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The Pre-Endosymbiont Hypothesis: A NewPerspective on the Origin and Evolutionof Mitochondria

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]

Mitochondrial DNA (mtDNA) is unquestionably the remnant of an a-proteobacterial ge-nome, yet only �10%–20% of mitochondrial proteins are demonstrably a-proteobacterialin origin (the “a-proteobacterial component,” or APC). The evolutionary ancestry of the non-a-proteobacterial component (NPC) is obscure and not adequately accounted for in currentmodels of mitochondrial origin. I propose that in the host cell that accommodated an a-proteobacterial endosymbiont, much of the NPC was already present, in the form of amembrane-bound metabolic organelle (the premitochondrion) that compartmentalizedmany of the non-energy-generating functions of the contemporary mitochondrion. Isuggest that this organelle also possessed a protein import system and various ion andsmall-molecule transporters. In such a scenario, an a-proteobacterial endosymbiont couldhave been converted relatively directly and rapidly into an energy-generating organelle thatincorporated the extant metabolic functions of the premitochondrion. This model (the “pre-endosymbiont hypothesis”) effectively represents a synthesis of previous, contending mito-chondrial origin hypotheses, with the bulk of the mitochondrial proteome (much of the NPC)having an endogenous origin and the minority component (the APC) having a xenogenousorigin.

Considering the central role played in all eu-karyotic cells by mitochondria or mito-

chondrion-related organelles (MROs, such ashydrogenosomes and mitosomes) (Hjort et al.2010; Shiflett and Johnson 2010; Muller et al.2012), the question of the origin and subse-quent evolution of the mitochondrion haslong captivated and challenged biologists. In arecent article in this series (Gray 2012), I dis-cussed in detail several aspects of mitochondrial

evolution, focusing particularly on how well theaccumulating molecular data can be accommo-dated in current models of mitochondrial ori-gin. In this context, the origin and evolution ofthe mitochondrial proteome, as opposed to theorigin and evolution of the mitochondrial ge-nome, were examined from the perspective ofcomparative mitochondrial proteomics. Some-what disconcertingly, as more data have becomeavailable, we find ourselves considerably less

Editors: Patrick J. Keeling and Eugene V. Koonin

Additional Perspectives on The Origin and Evolution of Eukaryotes available at www.cshperspectives.org

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certain about key aspects of how mitochondriaoriginated than we were (or thought we were)several decades ago.

Here, I summarize key points discussed inmore detail in the previous article before pre-senting a novel perspective on how the mito-chondrion might have originated. The newmodel proposed here, which represents a syn-thesis of both endogenous (“origin from with-in”) and xenogenous (“origin from outside”)modes, is advanced in an attempt to accountfor the inability of a purely endosymbiotic mod-el, whose strongest support has come fromstudies of the mitochondrial genome, to ade-quately accommodate data on the mitochondri-al proteome.

A RUMSFELDIAN TAKE ON THE ORIGINOF THE MITOCHONDRION

. . . there are known knowns; there are things we

know we know.

We also know there are known unknowns; that is tosay, we know there are some things we do not know.

But there are also unknown unknowns—the ones wedon’t know we don’t know.

—U.S. Secretary of Defense Donald Rumsfeld

Press briefing, February 12, 2002

This Rumsfeldian view provides a usefulframework for outlining key aspects of whatwe know and what we don’t know about theorigin and evolution of the mitochondrion.

Known Knowns

† We know from comparative mitochondrialgenomics—through the analysis of genescontained in mtDNA and the protein se-quences they specify—that the mitochondri-al genome is of (eu)bacterial origin. Thesesequences point us to members of a particu-lar bacterial phylum, a-proteobacteria (alsotermed Alphaproteobacteria), as the closestextant free-living relatives of mitochondria,and therefore the bacterial group from withinwhich the mitochondrial genome emerged(Gray and Doolittle 1982; Gray 1992).

† We know that the most bacteria-like (least-derived) and gene-rich mitochondrial ge-nomes are found within the core jakobids(Fig. 1) (Lang et al. 1997; Burger et al.2013), a group of flagellated protozoa thatare members of the proposed eukaryoticsupergroup Excavata (Hampl et al. 2009).Jakobid mtDNAs specify strikingly bacteria-like large subunit (LSU), small subunit (SSU)and 5S ribosomal RNAs (rRNAs) and trans-fer RNAs (tRNAs), as well as upward of 70proteins, including subunits of an a2bb

0sRNA polymerase, the enzyme used for tran-scription in Bacteria. Other notable featuresinclude genes encoding transfer-messengerRNA (tmRNA) and the RNA component ofRNase P; bacteria-like operon organization;and putative Shine–Dalgarno-like motifs in-volved in translation initiation in Bacteria.

† We know that certain components of theoriginal, bacteria-like systems used for repli-cation and transcription of mtDNA (e.g., themitochondrial RNA polymerase) have beenreplaced in some or virtually all eukaryotesby bacteriophage-like counterparts (Shuttand Gray 2006).

