relevant to continuing questionsregarding how mitochondria originated andhave since evolved.

WHAT DO GENETIC, GENOMIC, ANDPHYLOGENOMIC DATA TELL US ABOUTTHE ORIGIN OF MITOCHONDRIA?

The genetic function of mitochondrial DNA(mtDNA) was first fully revealed by completesequencing of the �16-kb mitochondrial ge-nome from several mammalian species (Ander-son et al. 1981, 1982; Bibb et al. 1981). This workestablished the paradigm that mtDNA encodes asmall number (13 in mammals) of protein sub-units of the mitochondrial electron transportchain and ATP synthase, as well as the ribosomalRNA (rRNA) and transfer RNA (tRNA) com-ponents of a mitochondrial translation system.The paradigm was revised when investigationsof mtDNA from non-animal species showedquite extraordinary variation in size, physicalform, coding capacity, organizational patterns,and modes of expression across the eukaryoticdomain (Gray et al. 1998). In some non-animaltaxa,additionalproteins areencodedin mtDNA,notably extra respiratory proteins and ribo-somal proteins (Table 1). On the other hand,some mitochondrial genomes have been re-duced drastically in size, losing many of the pro-tein genes encoded in animal mtDNA as wellas some or all mtDNA-encoded tRNA genes.At �6 kb in size, the mitochondrial genome ofPlasmodium falciparum (human malaria para-site) and related apicomplexans is the smallestknown, harboring only three protein genes,highly fragmented and rearranged small subunit(SSU) and large subunit (LSU) rRNA genes, andno tRNA genes (Feagin 2000). In marked con-trast, within land plants, mtDNA has expandedsubstantially in size (.200 kb) if not in coding

capacity, with the largest known mitochondrialgenome in this lineage (�11,000 kb) exceedingthe size of some bacterial and even some nucleargenomes (Sloan et al. 2012). What was evidenteven early on is that none of the initial mtDNAsinvestigated by detailed sequencing, includinganimal mtDNAs, looks anything like a typicalbacterial genome in the way in which genes areorganized and expressed.

Ribosomal RNA genes are among the fewgenes universally encoded by mtDNA acrosseukaryotes, and the corresponding rRNA se-quences were exploited early on in phylogeneticreconstructions aimed at elucidating their evo-lutionary origin. Although mitochondrial largesubunit (LSU) and small subunit (SSU) rRNAsare in general very different in size and second-ary structure compared with their cytosolic andprokaryotic counterparts, they retain a suffi-cient degree of primary sequence and secondarystructure correspondence that they can be in-corporated into the aligned sequence databaseson which these phylogenetic reconstructions arebased (Gray et al. 1984, 1989; Cedergren et al.1988). These initial phylogenetic trees showedthat mitochondrial rRNA sequences, and pre-sumably the genomes encoding them, emanatefrom within the domain Bacteria (eubacteria)and not from within Archaea (archaebacteria)or Eucarya (eukaryotes). Plant mitochondrialrRNAs are especially slowly evolving and bacte-ria-like (Schnare and Gray 1982; Spencer et al.1984) and were instrumental in pinpointingthe a-class of Proteobacteria (Alphaproteobac-teria) as the specific bacterial lineage from with-in which they originated (Yang et al. 1985). Sub-sequent phylogenetic reconstructions using amuch larger number of both mitochondrialand bacterial rRNA sequences have consistent-ly confirmed this affiliation and, moreover,pointed to the Rickettsiales, one of six or moreorders within Alphaproteobacteria (Williamset al. 2007)—and comprising obligate parasitessuch as Rickettsia, Anaplasma, and Ehrlichia—as especially close relatives of mitochondria(Gray and Spencer 1996; Gray 1998).

These phylogenetic conclusions were con-siderably bolstered by the sequencing in 1997of a minimally derived (ancestral) mitochon-

M.W. Gray

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drial genome from Reclinomonas americana,a member of an obscure group of protists(eukaryotic microbes) termed jakobid flagel-lates. The �69-kb, circular-mapping mtDNAof R. americana not only contains more genesthan any other characterized mitochondrialgenome (including genes specifying 5S rRNAand RNase P RNA) (Table 1), but it also dis-plays evident bacterial characteristics not seenin combination in any other mtDNA, such asoperon-like gene clusters, highly bacteria-likerRNA and tRNA secondary structures, andputative Shine–Dalgarno motifs upstream of

protein-coding genes. Indeed, R. americanamtDNA—so different from that of animal,plant, fungal, and many other protist mt-DNAs—has been dubbed “a eubacterial genomein miniature,” so striking is its resemblance to atypical bacterial genome (Lang et al. 1997).

