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PAPER Mosaic nature of the mitochondrial : COLLOQUIUM Implications for the origin and of mitochondria

Michael W. Gray1

Department of Biochemistry and Molecular and Centre for Comparative and Evolutionary , Dalhousie University, Halifax, NS, Canada B3H 4R2

Edited by Patrick J. Keeling, University of British Columbia, Vancouver, BC, Canada, and accepted by the Editorial Board March 11, 2015 (received for review December 23, 2014) Comparative studies of the mitochondrial proteome have identi- during mitochondrial evolution. This essay briefly highlights the fied a conserved core of descended from the α-proteo- results of studies that revealed the mosaic nature of the mito- bacterial that gave rise to the and chondrial proteome and discusses the implications of that obser- was the source of the mitochondrial in contemporary vation for models of mitochondrial origin and evolution. . A surprising result of phylogenetic analyses is the rel- atively small proportion (10–20%) of the mitochondrial proteome Proteins Encoded by Mitochondrial DNA displaying a clear α-proteobacterial ancestry. A large fraction of The mitochondrial genome specifies a miniscule but essential mitochondrial proteins typically has detectable homologs only in portion of the mitochondrial proteome, ranging from a low of 3 other eukaryotes and is presumed to represent proteins that proteins of defined function in apicomplexans such as Plasmo- emerged specifically within eukaryotes. A further significant frac- dium to a high of 66 in the excavate Andalucia godoyi, a member tion of the mitochondrial proteome consists of proteins with ho- of the core . The latter assemblage of contains mologs in , but without a robust phylogenetic signal the most ancestral (-like) mitochondrial known