† We know that mitochondrial genome evolu-tion has been characterized by extensive geneloss, with relocation of many of the genes inthe protomitochondrial genome to the nu-cleus and import of their protein productsback into the organelle. Such endosymbioticgene transfer (EGT) usually involves com-plete genes, but EGT may also relocate onlya portion of a mtDNA-encoded gene, withthe rest of that gene remaining in and ex-pressed from the mitochondrial genome(Burger et al. 2003).

† We know that the mitochondrion originatedonly once, that is, mitochondria in all eu-karyotic lineages descend from a commonancestor, the LMCA (last mitochondrialcommon ancestor). Commonality in key as-pects of genetic and biochemical function,the presence of shared mitochondrion-spe-cific deletions in bacteria-like operons pre-sent in some mtDNAs, and phylogenetic

M.W. Gray

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Figure 1. Genetic map of the strikingly bacteria-like mitochondrial genome of the core jakobid, Andaluciagodoyi: the most gene-rich mitochondrial genome known (Burger et al. 2013). (Solid rectangles) Genes, withthose on the outer circle transcribed in a clockwise direction, and those on the inner circle transcribed coun-terclockwise. Colors denote taxonomic gene distribution: (black) common genes found in most animal, fungal,and protist mtDNAs; (blue) expanded gene set, mostly present in protist and plant mtDNAs; (red) genespredominantly found in jakobid mtDNAs; (green) hypothetical protein-coding genes (ORFs). A. godoyimtDNA encodes 25 proteins involved in coupled electron transport–oxidative phosphorylation (nad, sdh,cob, cox, atp; complexes I–V, respectively); 12 SSU (rps) and 16 LSU (rpl) ribosomal proteins; one translationelongation factor (tuf ); four subunits of a bacteria-like multisubunit RNA polymerase (rpoA,B,C,D); eightproteins involved in protein import (ccmA,B,C; tatA,C) and protein maturation (cox11,15; ccmF); LSU (rnl),SSU (rns), and 5S (rrn5) rRNAs; 25 tRNAs—single uppercase letters: (Mf ) initiator methionine; (Me) elongatormethionine; transfer-messenger RNA (tmRNA) (ssrA); and the RNA component of RNase P (rnpB). Fiveadditional open reading frames (orf ) encode putative proteins of unknown function. (Arrows) Positions ofpredicted promoters. (Figure courtesy of G. Burger.)

The Pre-Endosymbiont Hypothesis

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tree reconstruction strongly support this in-ference (Gray et al. 1999).

† We know that despite an evident a-proteo-bacterial origin of the mitochondrial ge-nome, only �10%–20% of the mitochon-drial proteome—the collection of proteinsmaking up the organelle—can confidentlybe traced to a-proteobacteria (thisa-proteo-bacterial component is referred to here as theAPC). The 80%–90% non-a-proteobacte-rial component, or NPC, appears from phy-logenetic analysis either to be of diverse pro-karyotic (bacterial or archaeal) evolutionaryancestry or to have arisen specifically withineukaryotes (Karlberg et al. 2000; Kurland andAndersson 2000; Gray et al. 2001; Gabaldonand Huynen 2003, 2004, 2007; Szklarczykand Huynen 2010).

† We know that the core of the mitochondrialproteome was already in place in the LMCA,that is, the LMCAwas likely as complex in itsfundamental functions as a contemporaryaerobic mitochondrion. We also know thatsubsequent retailoring of this proteome, in-volving both gain and loss of protein com-ponents and functions, occurred to differentextents in diverse eukaryotic lineages, lead-ing to the emergence of lineage-specific mi-tochondrial proteins characterized by a re-stricted phylogenetic distribution (Huynenet al. 2013).

Known Unknowns

Despite what we are confident we know weknow, several key aspects of mitochondrial ori-gin and evolution remain unresolved:

† We do not know precisely which extant a-proteobacterial lineage is most likely to bethe descendant of the lineage that was theevolutionary source of the mitochondrial ge-nome (for more detailed discussion of thispoint and references, see Gray 2012; Lang andBurger 2012). Various studies have consis-tently pointed to Rickettsiales, one of six ormore orders within a-proteobacteria—andcomprising obligate intracellular parasites

such as Rickettsia, Anaplasma, and Ehrli-chia—asthe closest relatives of mitochondria.Rickettsiales comprises two distinct families,Rickettsiaceae and Anaplasmataceae, withseveral studies suggesting that mitochondriaare more closely related to the former family,containing various Rickettsia species, than tothe latter, which encompasses the genera Ana-plasma, Ehrlichia, and Wolbachia. However,there is currently no consensus in the litera-ture as towhether mitochondria branchwith-in or as sister to either of the two Rickettsialesfamilies, oroutside of Rickettsiales altogether.Several newly identified free-livinga-proteo-bacteria have recently been considered candi-dates for the title of “closest mitochondrialrelative” (Brindefalk et al. 2011; Georgiadeset al. 2011; Viklund et al. 2012), but method-ological issues complicate phylogenetic treereconstructions (Thrash et al. 2011; Rodrı-guez-Ezpeleta and Embley 2012), and thusthe jury is still out on this point.

