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The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and of Mitochondria

Michael W. Gray

Centre for Comparative Genomics and Evolutionary , Department of Biochemistry and Molecular 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-proteobacterial in 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 current models of mitochondrial origin. I propose that in the host that accommodated an a- proteobacterial endosymbiont, much of the NPC was already present, in the form of a membrane-bound metabolic (the premitochondrion) that compartmentalized many of the non-energy-generating functions of the contemporary . I suggest that this organelle also possessed a protein import system and various ion and small-molecule transporters. In such a scenario, an a-proteobacterial endosymbiont could have been converted relatively directly and rapidly into an energy-generating organelle that incorporated 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 xenogenous origin.

onsidering the central role played in all eu- evolution, focusing particularly on how well the Ckaryotic cells by mitochondria or mito- accumulating molecular data can be accommo- chondrion-related (MROs, such as dated in current models of mitochondrial ori- and ) (Hjort et al. gin. In this context, the origin and evolution of 2010; Shiflett and Johnson 2010; Mu¨ller et al. the mitochondrial proteome, as opposed to the 2012), the question of the origin and subse- origin and evolution of the mitochondrial ge- quent evolution of the mitochondrion has nome, were examined from the perspective of long captivated and challenged biologists. In a comparative mitochondrial proteomics. Some- recent article in this series (Gray 2012), I dis- what disconcertingly, as more data have become cussed in detail several aspects of mitochondrial available, we find ourselves considerably less

Editors: Patrick J. Keeling and Eugene V. Koonin Additional Perspectives on The Origin and Evolution of available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016097 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016097

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M.W. Gray

certain about key aspects of how mitochondria † We know that the most -like (least- originated than we were (or thought we were) derived) and -rich mitochondrial ge- several decades ago. nomes are found within the core jakobids Here, I summarize key points discussed in (Fig. 1) (Lang et al. 1997; Burger et al. more detail in the previous article before pre- 2013), a group of flagellated that senting a novel perspective on how the mito- are members of the proposed eukaryotic chondrion might have originated. The new supergroup Excavata (Hampl et al. 2009). model proposed here, which represents a syn- Jakobid mtDNAs specify strikingly bacteria- thesis of both endogenous (“origin from with- like large subunit (LSU), small subunit (SSU) in”) and xenogenous (“origin from outside”) and 5S ribosomal RNAs (rRNAs) and trans- modes, is advanced in an attempt to account fer RNAs (tRNAs), as well as upward of 70 0 for the inability of a purelyendosymbiotic mod- proteins, including subunits of an a2bb s el, whose strongest support has come from RNA polymerase, the enzyme used for tran- studies of the mitochondrial , to ade- scription in Bacteria. Other notable features quately accommodate data on the mitochondri- include encoding transfer-messenger al proteome. RNA (tmRNA) and the RNA component of RNase P; bacteria-like operon organization; and putative Shine–Dalgarno-like motifs in- A RUMSFELDIAN TAKE ON THE ORIGIN volved in initiation in Bacteria. OF THE MITOCHONDRION † We know that certain components of the ... there are known knowns; there are things we original, bacteria-like systems used for repli- know we know. cation and of mtDNA (e.g., the We also know there are known unknowns; that is to mitochondrial RNA polymerase) have been say, we know there are some things we do not know. replaced in some or virtually all eukaryotes But there are also unknown unknowns—the ones we by bacteriophage-like counterparts (Shutt don’t know we don’t know. and Gray 2006). —U.S. Secretary of Defense Donald Rumsfeld † We know that mitochondrial genome evolu- Press briefing, February 12, 2002 tion has been characterized by extensive gene This Rumsfeldian view provides a useful loss, with relocation of many of the genes in framework for outlining key aspects of what the protomitochondrial genome to the nu- we know and what we don’t know about the cleus and import of their protein products origin and evolution of the mitochondrion. back into the organelle. Such endosymbiotic gene transfer (EGT) usually involves com- plete genes, but EGT may also relocate only Known Knowns a portion of a mtDNA-encoded gene, with the rest of that gene remaining in and ex- † We know from comparative mitochondrial pressed from the mitochondrial genome genomics—through the analysis of genes (Burger et al. 2003). contained in mtDNA and the protein se- quences they specify—that the mitochondri- † We know that the mitochondrion originated al genome is of (eu)bacterial origin. These only once, that is, mitochondria in all eu- sequences point us to members of a particu- karyotic lineages descend from a common lar bacterial phylum, a- (also ancestor, the LMCA (last mitochondrial termed ), as the closest common ancestor). Commonality in key as- extant free-living relatives of mitochondria, pects of genetic and biochemical function, and therefore the bacterial group from within the presence of shared mitochondrion-spe- which the mitochondrial genome emerged cific deletions in bacteria-like operons pre- (Gray and Doolittle 1982; Gray 1992). sent in some mtDNAs, and phylogenetic