At the same time, sequencing of the firstRickettsiales genome, that of Rickettsia prowa-zekii, showed it to be a markedly reduced bacte-rial genome, superficially resembling mitochon-dria in its dependence on a host cell (Anderssonet al. 1998). However, the genomes of the mito-chondrion and members of Rickettsiales are

Table 1. Mitochondrial DNA-encoded genes and their functions

(1) Coupled electron transport–oxidative phosphorylation (ATP synthesis)Complex I (NADH:ubiquinone oxidoreductase) nad1, 2, 3, 4, 4L, 5, 6, 7, 8, 9, 10, 11Complex II (succinate:ubiquinone oxidoreductase) sdh 2, 3, 4Complex III (ubiquinol:cytochrome c oxidoreductase) cobComplex IV (cytochrome c:O2 oxidoreductase) cox1, 2, 3Complex V (F1F0 ATP synthase) atp1, 3, 4,a 6, 8,b 9

(2) TranslationRibosomal RNAs rnl (LSU), rns (SSU), rrr5 (5S)Ribosomal proteins

Small subunit (SSU) rps1, 2, 3, 4, 7, 8, 10, 11, 12, 13, 14, 19Large subunit (LSU) rpl1, 2, 5, 6, 10, 11, 14, 16, 18, 19, 20, 27, 31, 32, 34

Transfer RNAs trnA, C, . . . W, YElongation factor tufAtm RNA (unstalling of translation) ssrA

(3) TranscriptionCore RNA polymerase rpoA, B, CSigma factor rpoD

(4) RNA processingRNase P RNA (50 tRNA processing) rnpB

(5) Protein importABC transporter ccmA (yejV), ccmB (yejW)Heme delivery ccmC (yejU)SecY-type transporter secYSec-independent transporter tatA (mttA)c, tatC (mttB)

(6) Protein maturationCytochrome oxidase assembly cox11Heme c maturation ccmF (yejR)

A subset of the genes is encoded in the mtDNA of various eukaryotes; for example, those in bold are found in human and

other mammalian mitochondrial genomes. Only the most ancestral (gene-rich) mtDNAs, for example, those of jakobid

flagellates such as Reclinomonas americana, encode all or almost all of this gene set. Rare genes of uncertain origin and

function include dpo (plasmid-derived DNA polymerase), rpo (plasmid-derived RNA polymerase), rtl (reverse

transcriptase), mutS (DNA repair), and dam (methyltransferase). See Gray et al. (2004) for details.aAlso referred to as orf25 and ymf39 (Burger et al. 2003b).bAlso referred to as orfB and ymf19 (Gray et al. 1998).cJacob et al. (2004).


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clearly the products of independent evolution-ary reduction, implying that mitochondria werenot derived directly from a Rickettsiales taxon(Andersson et al. 1998; Gray 1998); rather, thesetwo groups share a more remote common an-cestor. Although the mitochondria–Rickett-siales connection has been a consistent phyloge-netic finding (Viale and Arakaki 1994; Gupta1995; Sicheritz-Ponten et al. 1998; Lang et al.1999), it is still not certain whether the two aresister groups, or whether mitochondria actual-ly branch within Rickettsiales, which is com-posed of two distinct families, Rickettsiaceaeand Anaplasmataceae (Williams et al. 2007).In several studies, mitochondria appear to bemore closely related to the former family, con-taining various Rickettsia species, than to thelatter, comprising the genera Anaplasma, Ehrli-chia, and Wolbachia (Karlin and Brocchieri2000; Emelyanov 2001a,b, 2003a,b).

The specific affiliation of mitochondria andRickettsiales in phylogenetic trees, although ro-bust, has been questioned (e.g., Esser et al. 2004)on the grounds that this inferred relationshipmight be a phylogenetic artifact due to thehigh rate of sequence divergence and elevatedAþT content of the genomes of Rickettsialestaxa and mitochondria—in other words, along-branch-attraction (LBA) artifact. For thatreason, there has been considerable interest inexpanding the database of both mitochondri-al and bacterial sequences and in applying amore comprehensive phylogenomics approach(based on many genes) to the reconstructionof phylogenetic trees, in combination with mul-tiple and more sophisticated algorithms, rig-orous statistical evaluation methods, and morerealistic evolutionary models.

Of particular interest in broadening taxonsampling has been the identification of free-living members of Alphaproteobacteria thatmay be specifically affiliated with the parasiticgroup Rickettsiales (Williams et al. 2007). Thesepreviously unknown a-proteobacterial lineagescame to light through the Global Oceanic Sur-vey, metagenomic data from which revealed thatoceanic a-Proteobacteria are abundant, withone particular clade (termed SAR11) compris-ing 30%–40% of total oceanic cell counts. The

1.3-Mb genome of one SAR11 member, Candi-datus Pelagibacter ubique, is the smallest knowngenome, encoding the fewest genes, of any free-living bacterium (Giovannoni et al. 2005).Several recent reports have suggested that theSAR11 clade including Ca. P. ubique shares acommon ancestor with mitochondria, the twotogether forming a sister group to Rickettsiales(Georgiades et al. 2011; Thrash et al. 2011).Other investigators reject this affiliation, con-cluding instead that Ca. P. ubique is most closelyrelated to soil and aquatic a-Proteobacteriawith large genomes (Viklund et al. 2012) andarguing that a rare oceanic group of a-Proteo-bacteria, termed Oceanic Mitochondria Affili-ated Clade (OMAC), represents the closest free-living relatives to mitochondria (Brindefalket al. 2011). Still other groups have suggestedthat free-living members of a-proteobacterialorders other than Rickettsiales should be con-sidered as possible sources of the mitochondrialprogenitor (Esser et al. 2004; Atteia et al. 2009).