† We do not know the evolutionary origin ofmost of the mitochondrial proteome (i.e.,the NPC). The general assumption hasbeen that these proteins were added to thea-proteobacterial symbiont during its trans-formation into an organelle, but where theseproteins came from and how and when theywere added to the evolving mitochondrion isnot spelled out in models of mitochondrialorigin. A substantial portion of the NPC ap-pears to be eukaryote-specific (i.e., there areno evident homologs within either of thedomains Bacteria or Archaea) and thus ispresumed to have arisen within the domainEucarya, although by what mechanism(s) isunclear.

Unknown Unknowns

We have no way of knowing what we might stilldiscover about mitochondrial evolution, al-though we may be confident that further inves-tigation of diverse mitochondrial genomes andproteomes will continue to surprise us. Unan-ticipated and increasingly bizarre forms of mi-tochondrial genome organization and expres-

M.W. Gray




sion continue to be reported, a recent examplebeing from the euglenozoon Diplonema papil-latum. Here, mitochondrial genes are fragment-ed, with the individual gene pieces distributedamong several small, circular molecules and thecorresponding transcripts processed at bothends before being joined together in the correctorder to generate a translatable mRNA, a pro-cess that also involves U insertion RNA editing(Vlcek et al. 2011; Kiethega et al. 2013). (Nota-bly, D. papillatum, whose mitochondrial geneticsystem is arguably one of the most radically di-vergent yet characterized, is a member of Exca-vata, which as noted above contains the corejakobids, the eukaryotic group featuring themost primitive [least-derived] mitochondrialgenomes known.) Investigation of the mito-chondrial genetic system has uncovered a wealthof unexpected phenomena, including variantgenetic codes, a bewildering array of RNA edit-ing processes, self-splicing introns, fragmentedrRNAs, and tRNA import (for review, see Grayet al. 1998; Gray 2003), as well as documentingclear examples of mitochondrion-to-nucleusgene transfer (Adams and Palmer 2003). Basedon the experience of the last three decades, wemay expect more of the same.

CONTENDING SYMBIOTIC MODELS FORTHE ORIGIN OF MITOCHONDRIA

Endosymbiotic models for the origin of mito-chondria (for review, see Martin et al. 2001) canbe grouped into two scenarios, depending onwhether the organelle is seen to have originatedafter or at the same time as the rest of the eukary-otic cell (Gray et al. 1999). These two scenarioshave been dubbed “archezoan” and “symbio-genesis,” respectively (Koonin 2010). The arche-zoan scenario hypothesizes that an amito-chondriate eukaryotic host cell phagocytosedan a-proteobacterium, which was subsequentlytransformed into the mitochondrion. In con-trast, the symbiogenesis scenario hypothesizesthat a physical and metabolic fusion betweenan a-proteobacterium and an archaeal cell gen-erated the ancestor of the eukaryotic cell, withthe a-proteobacterial partner subsequently be-coming the mitochondrion. The archezoan sce-

nario most closely approximates the classicalendosymbiont hypothesis of mitochondrial or-igin (Margulis 1970; Doolittle 1980), whereasthe hydrogen hypothesis (Martin and Muller1998) is an example of the symbiogenesis sce-nario (for more detailed discussion, see Koonin2010; Gray 2012).

The archezoan scenario takes its namefrom the “archezoa hypothesis” (Cavalier-Smith1987, 1989), which posited that a group ofputatively amitochondrial eukaryotes was, infact, primitively amitochondriate (i.e., neverpossessed mitochondria). As such, archezoanswere offered as potential modern representativesof the sort of host cell that would have taken upthe mitochondrial endosymbiont. The fact thatarchezoans branched early in SSU rRNA treessupported the idea that these eukaryotes were,indeed, primitive eukaryotes. However, the ar-chezoa hypothesis was abandoned after thediscovery that all putatively amitochondriateeukaryotes so far investigated contain a mito-chondrion-related organelle (MRO) and thatthe early branching position of archezoans is amethodological artifact attributable to a rela-tively high rate of sequence divergence in arche-zoan SSU rRNA genes, such that these divergentsequences are incorrectly pulled toward the baseof the eukaryotic tree (“long-branch attrac-tion”). The demise of the archezoa hypothesisremoves a base of support for the archezoan sce-nario of mitochondrial origin (admittedly, wecannot exclude the possibility that primitivelyamitochondriate eukaryotes still exist but havenot yet been discovered—albeit not for lack ofexhaustive searching—or that such lineages, ifthey once existed, have all become extinct). Onthe other hand, as critically discussed in moredetail elsewhere (Koonin 2010; Forterre 2011;Gray 2012; Lang and Burger 2012), symbiogen-esis models have their own problems both withrespect to the origin of the mitochondrion andthe origin of the eukaryotic cell per se. A partic-ular difficulty is the fact that collectively morebacterial-type genes in eukaryotes appear to de-rive from diverse non-a-proteobacterial line-ages or fail to affiliate robustly with any specificbacterial phylum (Pisani et al. 2007), whereasin a symbiogenesis scenario, one would antici-





pate that any bacterial genetic signal in the nu-clear genome would be predominantly a-pro-teobacterial.