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The Pre-Endosymbiont Hypothesis

rpl31 rns rrn5 rnl rpl34 rps1 rpl27 atp8 orf97 atp4 nad7 nad9 cob orf35 nad10 atp1 nad3 C atp3 rpl20 L rpl35 E P rpl11 rpl1 sdh3 I Me sdh4 K W rpl10 sdh2 S nad4L nad5 nad4 65 67/0 5 rpoB nad2 60 kbp rps2 R 10 VD ssrA FA 55 Andalucia orf240 godoyi 15 orf203 rpoC orf123 50 rps12 mtDNA orf146 20 rps7 ccmF 67,656 bp ccmC 45 nad11 ccmB 25 nad1 ccmA I G 40 cox11 RQ 30 rpl32 35 cox3 atp6 N tufA tatA rps10 cox2 S rp12 nad6 H nad8 T L Y rps19 cox1 Mf V G rps3 S rpoD L rpl16 cox15 rpl14 tatC rpl5 atp9 rpl19 rps14 rps4 rps8 rnpB rpl6 rpoA rpl18 rps11 rpl13

Figure 1. Genetic map of the strikingly bacteria-like mitochondrial genome of the core jakobid, Andalucia godoyi: the most gene-rich mitochondrial genome known (Burger et al. 2013). (Solid rectangles) Genes, with those 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 , fungal, and mtDNAs; (blue) expanded gene set, mostly present in protist and mtDNAs; (red) genes predominantly found in jakobid mtDNAs; (green) hypothetical protein-coding genes (ORFs). A. godoyi mtDNA 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 translation elongation factor (tuf ); four subunits of a bacteria-like multisubunit RNA polymerase (rpoA,B,C,D); eight proteins 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) elongator methionine; transfer-messenger RNA (tmRNA) (ssrA); and the RNA component of RNase P (rnpB). Five additional open reading frames (orf ) encode putative proteins of unknown function. (Arrows) Positions of predicted promoters. (Figure courtesy of G. Burger.)

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M.W. Gray

tree reconstruction strongly support this in- such as Rickettsia, Anaplasma, and Ehrli- ference (Gray et al. 1999). chia—asthe closestrelativesof mitochondria. Rickettsiales comprises two distinct families, † We know that despite an evident a-proteo- Rickettsiaceae and Anaplasmataceae, with bacterial origin of the mitochondrial ge- several studies suggesting that mitochondria nome, only 10%–20% of the mitochon- are more closely related to the former family, drial proteome—the collection of proteins containing various Rickettsia species, than to making up the organelle—can confidently thelatter,whichencompassesthegeneraAna- be traced to a-proteobacteria (this a-proteo- plasma, Ehrlichia, and . However, bacterial component is referred to here as the there is currently no consensus in the litera- APC). The 80%–90% non-a-proteobacte- ture astowhether mitochondria branchwith- rial component, or NPC, appears from phy- in or as sister to either of the two Rickettsiales logenetic analysis either to be of diverse pro- families,oroutsideofRickettsialesaltogether. karyotic (bacterial or archaeal) evolutionary Several newly identified free-living a-proteo- ancestry or to have arisen specifically within bacteria have recently been considered candi- eukaryotes (Karlberg et al. 2000; Kurland and dates for the title of “closest mitochondrial Andersson 2000; Gray et al. 2001; Gabaldo´n relative” (Brindefalk et al. 2011; Georgiades and Huynen 2003, 2004, 2007; Szklarczyk et al. 2011; Viklund et al. 2012), but method- and Huynen 2010). ological issues complicate † We know that the core of the mitochondrial reconstructions (Thrash et al. 2011; Rodrı´- proteome was already in place in the LMCA, guez-Ezpeleta and Embley 2012), and thus that is, the LMCAwas likely as complex in its the jury is still out on this point. fundamental functions as a contemporary † We do not know the evolutionary origin of aerobic mitochondrion. We also know that most of the mitochondrial proteome (i.e., subsequent retailoring of this proteome, in- the NPC). The general assumption has volving both gain and loss of protein com- been that these proteins were added to the ponents and functions, occurred to different a-proteobacterial symbiont during its trans- extents in diverse eukaryotic lineages, lead- formation into an organelle, but where these ing to the emergence of lineage-specific mi- proteins came from and how and when they tochondrial proteins characterized by a re- were added to the evolving mitochondrion is stricted phylogenetic distribution (Huynen not spelled out in models of mitochondrial et al. 2013). origin. A substantial portion of the NPC ap- pears to be -specific (i.e., there are Known Unknowns no evident homologs within either of the domains Bacteria or ) and thus is Despite what we are confident we know we presumed to have arisen within the domain know, several key aspects of mitochondrial ori- Eucarya, although by what mechanism(s) is gin and evolution remain unresolved: unclear. † We do not know precisely which extant a- proteobacterial lineage is most likely to be Unknown Unknowns the descendant of the lineage that was the evolutionary source of the mitochondrial ge- We have no way of knowing what we might still nome (for more detailed discussion of this discover about mitochondrial evolution, al- point and references, see Gray 2012; Lang and though we may be confident that further inves- Burger 2012). Various studies have consis- tigation of diverse mitochondrial and tently pointed to Rickettsiales, one of six or proteomes will continue to surprise us. Unan- more orders within a-proteobacteria—and ticipated and increasingly bizarre forms of mi- comprising obligate intracellular parasites tochondrial genome organization and expres-