These differing conclusions emphasize thechallenges inherent in these sorts of analyses,which have to contend with various types ofsystematic error (for review, see Thrash et al.2011), biased taxon sampling, and the highlyrestricted gene content of mitochondrial ge-nomes. At present, although we know a greatdeal regarding the mitochondrial family tree,we have to admit that the identity of the imme-diate next of kin remains elusive.

Phylogenetic and phylogenomic recon-structions strongly and consistently support amonophyletic mitochondrial assemblage, andtherefore a single origin of mitochondria. Twoother pieces of genomic evidence support theview that extant mitochondrial genomes share asingle common ancestor. First, the genes encod-ed by mitochondrial genomes are, with few ex-ceptions, a subset of the genes encoded by themost gene-rich mtDNA, that of R. americana.Because the mitochondrial genome is consid-ered to be a highly reduced version of a muchlarger a-proteobacterial progenitor genome, itis extremely unlikely that genome reduction inindependently acquired bacterial symbiontswould separately converge on the same smallrepertoire of respiratory-chain and ribosomal-

M.W. Gray




protein genes. Second, in plant and many an-cestral protist mtDNAs, ribosomal proteingenes are clustered in the same transcriptionalorder in which they appear in the correspondingbacterial operons. However, some genes presentin the bacterial operons are missing from thecorresponding mitochondrial gene clusters, ei-ther having been moved elsewhere in the mito-chondrial genome or to the nuclear genome, asa result of endosymbiotic gene transfer (EGT).The same gene deletions are seen in the homol-ogous mitochondrial ribosomal gene clusterswhere these occur throughout eukaryotes, argu-ing that these deletions were already present inthe common ancestor of extant mitochondrialgenomes (Gray et al. 1999). This conclusion isreinforced by consideration of various nucleus-encoded mitochondrial proteins that are com-ponents of the mitochondrial proteome (seebelow).

HOW DID THE MITOCHONDRIALSYMBIOSIS HAPPEN?

Endosymbiotic models for the origin of mito-chondria (for review, see Martin et al. 2001)are basically variations on two fundamentallydifferent themes that have been referred to, re-spectively, as the “archezoan scenario” and the“symbiogenesis scenario” (Koonin 2010). In thearchezoan scenario, “The host of the proto-mi-tochondrial endosymbiont was a hypotheticalprimitive amitochondrial eukaryote, termedarchezoan” (Koonin 2010). In contrast, in thesymbiogenesis scenario, “A single endosymbiot-ic event involving the uptake of ana-Proteobac-terium byan archaeal cell led to the generation ofthe mitochondria,” followed subsequently “bythe evolution of the nucleus and compartmen-talization of the eukaryotic cell” (Koonin 2010).The archezoan scenario most closely approxi-mates the classical endosymbiont hypothesis ofmitochondrial origin (Margulis 1970; Doolittle1980), whereas the hydrogen hypothesis (Martinand Muller 1998) is an example of the symbio-genesis scenario. A fundamental difference be-tween these two scenarios is whether the a-Pro-teobacterial endosymbiosis that gave rise to theproto-mitochondrion happened at the same

time as and was integral to the formation ofthe eukaryotic cell (symbiogenesis scenario) oroccurred subsequent to the formation of aprimitive, amitochondriate cell that served ashost and that was already essentially eukaryotic(archezoan scenario).

Archezoan Scenario

A major boost to the classical endosymbionthypothesis came from early phylogenetic re-constructions, based initially on SSU rRNA se-quences, that showed several eukaryotic lineagesbranching deeply within the eukaryotic domainof the resulting trees. These early-branching lin-eages, collectively termed “archezoa,” consistedof protists such as microsporidians, diplomo-nads, and parabasalids, living as parasites in an-aerobic environments and characterized by theabsence of recognizable mitochondria (Cava-lier-Smith 1987a, 1989). Members of archezoa,assumed to be primitively amitochondriate (i.e.,never having had mitochondria in their evolu-tionary history: the so-called “archezoa hypoth-esis”), could be seen as modern representativesof the sort of ancestral eukaryote that might haveplayed host to an a-Proteobacterial symbiont ina classical endosymbiosis scheme.

Two findings led to the ultimate abandon-ment of the archezoa hypothesis. First, morerigorous phylogenetic reconstructions com-bined with other sorts of data convincinglyshowed that the early-branching position ofarchezoan taxa in the eukaryotic tree is a meth-odological artifact, due to a relatively high rateof sequence divergence of the archezoan se-quences used in the analysis. This property givesrise to an LBA effect that incorrectly positionsthese taxa at the base of the eukaryotic clade,closest to the outgroup (prokaryotic) sequencesused to root the tree. Microsporidia, for exam-ple, were eventually recognized as secondarilydegenerate fungi rather than as primitive, ear-ly-branching eukaryotes (Hirt et al. 1999; Kee-ling et al. 2000). Indeed, the current eukaryotictree is more accurately characterized as a bush,with no one lineage clearly the earliest diverging(Keeling et al. 2005; Koonin 2010). Second, inevery apparently amitochondrial lineage that





has been carefully investigated, evolutionaryremnants of mitochondria (“mitochondrion-related organelles,” or MROs) have been identi-fied (see below). Thus, we currently know of noextant eukaryotic lineages that are convincinglyamitochondrial and that therefore might havebeen primitively amitochondriate (Embley andHirt 1998). This conclusion does not mean thatsuch lineages do not exist. We may simply nothave discovered them yet, or they may have ex-isted at some point in evolutionary history buthave now all become extinct. Nevertheless, re-jection of the archezoa hypothesis on phylo-genetic grounds coupled with the apparentabsence of any known amitochondriate eu-karyotic lineages considerably weakens the casefor an acquisition of mitochondria via the clas-sical endosymbiont route.