DECONSTRUCTING THE MITOCHONDRIALPROTEOME

The mosaic nature of the mitochondrial pro-teome (Karlberg et al. 2000; Szklarczyk andHuynen 2010) is proving increasingly difficultto accommodate within current schemes of mi-tochondrial origin. A specific contributionfrom a-proteobacteria is strongly indicated,comprising the mitochondrial genome, thefew proteins it encodes, and a limited numberof other proteins whose genes we infer onceresided in mtDNA but were subsequently relo-cated to the nucleus via EGT. This a-proteobac-terial component (APC) forms a crucial part ofthe evidence that has been offered in support ofan endosymbiotic origin of mitochondria; thus,it was a surprise to discover that the APC is aquantitatively minor (albeit qualitatively essen-tial) element of the contemporary oxygen-usingand ATP-producing organelle. The non-a-pro-teobacterial component (NPC), the evolution-ary origin of which appears both phylogeneti-cally diverse and largely obscure, dominates themitochondrial proteome. Any mitochondrionorigin scenario must take into account the ac-cumulating evidence that says that only a minorportion of the mitochondrial proteome is clear-ly of a-proteobacterial ancestry, whereas theevolutionary origin of the major portion of mi-tochondrial proteins is obscure. Indeed, it isimportant to emphasize that, strictly speaking,the data used to “prove” the endosymbiotic or-igin of the mitochondrion in essence only provethe origin of the mitochondrial genome per se.

In both classical endosymbiotic (arche-zoan) and symbiogenesis (fusion) scenariosfor the origin of the eukaryotic cell, an a-pro-teobacterial symbiont is assumed to have beenextensively retailored to create the modern or-ganelle, both by wholesale gene loss from themuch larger endosymbiont genome and bythe addition of new proteins. Comparativelyfew mitochondrial proteomes have been inves-tigated directly (e.g., via mass spectrometry),

yet the available data already show that keymitochondrial protein assemblages and path-ways existed in essentially their modern formin the last eukaryotic common ancestor(LECA). Lineage-specific modification throughboth loss and addition of mitochondrial pro-teins has certainly occurred, and specific casesof mitochondrial protein acquisition throughlateral gene transfer (LGT) can definitely beidentified, but the fact remains that the basicstructure and complex function of the mito-chondrion were likely already well establishedin the LECA. Thus, between the events that cre-ated both the modern mitochondrion and therest of the cell in which it resides, much reorga-nization of symbiont and host cell would havehad to occur, whether the acquisition of themitochondrion was through a symbiogenesispathway or an archezoan one.

Symbiont-to-mitochondrion transforma-tion is generally considered to have occurred ina stepwise fashion, involving both loss and ad-dition of proteins and functions. In comparing areconstructed protomitochondrial proteomewith the proteomes of contemporary a-proteo-bacteria and mitochondria (yeast and human),Gabaldon and Huynen (2007) inferred thatthe transformation of an a-proteobacterialsymbiont to an organelle involved loss of someprotomitochondrial metabolic pathways ortheir movement to other parts of the cell, aswell as acquisition of new proteins leading tothe gain of novel pathways, such as the proteinimport machinery and mitochondrial carriers.In addition, new proteins have been recruited toexisting mitochondrial complexes resulting, forexample, in the expansion of respiratory com-plex I from 17 subunits in a-proteobacteria tomore than 40 subunits in eukaryotes (Cardol2011). Such restructuring over a long period oftime would presumably have generated a seriesof intermediate organelles differing in the rangeof functions they possessed. A key question thatsuch a scenario poses is how the hundreds (per-haps thousands) of proteins making up themitochondrial proteome were introduced. Inparticular, what was the evolutionary source ofthe NPC? I argue here that neither the archezoannor the symbiogenesis scenario accommodates,

M.W. Gray




in a compelling way, the diverse functional andphylogenetic character of the NPC.

At this point, a word of caution is in orderwith respect to how we view the NPC. In par-ticular, it is possible (perhaps even likely) that aportion of the NPC consists of originallya-pro-teobacterial proteins that are too divergent insequence to be recognized as such, so that the10%–20% estimated a-proteobacterial contri-bution to the mitochondrial proteome is prob-ably an underestimate, and instead represents aminimal contribution from the a-proteobacte-rial endosymbiont. Marked differences in phy-logenetic resolution are to be expected in a pro-tein collection as complex as the mitochondrialproteome, even though it is notable that the a-proteobacterial signal in the recognizable APCis particularly strong (Lang and Burger 2012).Nevertheless, it does not seem probable that the“bacterial” portion of the NPC could all be ac-counted for by divergent proteins of originallya-proteobacterial origin.