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The Pre-Endosymbiont Hypothesis

sion continue to be reported, a recent example nario most closely approximates the classical being from the euglenozoon Diplonema papil- endosymbiont hypothesis of mitochondrial or- latum. Here, mitochondrial genes are fragment- igin (Margulis 1970; Doolittle 1980), whereas ed, with the individual gene pieces distributed the hydrogen hypothesis (Martin and Mu¨ller among several small, circular molecules and the 1998) is an example of the sce- corresponding transcripts processed at both nario (for more detailed discussion, see Koonin ends before being joined together in the correct 2010; Gray 2012). order to generate a translatable mRNA, a pro- The archezoan scenario takes its name cess that also involves U insertion RNA editing from the “archezoa hypothesis”(Cavalier-Smith (Vlcˇek et al. 2011; Kiethega et al. 2013). (Nota- 1987, 1989), which posited that a group of bly, D. papillatum, whose mitochondrial genetic putatively amitochondrial eukaryotes was, in system is arguably one of the most radically di- fact, primitively amitochondriate (i.e., never vergent yet characterized, is a member of Exca- possessed mitochondria). As such, archezoans vata, which as noted above contains the core were offered as potential modern representatives jakobids, the eukaryotic group featuring the of the sort of host cell that would have taken up most primitive [least-derived] mitochondrial the mitochondrial endosymbiont. The fact that genomes known.) Investigation of the mito- archezoans branched early in SSU rRNA trees chondrial genetic system has uncovered awealth supported the idea that these eukaryotes were, of unexpected phenomena, including variant indeed, primitive eukaryotes. However, the ar- genetic codes, a bewildering array of RNA edit- chezoa hypothesis was abandoned after the ing processes, self-splicing introns, fragmented discovery that all putatively amitochondriate rRNAs, and tRNA import (for review, see Gray eukaryotes so far investigated contain a mito- et al. 1998; Gray 2003), as well as documenting chondrion-related organelle (MRO) and that clear examples of mitochondrion-to-nucleus the early branching position of archezoans is a gene transfer (Adams and Palmer 2003). Based methodological artifact attributable to a rela- on the experience of the last three decades, we tively high rate of sequence divergence in arche- may expect more of the same. zoan SSU rRNA genes, such that these divergent sequences are incorrectly pulled toward the base of the eukaryotic tree (“long-branch attrac- CONTENDING SYMBIOTIC MODELS FOR tion”). The demise of the archezoa hypothesis THE ORIGIN OF MITOCHONDRIA removes a base of support for the archezoan sce- Endosymbiotic models for the origin of mito- nario of mitochondrial origin (admittedly, we chondria (for review, see Martin et al. 2001) can cannot exclude the possibility that primitively be grouped into two scenarios, depending on amitochondriate eukaryotes still exist but have whether the organelle is seen to have originated not yet been discovered—albeit not for lack of after or at the same time as the rest of the eukary- exhaustive searching—or that such lineages, if otic cell (Gray et al. 1999). These two scenarios they once existed, have all become extinct). On have been dubbed “archezoan” and “symbio- the other hand, as critically discussed in more genesis,” respectively (Koonin 2010). The arche- detail elsewhere (Koonin 2010; Forterre 2011; zoan scenario hypothesizes that an amito- Gray 2012; Lang and Burger 2012), symbiogen- chondriate eukaryotic host cell phagocytosed esis models have their own problems both with an a-proteobacterium, which was subsequently respect to the origin of the mitochondrion and transformed into the mitochondrion. In con- the origin of the eukaryotic cell per se. A partic- trast, the symbiogenesis scenario hypothesizes ular difficulty is the fact that collectively more that a physical and metabolic fusion between bacterial-type genes in eukaryotes appear to de- an a-proteobacterium and an archaeal cell gen- rive from diverse non-a-proteobacterial line- erated the ancestor of the eukaryotic cell, with ages or fail to affiliate robustly with any specific the a-proteobacterial partner subsequently be- bacterial phylum (Pisani et al. 2007), whereas coming the mitochondrion. The archezoan sce- in a symbiogenesis scenario, one would antici-