Symbiogenesis Scenario

An alternative view, that the host cell for themitochondrial endosymbiosis was a prokary-ote—and specifically an archaeon, not a eu-karyote—has recently gained in prominence(Koonin 2010). The “hydrogen hypothesis”(Martin and Muller 1998) is perhaps the best-known example of a symbiogenesis scenario.This scheme proposes that eukaryotes arose

. . .through symbiotic association of an anaero-bic, strictly hydrogen-dependent, strictly auto-trophic archaebacterium (the host) with a eu-bacterium (the symbiont) that was able torespire, but generated molecular hydrogen as awaste product of anaerobic heterotrophic me-tabolism. The host’s dependence upon molecu-lar hydrogen produced by the symbiont is putforward as the selective principle that forged thecommon ancestor of eukaryotic cells. (Martinand Muller 1998)

Thus, the hydrogen hypothesis “posits that theorigins of the heterotrophic organelle (the sym-biont) and the origins of the eukaryotic lineageare identical” (Martin and Muller 1998). A cor-ollary of the hydrogen hypothesis and othersymbiogenesis scenarios is that the complexityof the eukaryotic cell and its defining featuresemerged after the mitochondrial symbiosis,rather than before.

Several arguments can be advanced againsta symbiogenesis scenario for the origin of mi-tochondria (Koonin 2010). Endocytosis (a eu-karyotic hallmark) has long been considered anessential function for incorporating a bacterialsymbiont, although it is the case that bacterialendosymbioses (e.g., g-Proteobacteria insideb-Proteobacteria) have been documented (vonDohlen et al. 2001; Thao et al. 2002). In addi-tion, assuming an a-Proteobacterial symbiontas the mitochondrial progenitor in a partner-ship that simultaneously gave rise to this organ-elle and the rest of the eukaryotic cell, one mightexpect that any eubacterial genetic signal in thenuclear genome would be overwhelmingly a-Proteobacterial. However, although an a-Pro-teobacterial signal does, in fact, predominate(Pisani et al. 2007), in any given eukaryotic lin-eage collectively more bacterial-type genes ap-pear to derive from diverse non-a-Proteobacte-rial lineages or fail to affiliate robustly with anyspecific bacterial phylum. Nevertheless, it is pos-sible that ancestral lineages contributing to abacterial–archaeal symbiogenesis might havepossessed genomes already “scrambled” to a cer-tain extent by horizontal gene transfer (HGT).

A prominent feature of the hydrogen hy-pothesis is its claim to account simultaneouslyfor the origins of both aerobic and anaerobicenergy metabolism in eukaryotes, the assump-tion being that both pathways were contained inand contributed to the hybrid cell by the a-Pro-teobacterial partner. It is supposed that the twopathways would have been differentially ex-pressed in the free-living bacterial symbiontwhen it encountered the appropriate environ-mental conditions. The hypothesis further pos-its that genes for aerobic respiration were lostin those eukaryotic lineages in which the mi-tochondrion was converted to an MRO, sometypes of which (e.g., hydrogenosome) functionin anaerobic energy metabolism (see below).The hydrogen hypothesis predicts that genesof anaerobic energy metabolism in MROsshould have been inherited vertically through-out eukaryotes from a common ancestor,clustering as a monophyletic lineage togetherwith a-Proteobacteria in phylogenetic recon-structions. However, a rigorous study of the

M.W. Gray




phylogenetic distributions and histories of pro-teins involved in anaerobic pyruvate metabo-lism in eukaryotes has not provided supportfor this prediction (Hug et al. 2010). Rather,MROs and the enzymatic machinery they con-tain for anaerobic energy metabolism appear toreflect a high degree of independent and con-vergent evolution (see below).

Very recently, Lane and Martin (2010) haveargued, from a consideration of the energeticsof genome complexity, that because eukaryotesencode and express a substantially larger num-ber of proteins than do prokaryotes, this in-creased expression demands a level of cellularenergy that only the mitochondrion is able tosatisfy. Accordingly, these investigators view themitochondrion as the sine qua non to eukary-otic genomic and cellular complexity, conclud-ing, rather definitively, that “the host for mito-chondria was a prokaryote.”

On balance, it would seem that a symbio-genesis scenario (bacterial endosymbiont inan archaeal host) better accommodates theaccumulated data relevant to the question ofmitochondrion origin than does an archezoanscenario (bacterial endosymbiont in an ami-tochondriate but essentially eukaryotic host).However, as emphasized above, the latter sce-nario cannot be entirely discounted at this point.Each scenario raises its own set of issues that aredifficult to rationalize without resorting to adhoc explanations. In the end, each faces the co-nundrum that there is no straightforward andcompelling way to discern how similar the ge-nomes of the proposed prokaryotic ancestorsof the eukaryotic cell were to their modern-day descendants.