Another suggestion is that the a-proteobac-terial symbiont was itself chimeric as a conse-quence of prior and extensive LGT. In this case,the symbiont could have contributed genes en-coding proteins that appear to come from otherbacterial lineages. A problem with this sugges-tion is that this non-a-proteobacterial contribu-tion would have had to have been extensiveenough to quantitatively swamp the a-proteo-bacterial component in the contemporary mi-tochondrion, which would imply a highly chi-meric symbiont genome and/or differential lossof a-proteobacterial genes in the subsequentsymbiont-to-organelle transformation. More-over, it is not clear from this scenario why ex-tant a-proteobacterial genomes generally lackorthologs corresponding to this putative LGTcomponent. Thiegart et al. (2012) attributethis phylogenetic diversity to “a single originof mitochondria followed by subsequent LGTamong free-living prokaryotes.” However, thisexplanation would seem to demand that for mi-tochondrial homologs transferred in this way,the transferred genes would have to have evolvedmore slowly than donor (a-proteobacterial)ones, or would have to have been selectivelylost from a-proteobactrerial lineages. Other-

wise, they would still score in phylogenetic re-constructions as “a-proteobacterial.”

In yeast, about half of the prokaryote-likesequences in the mitochondrial proteome arecommon to Bacteria and Archaea as well as eu-karyotes, and thus may well have been present inthe last universal common ancestor (LUCA)(Karlberg et al. 2000). Of the remainder, fewaffiliate convincingly with specific non-a-proteobacterial bacterial lineages, whereas thea-proteobacterial signal is particularly robust(Lang and Burger 2012). Moreover, we stillhave to account for the large proportion (up to50%) of the mitochondrial proteome that isunique to eukaryotes and that has been assumedto have evolved within the eukaryotic lineage.Thus, even if we are liberal in assigning a per-centage to the APC (e.g., doubling it to 20%–40%), we are still left with upward of 60%–80%of the mitochondrial proteome that does notappear to have come from an a-proteobacterialsource.

In this context, a final possibility is that atleast some of the NPC proteins predated theendosymbiotic event, preexisting in whateverthe host cell was that accommodated the sym-biont. For example, in a recent review on plastidendosymbiosis, Keeling (2013a) has suggesteda “targeting rachet” model in which proteintargeting is established in the host before theendosymbiont is permanently integrated. Theidea is that this process would target preexistingtransporters to the membrane of transient sym-bionts, accelerating and facilitating the sym-biont-to-organelle transformation.

The possibility that some portion of whatwould become the mitochondrial NPC preex-isted in the host is attractive because it wouldeffectively “precondition” the host–symbiontinteraction. Rather than the NPC appearingon the scene after the acquisition of the en-dosymbiont (e.g., via LGT) or actually beingpart of the endosymbiont, it might be thatmuch of the NPC was already present in thehost cell that accommodated the a-proteobac-terial symbiont. If such were the case, rawmaterial for initiating the conversion of a bac-terium to an organelle would be readily avail-able.





The above proposal demands a rather differ-ent view of the eukaryotic cell and how it arosethan is suggested in current proposals. In par-ticular, the idea that the eukaryotic cell is funda-mentally a fusion between bacterial and archaealpartners, with a large number of novel proteinsarising subsequently and specifically within theeukaryotic lineage, is increasingly being ques-tioned (e.g., Koonin 2010; Forterre 2011; Langand Burger 2012). In proposing yet anothersymbiogenesis scenario (Planctomycetes, Ver-rucomicrobia, Chlamydiae [PVC] Bacteria—Thaumarchaeota—Virus,” or PTV), Forterre(2011) states that “the transformation of aFECA (first eukaryotic common ancestor)born from a fusion event into a reasonable an-cestor of modern eukarya remains difficult toimagine in a convincing way in the PTV scenarioas in all other fusion scenarios.” In fact, Forterreis led to conclude that eukaryotes “probablyevolved from a specific lineage, according tothe three domains scenario originally proposedby Carl Woese.”

In contrast to the prevailing symbiogenesisview, a radically different proposal for the originof the eukaryotic lineage has just been publishedby Harish et al. (2013), drawn from phylogeneticreconstructions based on genome content ofcompact protein domains, as defined in theSCOP (structural classification of proteins) da-tabase. These investigators identify Archaea andBacteria as sister domains in a lineage (“akar-yotes”) that descends in parallel, but indepen-dently, from an entity they term the “MRUCA”(most recent universal common ancestor),which “is not a bacterium.” According to theiranalysis, Harish et al. (2013) assert that MRUCAwould have contained 75% of the unique SFs(SCOP superfamilies) encoded by extant ge-nomes of the domains Bacteria, Archaea, andEucarya, each specifying a proteome that par-tially overlaps each of the other two: “the conse-quence of descent from a universal common an-cestor, MRUCA.” The authors state: “we cannotdraw the conclusion that MRUCAwas morpho-logically similar to a eukaryote,” but they doconclude that “elements and cohorts of SFsfrom its proteome are recognizable in the pro-teomes of modern eukaryotes. Thus, SFs ho-

mologous to those of modern mitochondria,chloroplasts, nuclei, spliceosomes, cytoskele-ton, and the like are evident in the proteomeof MRUCA.”