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M.W. Gray

pate that any bacterial genetic signal in the nu- yet the available data already show that key clear genome would be predominantly a-pro- mitochondrial protein assemblages and path- teobacterial. ways existed in essentially their modern form in the last eukaryotic common ancestor (LECA). Lineage-specific modification through DECONSTRUCTING THE MITOCHONDRIAL both loss and addition of mitochondrial pro- PROTEOME teins has certainly occurred, and specific cases The mosaic nature of the mitochondrial pro- of mitochondrial protein acquisition through teome (Karlberg et al. 2000; Szklarczyk and lateral gene transfer (LGT) can definitely be Huynen 2010) is proving increasingly difficult identified, but the fact remains that the basic to accommodate within current schemes of mi- structure and complex function of the mito- tochondrial origin. A specific contribution chondrion were likely already well established from a-proteobacteria is strongly indicated, in the LECA. Thus, between the events that cre- comprising the mitochondrial genome, the ated both the modern mitochondrion and the few proteins it encodes, and a limited number rest of the cell in which it resides, much reorga- of other proteins whose genes we infer once nization of symbiont and host cell would have resided in mtDNA but were subsequently relo- had to occur, whether the acquisition of the cated to the nucleus via EGT.This a-proteobac- mitochondrion was through a symbiogenesis terial component (APC) forms a crucial part of pathway or an archezoan one. the evidence that has been offered in support of Symbiont-to-mitochondrion transforma- an endosymbiotic origin of mitochondria; thus, tion is generally considered to have occurred in it was a surprise to discover that the APC is a a stepwise fashion, involving both loss and ad- quantitatively minor (albeit qualitatively essen- dition of proteins and functions. In comparing a tial) element of the contemporary oxygen-using reconstructed protomitochondrial proteome and ATP-producing organelle. The non-a-pro- with the proteomes of contemporary a-proteo- teobacterial component (NPC), the evolution- bacteria and mitochondria (yeast and human), ary origin of which appears both phylogeneti- Gabaldo´n and Huynen (2007) inferred that cally diverse and largely obscure, dominates the the transformation of an a-proteobacterial mitochondrial proteome. Any mitochondrion symbiont to an organelle involved loss of some origin scenario must take into account the ac- protomitochondrial metabolic pathways or cumulating evidence that says that only a minor their movement to other parts of the cell, as portion of the mitochondrial proteome is clear- well as acquisition of new proteins leading to ly of a-proteobacterial ancestry, whereas the the gain of novel pathways, such as the protein evolutionary origin of the major portion of mi- import machinery and mitochondrial carriers. tochondrial proteins is obscure. Indeed, it is In addition, new proteins have been recruited to important to emphasize that, strictly speaking, existing mitochondrial complexes resulting, for the data used to “prove” the endosymbiotic or- example, in the expansion of respiratory com- igin of the mitochondrion in essence only prove plex I from 17 subunits in a-proteobacteria to the origin of the mitochondrial genome per se. more than 40 subunits in eukaryotes (Cardol In both classical endosymbiotic (arche- 2011). Such restructuring over a long period of zoan) and symbiogenesis (fusion) scenarios time would presumably have generated a series for the origin of the eukaryotic cell, an a-pro- of intermediate organelles differing in the range teobacterial symbiont is assumed to have been of functions they possessed. A key question that extensively retailored to create the modern or- such a scenario poses is how the hundreds (per- ganelle, both by wholesale gene loss from the haps thousands) of proteins making up the much larger endosymbiont genome and by mitochondrial proteome were introduced. In the addition of new proteins. Comparatively particular, what was the evolutionary source of few mitochondrial proteomes have been inves- the NPC? I argue here that neither the archezoan tigated directly (e.g., via mass spectrometry), nor the symbiogenesis scenario accommodates,

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The Pre-Endosymbiont Hypothesis

in a compelling way, the diverse functional and wise, they would still score in phylogenetic re- phylogenetic character of the NPC. constructions as “a-proteobacterial.” At this point, a word of caution is in order In yeast, about half of the -like with respect to how we view the NPC. In par- sequences in the mitochondrial proteome are ticular, it is possible (perhaps even likely) that a common to Bacteria and Archaea as well as eu- portion of the NPC consists of originally a-pro- karyotes, and thus may well have been present in teobacterial proteins that are too divergent in the last universal common ancestor (LUCA) sequence to be recognized as such, so that the (Karlberg et al. 2000). Of the remainder, few 10%–20% estimated a-proteobacterial contri- affiliate convincingly with specific non-a- bution to the mitochondrial proteome is prob- proteobacterial bacterial lineages, whereas the ably an underestimate, and instead represents a a-proteobacterial signal is particularly robust minimal contribution from the a-proteobacte- (Lang and Burger 2012). Moreover, we still rial endosymbiont. Marked differences in phy- have to account for the large proportion (up to logenetic resolution are to be expected in a pro- 50%) of the mitochondrial proteome that is tein collection as complex as the mitochondrial unique to eukaryotes and that has been assumed proteome, even though it is notable that the a- to have evolved within the eukaryotic lineage. proteobacterial signal in the recognizable APC Thus, even if we are liberal in assigning a per- is particularly strong (Lang and Burger 2012). centage to the APC (e.g., doubling it to 20%– Nevertheless, it does not seem probable that the 40%), we are still left with upward of 60%–80% “bacterial” portion of the NPC could all be ac- of the mitochondrial proteome that does not counted for by divergent proteins of originally appear to have come from an a-proteobacterial a-proteobacterial origin. source. Another suggestion is that the a-proteobac- In this context, a final possibility is that at terial symbiont was itself chimeric as a conse- least some of the NPC proteins predated the quence of prior and extensive LGT. In this case, endosymbiotic event, preexisting in whatever the symbiont could have contributed genes en- the host cell was that accommodated the sym- coding proteins that appear to come from other biont. For example, in a recent review on bacterial lineages. A problem with this sugges- endosymbiosis, Keeling (2013a) has suggested tion isthat this non-a-proteobacterial contribu- a “targeting rachet” model in which protein tion would have had to have been extensive targeting is established in the host before the enough to quantitatively swamp the a-proteo- endosymbiont is permanently integrated. The bacterial component in the contemporary mi- idea is that this process would target preexisting tochondrion, which would imply a highly chi- transporters to the membrane of transient sym- meric symbiont genome and/or differential loss bionts, accelerating and facilitating the sym- of a-proteobacterial genes in the subsequent biont-to-organelle transformation. symbiont-to-organelle transformation. More- The possibility that some portion of what over, it is not clear from this scenario why ex- would become the mitochondrial NPC preex- tant a-proteobacterial genomes generally lack isted in the host is attractive because it would orthologs corresponding to this putative LGT effectively “precondition” the host–symbiont component. Thiegart et al. (2012) attribute interaction. Rather than the NPC appearing this phylogenetic diversity to “a single origin on the scene after the acquisition of the en- of mitochondria followed by subsequent LGT dosymbiont (e.g., via LGT) or actually being among free-living .” However, this part of the endosymbiont, it might be that explanation would seem to demand that for mi- much of the NPC was already present in the tochondrial homologs transferred in this way, host cell that accommodated the a-proteobac- the transferred genes would have to have evolved terial symbiont. If such were the case, raw more slowly than donor (a-proteobacterial) material for initiating the conversion of a bac- ones, or would have to have been selectively terium to an organelle would be readily avail- lost from a-proteobactrerial lineages. Other- able.