MITOCHONDRION-RELATEDORGANELLES (MROs)

An extreme form of mitochondrial genomereduction is found in two types of MROs, hy-drogenosomes and mitosomes, which entirelylack mtDNA. These two MRO types are distin-guished by the fact that hydrogenosomes retainATP-generating capacity, whereas mitosomesdo not. The hydrogenosome, a double-mem-brane-bound organelle originally discovered in

the parabasalid Trichomonas vaginalis (Lind-mark and Muller 1973), was subsequently iden-tified in various other anaerobic eukaryotes.The T. vaginalis hydrogenosome not only lacksa genome but has an incomplete tricarboxylicacid cycle and electron transport chain and nocytochromes. ATP is generated from pyruvatevia a substrate-level pathway comprising a char-acteristic suite of enzymes, including pyruvate:ferredoxin oxidoreductase and an iron–ironhydrogenase. The organelle takes its namefrom the fact that molecular hydrogen (H2) isone of the end products of this pathway. Theanaerobic metabolism performed by the hydro-genosome initially suggested that the organellemight have originated through an endosymbi-osis with an anaerobic bacterium such as Clos-tridium (Whatley et al. 1979). However, subse-quent studies have revealed that the T. vaginalishydrogenosome contains several proteins typ-ical of mitochondria, including chaperonins(Bui et al. 1996), the NADH dehydrogenasemodule of electron transport complex I (Hrdyet al. 2004), and components of the ISC biosyn-thesis pathway, the mitochondrial machineryfor synthesis of iron–sulfur (Fe–S) clusters(Sutak et al. 2004). These results strongly sup-port the view that the T. vaginalis hydrogeno-some is a relict mitochondrion.

A second group of double-membrane-bound MROs, in this case unable to generateATP, has been discovered in a number of anaer-obic, parasitic protists that were initially consid-ered to be amitochondriate, including the amoe-bozoons Entamoeba histolytica (Clark and Roger1995; Mai et al. 1999; Tovar et al. 1999) and Mas-tigamoeba balamuthi (Gill et al. 2007), the mi-crosporidians Trachipleistophora hominis (Wil-liams et al. 2002) and Encephalitozoon cuniculi(Goldberg et al. 2008; Tsaousis et al. 2008), andthe diplomonad Giardia lamblia (Tovar et al.2003). The MROs in these protists are collec-tively termed “mitosomes” (Embley et al. 2003;Embley 2006; Hjort et al. 2010). Here again,identification of typical mitochondrial proteinsin this MRO argues that mitosomes, like hydro-genosomes, are evolutionary derivatives of con-ventional mitochondria, but are even morehighly reduced than hydrogenosomes (see Hjort





et al. 2010 for a detailed listing and discussion ofrelevant data). Very limited metabolic capacityis retained by the mitosome, of which Fe–Scluster formation stands out. This pathway isubiquitous in both conventional mitochondriaand MROs, suggesting that Fe–S cluster forma-tion, rather than oxidative phosphorylation,may be the raison d’etre of the mitochondrionand its evolutionary derivatives (Tovar et al.2003).

The discovery of what appear to be transi-tional forms of MRO in certain anaerobic eu-karyotes has recently blurred the distinctionbetween mitochondria, on the one hand, andhydrogenosomes and mitosomes, on the other(Gray 2011). These novel MROs, like conven-tional hydrogenosomes, are able to generate H2

via an ATP-producing, hydrogenase-mediatedpathway; however, they retain a genome, albeitlacking several typical mtDNA-encoded genes,notably ones specifying components of respira-tory complexes III, IV, and V (see Table 1). Thus,these MROs lack the ability to generate ATPvia coupled electron transport and oxidativephosphorylation; however, they support a con-siderably more complex biochemistry than ei-ther hydrogenosomes or mitosomes. MROs ofthis type have been identified in the anaerobicciliate Nyctotherus ovalis (Boxma et al. 2005; deGraaf et al. 2011) and the anaerobic strameno-piles Blastocystis sp. (Perez-Brocal and Clark2008; Stechmann et al. 2008; Wawrzyniak et al.2008) and Proteromonas lacertae (Perez-Brocalet al. 2010), relatives of brown algae, diatoms,and oomycetes. In these cases, the MRO ge-nome specifies components of an organellartranslation system (rRNAs, tRNAs, ribosomalproteins) as well as subunits of electron trans-port complexes I and II, suggesting the presenceof a partial electron transport chain.

The sporadic phylogenetic distribution ofanaerobic MRO-containing eukaryotes andtheir interspersion with aerobic taxa within par-ticular lineages strongly indicate that MROshave originated independently, multiple times,throughout the eukaryotic domain (Roger andSilberman 2002; Embley et al. 2003; Embley2006; Hjort et al. 2010). Consequently, manyof the characteristics shared between MROs

(e.g., between those of Nyctotherus and Blasto-cystis) are almost certainly due to convergentevolution rather than vertical inheritance. Thecontinued study of variously evolved MROs willbe important for understanding both the path-ways and mechanisms involved in the evolu-tionary conversion of a conventional mitochon-drion to an MRO. At the same time, we will gaina better appreciation of the evolutionary flexi-bility of mitochondria, a theme considered be-low in the discussion of mitochondrial prote-ome evolution.