A notable implication of the proposal byHarish et al. (2013) is that the LECA couldwell have harbored a complex proteome thatalready included much of what was destinedto become the mitochondrial NPC. This infer-ence would be consistent with a new model ofmitochondrial evolution outlined below, whichhypothesizes that essential elements of the NPCalready existed in the host cell that formed anassociation with the APC-contributing a-pro-teobacterium.

THE “PREMITOCHONDRION”: APUTATIVE ORGANELLE PREDATING THEa-PROTEOBACTERIAL ENDOSYMBIOSIS

If we accept the possibility that much of themitochondrial proteome was already presentin the eukaryotic host cell before the a-proteo-bacterial symbiont arrived, what might it havebeen doing? To address this question, I proposea new model (the “pre-endosymbiont hypoth-esis”) (Fig. 2) that envisages an endogenouslyevolved organelle that we might term the “pre-mitochondrion,” which I suggest preexisted inwhat was in many respects already a eukaryoticcell (the “pre-eukaryote”). According to thisscheme, the premitochondrion was essentiallya metabolic rather than an energy-generatingorganelle: a membrane-bound structure com-partmentalizing many (perhaps most) of thepathways and reactions characteristic of extantmitochondria, such as amino acid, nucleotide,and lipid metabolism; coenzyme biosynthesis;iron–sulfur cluster formation, and so on. Thepremitochondrion would already have mem-brane structural proteins and a protein importsystem, as well as transporters for various ionsand metabolites, including amino acids and nu-cleotides. However, because the premitochon-drion would be energy-consuming rather thanenergy-generating, it would possess a nucleo-tide carrier system to import ATP and exportADP, the reverse of what occurs in the contem-porary mitochondrion.

M.W. Gray




In this scenario, a large part of the mito-chondrial proteome (much of the NPC) wouldbe of endogenous origin, having evolved withinthe pre-eukaryotic cell, along with its othersubcellular components and compartments. Insome respects, the premitochondrion wouldresemble non-DNA-containing MROs such ashydrogenosomes and mitosomes, which per-form a variable subset of normal mitochondrialmetabolic functions but lack the capacity togenerate ATP via coupled electron transport–oxidative phosphorylation. Such MROs couldbe regarded as representing a reversion to anancestral form.

Transformation of an a-proteobacterialsymbiont into an integrated organelle wouldoccur as conventionally envisaged, but it wouldbe substantially simplified if, in fact, the bulk ofthe proteins required for such a radical reorga-nization were already present, in the form ofa functional entity. Because coupled oxidative

phosphorylation would have existed and func-tioned in the symbiont to generate both a pro-ton gradient and ATP, it is likely that it was thesymbiont that was modified by premitochon-drial components to become the mitochondri-on, rather than the symbiont genome beingintroduced somehow into and becoming func-tional within the premitochondrion.

The pre-eukaryote cell would thus resemblethe proposed host cell in the classical serialendosymbiont hypothesis (Taylor 1987) exceptthat a progenitor of the mitochondrion (the pre-mitochondrion) was already present. Subse-quent transformation of the a-proteobacterialsymbiont, via incorporation of structural andfunctional elements of the premitochondrion,would effectively add energy-generating capac-ity, in the form of electron transport and coupledoxidative phosphorylation, to the varied meta-bolic capabilities already possessed by thepremitochondrion. Indeed, it is important to

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ATP

ATP

Figure 2. A schematic view of the pre-endosymbiont hypothesis. The premitochondrion is seen as a membrane-bound entity endowed with a protein import system and various ion/small-molecule transporters, compart-mentalizing many of the metabolic functions of the mitochondrion. The premitochondrion is assumed to haveevolved endogenously within the pre-eukaryote cell from proteins that now constitute a major portion of theNPC (non-a-proteobacterial component) of the contemporary mitochondrial proteome. Ana-proteobacteria-like endosymbiont is converted to the ancestral mitochondrion, effectively “capturing” the protein componentsand functions of the premitochondrion. The endosymbiont contributes the APC (a-proteobacterial compo-nent) of the mitochondrial proteome, which is largely directed toward specification of energy-generatingcapacity (in the form of coupled electron transfer/oxidative phosphorylation). The premitochondrion !ancestral mitochondrion conversion is greatly facilitated by the existence of the reservoir of premitochondrialproteins in the host cell. Endosymbiont-to-nucleus gene transfer (EGT) coupled with rationalization of redun-dant pathways results in the formation of evolutionarily chimeric enzymatic pathways and protein complexes inthe ancestral mitochondrion, as well as functional relocation of the products of some transferred a-proteobac-terial genes to other cellular compartments. The premitochondrion is assumed to be a non-energy-generatingorganelle that imports ATP; a key innovation in the transition to the contemporary mitochondrion is thegeneration/acquisition of an ADP/ATP transporter that reverses this flow, ultimately allowing the mitochon-drion to become the primary site of ATP generation for cellular functions.





emphasize that the essential function of the con-temporary mitochondrial genome is the speci-fication of critical protein subunits of the fourmembrane-bound complexes (I–IV) of the mi-tochondrial electron transport chain and theATP synthase (complex V), whereas otherAPC proteins are largely involved in supportingmitochondrial genome expression. In essence,the pre-endosymbiont hypothesis incorporatesaspects of both endogenous and xenogenousscenarios for the origin of the mitochondrion.