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M.W. Gray

The above proposal demands a rather differ- mologous to those of modern mitochondria, ent view of the eukaryotic cell and how it arose , nuclei, spliceosomes, cytoskele- than is suggested in current proposals. In par- ton, and the like are evident in the proteome ticular, the idea that the eukaryotic cell is funda- of MRUCA.” mentally a fusion between bacterial and archaeal A notable implication of the proposal by partners, with a large number of novel proteins Harish et al. (2013) is that the LECA could arising subsequently and specifically within the well have harbored a complex proteome that eukaryotic lineage, is increasingly being ques- already included much of what was destined tioned (e.g., Koonin 2010; Forterre 2011; Lang to become the mitochondrial NPC. This infer- and Burger 2012). In proposing yet another ence would be consistent with a new model of symbiogenesis scenario (Planctomycetes, Ver- mitochondrial evolution outlined below, which rucomicrobia, Chlamydiae [PVC] Bacteria— hypothesizes that essential elements of the NPC Thaumarchaeota—,” or PTV), Forterre already existed in the host cell that formed an (2011) states that “the transformation of a association with the APC-contributing a-pro- FECA (first eukaryotic common ancestor) teobacterium. born from a fusion event into a reasonable an- cestor of modern eukarya remains difficult to imagine in aconvincing way in the PTV scenario THE “PREMITOCHONDRION”: A PUTATIVE ORGANELLE PREDATING THE as in all other fusion scenarios.” In fact, Forterre a-PROTEOBACTERIAL ENDOSYMBIOSIS is led to conclude that eukaryotes “probably evolved from a specific lineage, according to If we accept the possibility that much of the the three domains scenario originally proposed mitochondrial proteome was already present by Carl Woese.” in the eukaryotic host cell before the a-proteo- In contrast to the prevailing symbiogenesis bacterial symbiont arrived, what might it have view, a radically different proposal for the origin been doing? To address this question, I propose of the eukaryotic lineage has just been published a new model (the “pre-endosymbiont hypoth- by Harish et al. (2013), drawn from phylogenetic esis”) (Fig. 2) that envisages an endogenously reconstructions based on genome content of evolved organelle that we might term the “pre- compact protein domains, as defined in the mitochondrion,” which I suggest preexisted in SCOP (structural classification of proteins) da- what was in many respects already a eukaryotic tabase. These investigators identify Archaea and cell (the “pre-eukaryote”). According to this Bacteria as sister domains in a lineage (“akar- scheme, the premitochondrion was essentially yotes”) that descends in parallel, but indepen- a metabolic rather than an energy-generating dently, from an entity they term the “MRUCA” organelle: a membrane-bound structure com- (most recent universal common ancestor), partmentalizing many (perhaps most) of the which “is not a bacterium.” According to their pathways and reactions characteristic of extant analysis, Harish et al. (2013) assert that MRUCA mitochondria, such as amino acid, nucleotide, would have contained 75% of the unique SFs and lipid ; coenzyme biosynthesis; (SCOP superfamilies) encoded by extant ge- iron–sulfur cluster formation, and so on. The nomes of the domains Bacteria, Archaea, and premitochondrion would already have mem- Eucarya, each specifying a proteome that par- brane structural proteins and a protein import tially overlaps each of the other two: “the conse- system, as well as transporters for various ions quence of descent from a universal common an- and metabolites, including amino acids and nu- cestor, MRUCA.” The authors state: “we cannot cleotides. However, because the premitochon- draw the conclusion that MRUCAwas morpho- drion would be energy-consuming rather than logically similar to a eukaryote,” but they do energy-generating, it would possess a nucleo- conclude that “elements and cohorts of SFs tide carrier system to import ATP and export from its proteome are recognizable in the pro- ADP, the reverse of what occurs in the contem- teomes of modern eukaryotes. Thus, SFs ho- porary mitochondrion.

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The Pre-Endosymbiont Hypothesis

ATP Premitochondrion (NPC: endogenous)

ATP

α-Proteobacterial endosymbiont Ancestral (APC: xenogenous) mitochondrion

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 have evolved endogenously within the pre-eukaryote cell from proteins that now constitute a major portion of the NPC (non-a-proteobacterial component) of the contemporary mitochondrial proteome. An a-proteobacteria- like endosymbiont is converted to the ancestral mitochondrion, effectively “capturing” the protein components and 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-generating capacity (in the form of coupled electron transfer/oxidative phosphorylation). The premitochondrion ! ancestral mitochondrion conversion is greatly facilitated by the existence of the reservoir of premitochondrial proteins 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 in the 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-generating organelle that imports ATP; a key innovation in the transition to the contemporary mitochondrion is the generation/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.