ORIGIN AND EVOLUTION OF THEMITOCHONDRIAL PROTEOME

Only 27 mitochondrial proteins are encoded bythe gene-rich R. americana mitochondrial ge-nome, and only three by the most gene-poormtDNA, that of apicomplexan parasites suchas P. falciparum. The vast majority of mitochon-drial proteins are encoded in the nucleus, syn-thesized in the cytosol, and imported back intothe organelle. Overall, the number of mitochon-drial proteins is estimated to range from severalhundred in P. falciparum to more than 3000 invertebrate animals (Richly et al. 2003). Reloca-tion of functional genes from the mitochondri-on to the nucleus via EGT has contributed sig-nificantly to the nucleus-encoded cohort (Gray1999; Adams and Palmer 2003; Timmis et al.2004). Occasionally, such genes are transferredin pieces (Adams et al. 2001; Perez-Martınezet al. 2001; Waller and Keeling 2006; Gawrylukand Gray 2009; Morales et al. 2009), sometimeswith a portion remaining in the mitochon-drial genome (Oudot et al. 1999; Kuck et al.2000; Nedelcu et al. 2000; Gawryluk and Gray2010a). Notably, only a minority of the proteinproducts of these relocated genes are importedback into the organelle; most now function inother subcellular compartments (Gabaldon andHuynen 2007). Extensive retailoring of the mi-tochondrial proteome in the course of evolu-tion has also involved addition of novel proteinsand new functions, as discussed below.

Identification of the proteins contributed tothe eukaryotic cell by the proto-mitochondrialendosymbiont has been approached through

M.W. Gray




comparisons of the gene contents of sequenceda-Proteobacterial and eukaryotic genomes.This strategy has identified at least 840 orthol-ogous groups that bear a cleara-Proteobacterialsignature—that is, a close and specific evolu-tionary relationship to a-Proteobacterial ho-mologs, without evidence of recent HGT (Ga-baldon and Huynen 2003, 2007; Szklarczyk andHuynen 2010). Comparisons among a-Proteo-bacterial genomes suggest that the free-livingbacterial ancestor of mitochondria containedabout 3000–5000 genes (Boussau et al. 2004),with an upper bound of approximately 1700ancestral clusters of orthologous genes in theproto-mitochondrial genome (Szklarczyk andHuynen 2010). These estimates indicate thatabout 1000–3000 genes were lost in the transi-tion from bacterial symbiont to proto-organ-elle. Significantly, of the more than 800 humangenes that display an a-Proteobacterial signa-ture, only about 200 are found in the humanmitochondrial proteome, indicating that theproto-mitochondrial contribution to eukaryot-ic cell evolution and function extends beyondthe mitochondrion itself, to encompass othercellular compartments, as well.

In the proto-mitochondrion, the completeelectron transport chain and complete pathwayforb-oxidation of fatty acids (providing NADHand FADH2 to the former) were likely present,indicating that the mitochondrial endosymbi-ont had an aerobic metabolism. Also promi-nently represented were pathways for the syn-thesis of lipids, biotin, heme, and Fe–S clusters,as well as an abundance of cation transporters.In all, the reconstructed metabolism suggeststhat the proto-mitochondrion was capable ofat least facultative aerobic respiration (Szklar-czyk and Huynen 2010). More than half ofwhat remains of this proto-mitochondrialmetabolism in modern mitochondria com-prises proteins that function in oxidative phos-phorylation and translation, including posttrans-lational modifications, a clear trend towardspecialization in energy metabolism. In short,mitochondrial proteome evolution has beencharacterized by loss of many original functions,retargeting of others to different cellular loca-tions, and wholesale addition of host-derived

proteins—a veritable “hijacking of mitochondri-al protein synthesis and metabolism” by the eu-karyotic cell (Gabaldon and Huynen 2007).

Initial delineation of the mitochondrialproteome relied on various bioinformatics ap-proaches, including homology searches as wellas techniques developed to identify amino-ter-minal mitochondrial targeting signals (MTSs)(e.g., Claros and Vincens 1996; Emanuelssonet al. 2000). Not all imported mitochondrialproteins feature MTSs identifiable in this way,and the accuracy and sensitivity of the searchalgorithms may be compromised where proteinsequences are highly divergent (Richly et al.2003). An alternative technique that has provenuseful is high-throughput immunolocalizationof tagged gene products to determine their sub-cellular localization. Based on these approaches,the yeast mitochondrial proteome has been es-timated to comprise about 400–800 proteins,or between �7% and �13% of the total yeastproteome of approximately 6100 proteins(Karlberg et al. 2000; Marcotte et al. 2000; Ku-mar et al. 2002).