SOME IMPLICATIONS ANDCONSIDERATIONS

Internal membrane-bound compartments suchas the endoplasmic reticulum, Golgi body, andother components of the endomembrane sys-tem are assumed to have emerged endogenouslywithin the evolving eukaryotic cell. Thus, it isnot inconceivable that an organelle such as thepremitochondrion could likewise have evolvedendogenously. Indeed, the premitochondrionis conceptually similar to the peroxisome, amembrane-bound, non-DNA-containing eu-karyotic organelle that is generally consideredto have had an endogenous origin (Gabaldon2013). One might imagine that the selectiveadvantage for such compartmentation wouldbe the concentration of enzymes and reactantsin various metabolic pathways, in the face of asteady increase in cell size, one of the hallmarksof the eukaryotic cell.

Symbiotic models of mitochondrial originusually invoke the introduction of aerobic (ox-ygen-dependent) metabolism into an essen-tially anaerobic host cell (Margulis 1970), theinitial selective advantage being either more ef-ficient ATP generation in an increasingly oxy-gen-rich environment or oxygen detoxification(the “ox-tox” hypothesis) (Andersson and Kur-land 1999). However, there are good argumentsto consider that the ancestral eukaryotic cellwas, in fact, already capable of aerobic metabo-lism (Raff and Mahler 1972). In that case, as-suming that an aerobic a-proteobacterium wastaken up by an aerobic pre-eukaryote via phago-cytosis (another eukaryotic hallmark), thereneed have been no selective advantage initially

that depended on the symbiont being an aerobe.Moreover, the relationship of the ingestee toingester might be less one of food (as generallyassumed) and more one of a coexisting long-term inhabitant, as is the situation in contem-porary eukaryotes that harbor other organisms,such as the amoebozoan protist Acanthamoebacastellanii. Although this phagotroph preys onother microbes for food, it also hosts a widevariety of Bacteria (including various types ofa-proteobacteria) in stable intracellular associ-ations (Marciano-Cabral 2004). This type of re-lationship would seem much more conducive topromoting a symbiont-to-organelle transfor-mation in the long term than one in which thesymbiont happens to be a rare escapee fromdigestion. As Lang and Burger (2012) havecommented, “While ancient symbiotic eventsare often viewed as driven by selective advantagefor both partners, it is equally possible that theyhave started out by pure accident. . . .”

If we accept that the host cell was capableof aerobic metabolism, what type of energy-generating system might it have used? Giventhe ubiquity and antiquity of aerobic metabo-lism based on cytochrome oxidase (Castresanaet al. 1994), it is possible that both host cell andsymbiont had parallel systems for energy gener-ation. Here again, no selective advantage wouldaccrue to the host from a symbiont bringingsuch a system into it. Initially, the energy re-quirements of host and symbiont would bemet by their respective energy-generating sys-tems. However, the equation would shift dra-matically once ADP import into the symbiontand ATP export from it evolved. That changemight have occurred through a “reverse flow”mutation of the ATP-importing/ADP-export-ing translocase postulated here to have beenpresent in the premitochondrion, or throughelaboration of a completely new transporter.Evidently, the required transporter was not con-tributed by the symbiont (Karlberg et al. 2000).

The emergence of an ADP-importing/ATP-exporting translocase is a fundamental elementin all models of mitochondrial evolution. Sub-sequent intracellular proliferation of the evolv-ing mitochondrion would lead to a pronouncedincrease in bioenergetic membranes supporting

M.W. Gray




coupled electron transport and oxidative phos-phorylation, greatly increasing the productionof ATP available to the cell as a whole. Indeed,Lane and Martin (2010) argue persuasively thatthe increased energy provided by specializationof the endosymbiont into an ATP-generatingorganelle and increasing organelle copy numberwas “a prerequisite to eukaryote complexity: thekey innovation to multicellular life.”

Even before the elaboration of an ADP-im-porting/ATP-exporting translocase, acquisitionby the symbiont of the protein import systemand other transporters of the premitochondri-on would set the stage for relocation to thesymbiont of many of the metabolic pathwaysof the premitochondrion. The advantage ofperforming these activities in the symbiontrather than in the premitochondrion wouldbe that the symbiont would have generated theATP in situ for biosynthetic pathways requiringit, whereas the premitochondrion would havehad to import ATP from the cytosol for thesesame reactions. Once ATP export from evolvingorganelle to cytosol was established, the selec-tive advantage would be greatly enhanced,because the evolving mitochondria could pro-liferate and thereby sequester an increasinglylarger proportion of the structural and enzy-matic components required to make the func-tional premitochondrion. Intracellular compe-tition of these sorts would inevitably lead to thedisappearance of the premitochondrion as aseparate entity and the fixation of its successor,the modern mitochondrion.