In this scenario, a large part of the mito- phosphorylation would have existed and func- chondrial proteome (much of the NPC) would tioned in the symbiont to generate both a pro- be of endogenous origin, having evolved within ton gradient and ATP, it is likely that it was the the pre-eukaryotic cell, along with its other symbiont that was modified by premitochon- subcellular components and compartments. In drial components to become the mitochondri- some respects, the premitochondrion would on, rather than the symbiont genome being resemble non-DNA-containing MROs such as introduced somehow into and becoming func- hydrogenosomes and mitosomes, which per- tional within the premitochondrion. form a variable subset of normal mitochondrial The pre-eukaryote cell would thus resemble metabolic functions but lack the capacity to the proposed host cell in the classical serial generate ATP via coupled electron transport– endosymbiont hypothesis (Taylor 1987) except oxidative phosphorylation. Such MROs could that a progenitorof the mitochondrion (the pre- be regarded as representing a reversion to an mitochondrion) was already present. Subse- ancestral form. quent transformation of the a-proteobacterial Transformation of an a-proteobacterial symbiont, via incorporation of structural and symbiont into an integrated organelle would functional elements of the premitochondrion, occur as conventionally envisaged, but it would would effectively add energy-generating capac- be substantially simplified if, in fact, the bulk of ity,in the form of electron transport and coupled the proteins required for such a radical reorga- oxidative phosphorylation, to the varied meta- nization were already present, in the form of bolic capabilities already possessed by the a functional entity. Because coupled oxidative premitochondrion. Indeed, it is important to

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M.W. Gray

emphasize that the essential function of the con- that depended on the symbiont being an aerobe. temporary mitochondrial genome is the speci- Moreover, the relationship of the ingestee to fication of critical protein subunits of the four ingester might be less one of food (as generally membrane-bound complexes (I–IV) of the mi- assumed) and more one of a coexisting long- tochondrial electron transport chain and the term inhabitant, as is the situation in contem- ATP synthase (complex V), whereas other porary eukaryotes that harbor other , APC proteins are largely involved in supporting such as the amoebozoan protist mitochondrial genome expression. In essence, castellanii. Although this phagotroph preys on the pre-endosymbiont hypothesis incorporates other microbes for food, it also hosts a wide aspects of both endogenous and xenogenous variety of Bacteria (including various types of scenarios for the origin of the mitochondrion. a-proteobacteria) in stable intracellular associ- ations (Marciano-Cabral 2004). This type of re- lationship would seem much more conducive to SOME IMPLICATIONS AND promoting a symbiont-to-organelle transfor- CONSIDERATIONS mation in the long term than one in which the Internal membrane-bound compartments such symbiont happens to be a rare escapee from as the , Golgi body, and . As Lang and Burger (2012) have other components of the endomembrane sys- commented, “While ancient symbiotic events tem are assumed to have emerged endogenously are often viewed as driven by selective advantage within the evolving eukaryotic cell. Thus, it is for both partners, it is equally possible that they not inconceivable that an organelle such as the have started out by pure accident. ...” premitochondrion could likewise have evolved If we accept that the host cell was capable endogenously. Indeed, the premitochondrion of aerobic metabolism, what type of energy- is conceptually similar to the , a generating system might it have used? Given membrane-bound, non-DNA-containing eu- the ubiquity and antiquity of aerobic metabo- karyotic organelle that is generally considered lism based on cytochrome oxidase (Castresana to have had an endogenous origin (Gabaldo´n et al. 1994), it is possible that both host cell and 2013). One might imagine that the selective symbiont had parallel systems for energy gener- advantage for such compartmentation would ation. Here again, no selective advantage would be the concentration of enzymes and reactants accrue to the host from a symbiont bringing in various metabolic pathways, in the face of a such a system into it. Initially, the energy re- steady increase in cell size, one of the hallmarks quirements of host and symbiont would be of the eukaryotic cell. met by their respective energy-generating sys- Symbiotic models of mitochondrial origin tems. However, the equation would shift dra- usually invoke the introduction of aerobic (ox- matically once ADP import into the symbiont ygen-dependent) metabolism into an essen- and ATP export from it evolved. That change tially anaerobic host cell (Margulis 1970), the might have occurred through a “reverse flow” initial selective advantage being either more ef- mutation of the ATP-importing/ADP-export- ficient ATP generation in an increasingly oxy- ing translocase postulated here to have been gen-rich environment or oxygen detoxification present in the premitochondrion, or through (the “ox-tox” hypothesis) (Andersson and Kur- elaboration of a completely new transporter. land 1999). However, there are good arguments Evidently, the required transporter was not con- to consider that the ancestral eukaryotic cell tributed by the symbiont (Karlberg et al. 2000). was, in fact, already capable of aerobic metabo- The emergence of an ADP-importing/AT P- lism (Raff and Mahler 1972). In that case, as- exporting translocase is a fundamental element suming that an aerobic a-proteobacterium was in all models of mitochondrial evolution. Sub- taken up by an aerobic pre-eukaryote via phago- sequent intracellular proliferation of the evolv- cytosis (another eukaryotic hallmark), there ing mitochondrion would lead to a pronounced need have been no selective advantage initially increase in bioenergetic membranes supporting