Considering the cleara-Proteobacterial sig-nature of the mitochondrial genome, we mighthave anticipated that the mitochondrial prote-ome would consist largely of a-Proteobacteria-like proteins plus proteins exclusive to the eu-karyotic domain, “invented” during the evolu-tionary transition from bacterial endosymbiontto organelle. Somewhat surprisingly, this expec-tation has not been met. In surveys of the yeastmitochondrial proteome (Karlberg et al. 2000;Marcotte et al. 2000), only �10%–15%—amuch smaller proportion than might havebeen anticipated—proved to originate clearlyfrom the a-Proteobacterial lineage. A larger, ge-nerically “prokaryotic” proportion (�40%–50%) consists of proteins that appear to origi-nate outside a-Proteobacteria but are not nec-essarily affiliated with any specific bacterial orarchaeal clade. Another, “eukaryotic” fraction(�20%–30%) contains proteins having noobvious homologs in either Archaea or Bacte-ria. A final, “unique” subset (�20%) comprisesseemingly yeast-specific proteins having noidentifiable homologs in other eukaryotes orin prokaryotes. These results indicate that the





yeast mitochondrial proteome has multipleevolutionary origins and a complex evolution-ary history (Kurland and Andersson 2000; Grayet al. 2001), a conclusion now firmly establishedfor the mitochondria of other eukaryotes(Gabaldon and Huynen 2004; Szklarczyk andHuynen 2010). Bacteriophage-like proteins (no-tably the mitochondrial RNA polymerase inmost eukaryotes) make an additional small con-tribution to the evolutionary mix (Shutt andGray 2006).

Comparative mitochondrial genomics,based on complete sequencing of mtDNAs,has told us much regarding the nature of theancestral mitochondrial genome and about pat-terns and mechanisms of mitochondrial ge-nome evolution (Gray et al. 1998; Gray 1999).In the same way, comparative mitochondrialproteomics, based on a combination of bio-informatics data mining and direct analysisof whole mitochondria or submitochondrialfractions and complexes (Dreger 2003; Yanet al. 2009), is proving to be an equally incisiveapproach for unraveling the evolution of themitochondrial proteome. Investigations usingmass spectrometry (MS) have confirmed andextended the initial, bioinformatics-based find-ings that indicated a mosaic evolutionary originof the mitochondrial proteome.

MS provides a powerful adjunct to bioinfor-matics-based approaches in its capacity to re-veal novel mitochondrial proteins that have noidentifiable sequence homologs and that lackMTSs. At least 13 novel, “ciliate-specific” pro-teins identified during MS analysis of mito-chondria from Tetrahymena pyriformis (Smithet al. 2007) were subsequently found as compo-nents of the purified mitochondrial F1F0-ATPsynthase (complex V) of this protist (Nina et al.2010). These observations highlight an emerg-ing theme of taxon-specific retooling of mito-chondrial complexes such as electron transportchain assemblies and ribosomes, only the corea-Proteobacteria-like components of which ap-pear to have been contributed by the proto-mi-tochondrion. A feature of this retailoring isthe addition of novel proteins of generally un-known function, sometimes accompanied byloss of otherwise conserved components. An

example is the ATP synthase (complex V) ofthe chlorophycean green alga, Chlamydomonasreinhardtii, in which nine novel subunits of un-known evolutionary origin replace eight sub-units that are otherwise conserved in the ATPsynthase of other non-chlorophycean green al-gae, as well as in plants, animals, and fungi (La-paille et al. 2010).

Other mitochondrial respiratory complex-es, such as complex I (CI; NADH:ubiquinoneoxidoreductase), clearly exemplify this retailor-ing theme. Bacterial CI comprises 14 subunits,all of which are found in the correspondingmammalian complex, with seven of these sub-units encoded in mammalian mtDNA (Table1). A further 18 subunits in mammalian CIare assumed to be eukaryote-specific additionsalready present in the last eukaryotic commonancestor, because they are ubiquitous through-out eukaryotes but are not found in bacteria(but see below). Thirteen other mammalianCI subunits appeared to display a narrow phy-logenetic distribution, being identified initiallyonly in metazoan animals (Brandt 2006).

In attempting to define the ancestral state ofselected mitochondrial components and path-ways, the importance of taking a comprehensivephylogenetic approach (examining mitochon-drial proteomes from as wide a range of eu-karyotes as possible) needs to be recognized.As noted above, mitochondrial CI has an addi-tional 18 “eukaryote-specific” subunits that areconsidered to have been incorporated at the ear-liest stages of mitochondrial CI evolution (Ga-baldon et al. 2005; Brandt 2006). In plants(Heazlewood et al. 2003; Parisi et al. 2004)and green algae (Cardol et al. 2004), mitochon-drial CI additionally contains proteins withhigh similarity to g-type carbonic anhydrases(gCAs). Comparative studies over a relativelynarrow phylogenetic range initially suggestedthat these proteins represented specific addi-tions in the plant lineage (Parisi et al. 2004).However, a more recent study focusing on pro-tists—eukaryotic microbes, wherein lies most ofthe evolutionary diversity within the domainEucarya—has revealed a much broader distri-bution of mitochondrial gCAs, either shown orpresumed to be associated with mitochondrial