Finally, because host and symbiont genomeswould initially encode a number of the same orvery similar metabolic pathways, a process ofgenome reduction would ensue whereby dupli-cate genes were lost from one or the other ge-nome. Most of this reduction appears to haveinvolved the endosymbiont genome, with manygenes being lost or transferred to the nucleus(Gabaldon and Huynen 2007). In the lattercase, relocated genes would have had to acquiresignals for nuclear expression and mitochondri-al targeting, although some a-proteobacterialsymbiont genes have evidently been retargetedto other cellular locations, where they currentlyfunction (Gabaldon and Huynen 2007). Con-

versely, host genes have evidently substitutedfor symbiont genes in specifying componentsof many mitochondrial pathways.

A particularly notable example in this regardis the tricarboxylic acid (TCA) cycle, genes forwhich were presumably contained originally inthe symbiont genome but moved to the nucleargenome. However, not all of the mitochondrialTCA cycle enzymes are decidedly a-proteobac-terial in origin (e.g., Kurland and Andersson2000; Schnarrenberger and Martin 2002). Cur-rent models of mitochondrial evolution gener-ally assume that LGT from another bacterialsource led to replacement of the originally a-proteobacterial gene: an assumption that re-quires many separate LGTevents to account forthe large number of non-a-proteobacterial con-stituents of the mitochondrial proteome. Thescenario presented here suggests a simplified al-ternative: that the non-a-proteobacterial com-ponents of the TCA cycle (and other mitochon-drial pathways) are encoded by genes that wereoriginally present in the ancestral eukaryotichost before the arrival of the mitochondrial en-dosymbiont, and that they replaced the homol-ogous endosymbiont genes at an early stage inthe symbiont-to-organelle transition.

CONCLUDING REMARKS

The new model of mitochondrial origin andevolution outlined here (Fig. 2) is an attemptto integrate conflicting data that are not read-ily accommodated by current hypotheses. Inparticular, the pre-endosymbiont hypothesisaccounts in a relatively straightforward wayfor the presence and characteristics of botha-proteobacterial and non-a-proteobacterialcomponents (APC and NPC, respectively) ofthe mitochondrial proteome. The existence ofan ancestral metabolic organelle (the premito-chondrion) would effectively “precondition”the host cell for the successful conversion of abacterial endosymbiont into an integrated or-ganelle because much of the material necessaryfor this transformation would already be avail-able in the host, in the form of much of theNPC. Furthermore, the hypothesis presentedhere provides an explanation for what the pre-





existing portion of the NPC would be doingbefore the endosymbiont arrived; moreover, ithelps to explain not only the mosaic nature ofthe mitochondrial proteome overall, but alsothe mosaic character of the enzymatic pathwayscontained therein.

The pre-endosymbiont hypothesis elab-orated here effectively represents a synthesis ofprevious, contending mitochondrial origin hy-potheses, with much of the mitochondrial pro-teome (represented within the NPC) having anendogenous origin and the minority compo-nent (the APC) having a xenogenous origin. Insome respects, this hypothesis hearkens back tothe (nonsymbiotic) model for the origin of mi-tochondria published more than 40 years ago byRaff and Mahler (1972). That publication pre-dated the molecular evidence that convincinglyestablished the a-proteobacterial nature of themitochondrial genome: data that had a pro-found influence on the demise of nonsymbioticmodels and the ascendency of endosymbioticmodels of mitochondrial origin (Gray andDoolittle 1982; Gray 1992). The moleculardata, drawn in large part from analyses ofmtDNA and the genes it contains, leave littledoubt that bacterial endosymbiosis played a cru-cial role in the origin of the mitochondrion, al-though I would contend that that role is consid-erably more circumscribed than we initiallyimagined. Because the mitochondrion was orig-inally viewed as a singular entity rather than as amosaic, proof for the origin of the mitochondri-al genome was generally accepted as constitutingproof for the origin of the organelle as a whole.In retrospect, that extrapolation turned out to beoverly simplistic. In the context of a discussionelsewhere in this series (Keeling 2013b), it isworth contemplating how different the histori-cal record of our views about mitochondrialorigin and evolution might have been had ourdetailed knowledge and understanding of themitochondrial proteome predated that of themitochondrial genome.

ACKNOWLEDGMENTS

This essay is dedicated to my PhD supervisor,Professor Byron G. Lane, on the occasion of his

80th birthday, in recognition and deep appreci-ation of 50 years of mentorship and friendship. Iam grateful to P.J. Keeling, J.M. Archibald, andA.J. Roger for critical comment on an early draftof this article; to C.G. Kurland for stimulatingdiscussion about mitochondrial evolution; andto G. Burger for provision of the A. godoyimtDNA gene map shown in Figure 1.

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2014; doi: 10.1101/cshperspect.a016097Cold Spring Harb Perspect Biol Michael W. Gray and Evolution of MitochondriaThe Pre-Endosymbiont Hypothesis: A New Perspective on the Origin

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