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The Pre-Endosymbiont Hypothesis

coupled electron transport and oxidative phos- versely, host genes have evidently substituted phorylation, greatly increasing the production for symbiont genes in specifying components of ATP available to the cell as a whole. Indeed, of many mitochondrial pathways. Lane and Martin (2010) argue persuasively that A particularly notable example in this regard the increased energy provided by specialization is the tricarboxylic acid (TCA) cycle, genes for of the endosymbiont into an ATP-generating which were presumably contained originally in organelle and increasing organelle copy number the symbiont genome but moved to the nuclear was “a prerequisite to eukaryote complexity: the genome. However, not all of the mitochondrial key innovation to multicellular .” TCA cycle enzymes are decidedly a-proteobac- Even before the elaboration of an ADP-im- terial in origin (e.g., Kurland and Andersson porting/ATP-exporting translocase, acquisition 2000; Schnarrenberger and Martin 2002). Cur- by the symbiont of the protein import system rent models of mitochondrial evolution gener- and other transporters of the premitochondri- ally assume that LGT from another bacterial on would set the stage for relocation to the source led to replacement of the originally a- symbiont of many of the metabolic pathways proteobacterial gene: an assumption that re- of the premitochondrion. The advantage of quires many separate LGTevents to account for performing these activities in the symbiont the large number of non-a-proteobacterial con- rather than in the premitochondrion would stituents of the mitochondrial proteome. The be that the symbiont would have generated the scenario presented here suggests a simplified al- ATP in situ for biosynthetic pathways requiring ternative: that the non-a-proteobacterial com- it, whereas the premitochondrion would have ponents of the TCA cycle (and other mitochon- had to import ATP from the for these drial pathways) are encoded by genes that were same reactions. Once ATPexport from evolving originally present in the ancestral eukaryotic organelle to cytosol was established, the selec- host before the arrival of the mitochondrial en- tive advantage would be greatly enhanced, dosymbiont, and that they replaced the homol- because the evolving mitochondria could pro- ogous endosymbiont genes at an early stage in liferate and thereby sequester an increasingly the symbiont-to-organelle transition. larger proportion of the structural and enzy- matic components required to make the func- CONCLUDING REMARKS tional premitochondrion. Intracellular compe- tition of these sorts would inevitably lead to the The new model of mitochondrial origin and disappearance of the premitochondrion as a evolution outlined here (Fig. 2) is an attempt separate entity and the fixation of its successor, to integrate conflicting data that are not read- the modern mitochondrion. ily accommodated by current hypotheses. In Finally, because host and symbiont genomes particular, the pre-endosymbiont hypothesis would initially encode a number of the same or accounts in a relatively straightforward way very similar metabolic pathways, a process of for the presence and characteristics of both genome reduction would ensue whereby dupli- a-proteobacterial and non-a-proteobacterial cate genes were lost from one or the other ge- components (APC and NPC, respectively) of nome. Most of this reduction appears to have the mitochondrial proteome. The existence of involved the endosymbiont genome, with many an ancestral metabolic organelle (the premito- genes being lost or transferred to the nucleus chondrion) would effectively “precondition” (Gabaldo´n and Huynen 2007). In the latter the host cell for the successful conversion of a case, relocated genes would have had to acquire bacterial endosymbiont into an integrated or- signals for nuclear expression and mitochondri- ganelle because much of the material necessary al targeting, although some a-proteobacterial for this transformation would already be avail- symbiont genes have evidently been retargeted able in the host, in the form of much of the to other cellular locations, where they currently NPC. Furthermore, the hypothesis presented function (Gabaldo´n and Huynen 2007). Con- here provides an explanation for what the pre-