M.W. Gray




CI (Gawryluk and Gray 2010b), than previouslysuspected. It appears likely that gCAs were an-cestral components of mitochondrial CI andthat they were subsequently lost from CI specif-ically in the evolutionary line leading to animalsand fungi (opisthokonts), rather than added toCI specifically in the line leading to plants andalgae. These results emphasize the importanceof comprehensive taxon coverage in drawingconclusions regarding mitochondrial proteomeevolution. In fact, wider taxon sampling hasrecently demonstrated that almost all of the 18supposedly metazoan-specific CI subunits werelikely already present in CI of the last eukaryoticcommon ancestor (Cardol 2011; Gawryluk et al.2012). In the same way, a more phylogeneticallybroad analysis of homologous bacterial com-plexes appears warranted, judging by the recentdemonstration that CI from the a-Proteobacte-rium Paracoccus dentrificans contains three ofthe “eukaryote-specific” CI proteins that hadlong been considered to be absent from bac-terial CI (Yip et al. 2011). (In this regard, itis notable that 35 years ago [before the ad-vent of sequence-based phylogenetic recon-structions], John and Whatley [1975] pointedout that P. denitrificans “resembles a mitochon-drion more closely than do other bacteria, inthat it effectively assembles in a single organismthose features of the mitochondrial respiratorychain and oxidative phosphorylation which areotherwise distributed at random among mostother aerobic bacteria.”)

Another mitochondrial complex that showsa profound degree of evolutionary retailoring isthe mitochondrial ribosome. In many eukary-otes (e.g., most animals), the mitochondrialLSU and SSU rRNAs have become much small-er than their bacterial counterparts, while atthe same time many new ribosomal proteinshave been added, so that what was originallyan RNA-rich complex is now a protein-richone (O’Brien 2002). The most extreme exampleof this ribosome retailoring is seen in the kinet-oplastid protozoa, such as Trypanosoma brucei(Zikova et al. 2008) and Leishmania tarentolae(Sharma et al. 2009). The T. brucei mitochon-drial ribosome, for example, contains 56 SSUand 77 LSU proteins, compared with the 21

SSU and 34 LSU proteins present in the Escher-ichia coli ribosome. The novel mitoribosomalproteins identified in these analyses do nothave detectable homologs outside of the kinet-oplastid protozoa and display only a low degreeof sequence conservation within this lineage.These observations reinforce the importanceof direct MS analyses of isolated mitochondrialcomplexes in order to accurately determinetheir composition, given that so many of thesecomponents appear to be new, lineage-specificinventions.

Other studies have shown that the ancestralmitochondrial ribosome in the last eukaryoticcommon ancestor was already much larger thanits bacterial ancestor, containing some 19 addi-tional eukaryote-specific proteins (Smits et al.2007; Desmond et al. 2011). The fact that thesenovel mitoribosomal proteins are found in allof the currently recognized eukaryotic super-groups is yet another strong argument in favorof a monophyletic origin of contemporary mi-tochondria, a conclusion in this case based oneukaryote-specific rather than prokaryote-spe-cific features.

CONCLUDING REMARKS

Comparative mitochondrial genomics has of-fered us a glimpse of what the ancestral mito-chondrial genome was like, and what genes itcontained. A monophyletic origin of the mito-chondrial genome from within a-Proteobacte-ria is strongly supported, with Rickettsialesmost often identified as the a-Proteobacterialorder most closely related to mitochondria.This phylogenetic-based approach has revealedthat mitochondrial genome evolution has beencharacterized by massive expansion in some lin-eages and extreme reduction and compaction inothers, with endosymbiotic gene transfer relo-cating much of the initial genetic informationin the proto-mitochondrial genome to the nu-cleus. Comparative mitochondrial proteomicshas provided evidence of a mosaic evolutionaryorigin of the protein complement of this organ-elle, with a much smaller proportion of the pro-teome than might have been anticipated clearlyderiving from an a-Proteobacterial progenitor.





Mitochondri-specific proteins, pathways, andfunctions emerging within the eukaryotic line-age subsequent to the a-Proteobacterial endo-symbiosis are being identified. Retailoring ofkey mitochondrial complexes relative to theira-Proteobacterial antecedents is seen to haveoccurred through addition of novel proteincomponents, often in a narrow, lineage-specificmanner. We may anticipate that the unabateddeluge of genomic and proteomic data will con-front us with currently unappreciated aspects ofmitochondrial structure and function across thebroad range of eukaryotes, with new data andinsights continually reshaping and refining ourideas regarding mitochondrial evolution.

ACKNOWLEDGMENTS

This essay is dedicated to the memory of Pro-fessor Lynn Margulis (1938–2011), a passion-ate advocate for the idea that endosymbiosis is adriving force in cell evolution and was the pro-cess that led to the origin of mitochondria andplastids. I sincerely thank the agencies that haveenabled my research in this area over the pastfour decades through operating and equipmentgrants and salary support, most particularly theCanadian Institutes of Health Research (for-merly the Medical Research Council of Cana-da), Natural Sciences and Engineering ResearchCouncil of Canada, Canadian Institute for Ad-vanced Research (Program in Evolutionary Bi-ology), Canada Research Chairs Program, Can-ada Foundation for Innovation, and GenomeCanada.

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Artigo 1 - Mitochondrial Evolution

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