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M.W. Gray

existing portion of the NPC would be doing 80th birthday, in recognition and deep appreci- before the endosymbiont arrived; moreover, it ation of 50 years of mentorship and friendship. I helps to explain not only the mosaic nature of am grateful to P.J. Keeling, J.M. Archibald, and the mitochondrial proteome overall, but also A.J. Roger for critical comment on an early draft the mosaic character of the enzymatic pathways of this article; to C.G. Kurland for stimulating contained therein. discussion about mitochondrial evolution; and The pre-endosymbiont hypothesis elab- to G. Burger for provision of the A. godoyi orated here effectively represents a synthesis of mtDNA gene map shown in Figure 1. previous, contending mitochondrial origin hy- potheses, with much of the mitochondrial pro- teome (represented within the NPC) having an REFERENCES endogenous origin and the minority compo- Reference is also in this collection. nent (the APC) having a xenogenous origin. In Adams KL, Palmer JD. 2003. Evolution of mitochondrial some respects, this hypothesis hearkens back to gene content: Gene loss and transfer to the nucleus. the (nonsymbiotic) model for the origin of mi- Mol Phylogenet Evol 29: 380–395. tochondria published more than 40 years ago by Andersson SGE, Kurland CG. 1999. Origins of mitochon- Raff and Mahler (1972). That publication pre- dria and hydrogenosomes. Curr Opin Microbiol 2: 535– 541. dated the molecular evidence that convincingly Brindefalk B, Ettema TJG, Viklund J, Thollesson M, An- established the a-proteobacterial nature of the dersson SGE. 2011. A phylometagenomic exploration mitochondrial genome: data that had a pro- of oceanic Alphaproteobacteria reveals mitochondrial rel- found influence on the demise of nonsymbiotic atives unrelated to the SAR11 . PLoS ONE 6: e24457. Burger G, Gray MW, Lang BF. 2003. Mitochondrial ge- models and the ascendency of endosymbiotic nomes: Anything goes. Trends Genet 19: 709–716. models of mitochondrial origin (Gray and Burger G, Gray MW, Forget L, Lang BF. 2013. Strikingly Doolittle 1982; Gray 1992). The molecular bacteria-like and gene-rich mitochondrial genomes data, drawn in large part from analyses of throughout jakobid . Genome Biol Evol 5: 418– 438. mtDNA and the genes it contains, leave little Cardol P.2011. Mitochondrial NADH:ubiquinone oxidore- doubt that bacterial endosymbiosis played acru- ductase (complex I) in eukaryotes: A highly conserved cial role in the origin of the mitochondrion, al- subunit composition highlighted by mining of protein though I would contend that that role is consid- databases. Biochim Biophys Acta 1807: 1390–1397. Castresana J, Lu¨bben M, Saraste M, Higgins DG. 1994. Evo- erably more circumscribed than we initially lution of cytochrome oxidase, an enzyme older than at- imagined. Because the mitochondrion was orig- mospheric oxygen. EMBO J 13: 2516–2525. inally viewed as a singular entity rather than as a Cavalier-Smith T. 1987. Eukaryotes with no mitochondria. mosaic, proof for the origin of the mitochondri- Nature 326: 332–333. Cavalier-Smith T. 1989. Archaebacteria and archezoa. al genome was generallyaccepted as constituting Nature 339: 100–101. proof for the origin of the organelle as a whole. Doolittle WF.1980. Revolutionary concepts in evolutionary In retrospect, that extrapolation turned out to be biology. Trends Biochem Sci 5: 146–149. overly simplistic. In the context of a discussion Forterre P. 2011. A new fusion hypothesis for the origin of elsewhere in this series (Keeling 2013b), it is Eukarya: Better than previous ones, but probably also wrong. Res Microbiol 162: 77–91. worth contemplating how different the histori- Gabaldo´n T.2013. A metabolic scenario for the evolutionary cal record of our views about mitochondrial origin of from the . origin and evolution might have been had our Cell Mol Life Sci doi: 10.1007/s00018-013-1424-z. detailed knowledge and understanding of the Gabaldo´n T, Huynen MA. 2003. Reconstruction of the proto-mitochondrial metabolism. Science 302: 609. mitochondrial proteome predated that of the Gabaldo´n T, Huynen MA. 2004. Shaping the mitochondrial mitochondrial genome. proteome. FEBS Lett 1659: 212–220. Gabaldo´n T, Huynen MA. 2007. From endosymbiont to host-controlled organelle: The hijacking of mitochondri- ACKNOWLEDGMENTS al protein synthesis and metabolism. PLoS Comput Biol 3: e219. This essay is dedicated to my PhD supervisor, Georgiades K, Madoui M-A, Le P,Robert C, Raoult D. 2011. Professor Byron G. Lane, on the occasion of his Phylogenomic analysis of Odyssella thessalonicensis forti-

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The Pre-Endosymbiont Hypothesis

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The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria

Michael W. Gray

Cold Spring Harb Perspect Biol 2014; doi: 10.1101/cshperspect.a016097

Subject Collection The Origin and Evolution of Eukaryotes

The Persistent Contributions of RNA to Eukaryotic Origins: How and When Was the Eukaryotic Gen(om)e Architecture and Cellular Mitochondrion Acquired? Function Anthony M. Poole and Simonetta Gribaldo Jürgen Brosius Green and the Origins of Multicellularity in Bacterial Influences on Animal Origins the Plant Rosanna A. Alegado and Nicole King James G. Umen The Archaeal Legacy of Eukaryotes: A Missing Pieces of an Ancient Puzzle: Evolution of Phylogenomic Perspective the Eukaryotic Membrane-Trafficking System Lionel Guy, Jimmy H. Saw and Thijs J.G. Ettema Alexander Schlacht, Emily K. Herman, Mary J. Klute, et al. Origin and Evolution of the Self-Organizing The Neomuran Revolution and Phagotrophic in the Network of Eukaryotic Origin of Eukaryotes and Cilia in the Light of Organelles Intracellular Coevolution and a Revised Tree of Gáspár Jékely Life Thomas Cavalier-Smith On the Age of Eukaryotes: Evaluating Evidence Protein Targeting and Transport as a Necessary from Fossils and Molecular Clocks Consequence of Increased Cellular Complexity Laura Eme, Susan C. Sharpe, Matthew W. Brown, Maik S. Sommer and Enrico Schleiff et al. Origin of Spliceosomal Introns and Alternative How Natural a Kind Is ''Eukaryote?'' Splicing W. Manuel Irimia and Scott William Roy

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Protein and DNA Modifications: Evolutionary What Was the Real Contribution of Imprints of Bacterial Biochemical Diversification Endosymbionts to the Eukaryotic Nucleus? and Geochemistry on the Provenance of Insights from Photosynthetic Eukaryotes Eukaryotic Epigenetics David Moreira and Philippe Deschamps L. Aravind, A. Maxwell Burroughs, Dapeng Zhang, et al. The Eukaryotic Tree of Life from a Global Bioenergetic Constraints on the Evolution of Phylogenomic Perspective Complex Life Fabien Burki Nick Lane

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