affiliating them with specific prokaryotic lineages. The presump- (22, 23), with mitochondrial- content varying only slightly EVOLUTION tive evolutionary source of these proteins is quite different in con- within the group. Among them, mtDNAs encode 67 pro- tending models of mitochondrial origin. teins: 25 components of the mitochondrial respiratory chain, 29 proteins (mostly ribosomal proteins), 9 proteins involved mitochondria | proteome | endosymbiont | phagotrophy | syntrophy in import and maturation, and 4 proteins constituting a multisubunit bacteria-like α2ββ′σ RNA polymerase. Mitochondrial- nderstanding the origin and evolution of the mitochon- gene content, although extremely variable among eukaryotes, is Udrion remains a challenge, despite the flood of relevant bio- largely a subset of that in the core jakobids (15), whose mitochon- chemical, and molecular biological, and phylogenetic data drial genomes lack only for small subunit ribosomal protein and insights that have accumulated in the almost five decades S16 (rps16), recently identified in the mtDNAs of Acanthamoeba since the modern resurrection (1, 2) of the long-standing endo- castellanii and Vermamoeba vermiformis (6, 24) and large subunit symbiont hypothesis: the idea that this is a tamed and ribosomal protein L36 (rpl36), annotated in the mtDNA sequence highly reworked endosymbiotic bacterium. The abundance of of Malawimonas jakobiformis (www.ncbi.nlm.nih.gov/gene?cmd= information bearing on eukaryotic cell evolution (particularly Retrieve&dopt=full_report&list_uids=2695583). On that basis, and most recently sequence data) and differences over how the we can infer that the mitochondrial genome of the last eukaryotic data are analyzed and interpreted have prompted a plethora of common ancestor (LECA) encoded a minimum of ∼70 proteins often-conflicting ideas about when and how, within an endosymbi- (6, 23). In extant eukaryotes, the role of the mitochondrial genome otic context, the mitochondrion originated (3, 4). Considering the is a highly circumscribed and critical one: It supports the trans- spate of recent papers dealing with various aspects of mitochondrial lation and maturation of a small number of proteins that are origin and evolution either explicitly (e.g., refs. 5–10)oraspartofa components of complexes I to IV (CI–CIV) of the broader view of eukaryotic cell evolution (e.g., refs. 11–14), interest transport chain (ETC) as well as complex V (CV; ATP synthase). in this subject is unlikely to wane any time soon. These mtDNA-encoded proteins are assembled with nucleus- That the mitochondrial genome is of bacterial (specifically encoded partner proteins to generate a functional respiratory α-proteobacterial) origin is now indisputable (15, 16), and that fact chain capable of carrying out coupled electron transport–oxidative has underpinned the current wide acceptance of a xenogenous/ . exogenous (endosymbiotic) origin of the mitochondrion (17, 18), effectively vanquishing autogenous/endogenous (nonsymbiotic) Composition of the Mitochondrial Proteome origin hypotheses (e.g., refs. 19–21). However, the mitochondrial The basic approach to identifying bona fide mitochondrial pro- proteome, most of which is nucleus-encoded, tells another, teins currently comprises a combination of direct subcellular murkier story. It was expected that many mitochondrial proteins would not have obvious bacterial homologs because they would have emerged specifically within eukaryotes, as part of the This paper results from the Arthur M. Sackler Colloquium of the National Academy of endosymbiont-to-organelle retailoring process. What was not Sciences, “Symbioses Becoming Permanent: The Origins and Evolutionary Trajectories of ,” held October 15–17, 2014, at the Arnold and Mabel Beckman Center of the anticipated was how relatively few mitochondrial proteins with National Academies of Sciences and Engineering in Irvine, CA. The complete program bacterial homologs would group specifically with α-Proteobac- and video recordings of most presentations are available on the NAS website at www. teria in phylogenetic reconstructions: At most, only 10–20% of nasonline.org/Symbioses. any of the mitochondrial examined so far display a Author contributions: M.W.G. wrote the paper. robust α-proteobacterial signal. This minority group includes The author declares no conflict of interest. proteins encoded by the mitochondrial genome as well as nucleus- This article is a PNAS Direct Submission. P.J.K. is a Guest Editor invited by the Editorial encoded mitochondrial proteins whose genes we infer were Board. transferred to the nucleus via endosymbiotic gene transfer (EGT) 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1421379112 PNAS | August 18, 2015 | vol. 112 | no. 33 | 10133–10138 Downloaded by guest on September 27, 2021 proteomics techniques [tandem mass spectrometry (MS/MS) was essentially complete before divergence of the main eukary- applied to purified mitochondria or submitochondrial fractions] otic supergroups, with novel nucleus-encoded subunits in place in combination with indirect bioinformatics-based prediction of and many of the original endosymbiont-encoded ribosomal proteins exhibiting N-terminal mitochondrial targeting se- protein genes having already undergone EGT to the nucleus. quences (25–27). The two techniques have their complementary Subsequent evolution involved additional mitochondrion- strengths and limitations, with MS/MS capable of revealing novel to-nucleus gene relocation, as well as lineage-specific gains and mitochondrial proteins (ones having no known homologs) but losses (41, 42). The most extreme examples of mitoribosome often missing low-abundance proteins that may be identified by retailoring are seen in the kinetoplastid protists, Trypanosoma in silico analyses, which, however, fail to retrieve mitochondrial brucei (43) and tarentole (44). In T. brucei, the ri- proteins that lack well-defined N-terminal mitochondrial tar- bosomal SSU and LSU contain 56 and 77 proteins, compared geting signals. Delineation of the mitochondrial proteome in any with 21 and 34, respectively, in a bacterial (e.g., Escherichia coli) requires a list of cellular proteins predicted from , with the novel mitochondrial proteins displaying very complete nuclear and mitochondrial genome sequences, as well limited sequence similarity with their counterparts within as assessment of possible nonmitochondrial contamination. Kinetoplastidae and no detectable similarity outside this lineage. Given these requirements and constraints, it is hardly surprising These focused studies highlight the dynamics of mitochondrial- that a relatively small number of largely complete and robustly proteome evolution, as well as emphasizing the importance of validated mitochondrial proteomes have been determined to direct MS/MS analysis in revealing novel mitochondrial proteins date, primarily from multicellular (notably human and that cannot be identified through sequence comparisons. mouse), fungi (e.g., the Saccharomyces cerevisiae), and As the database for mitochondrial-proteome comparisons has (e.g., Arabidopsis thaliana, rice). Although eukaryotic expanded, it has become apparent that the LMCA was already as microbes (protists) constitute most of the evolutionary diversity complex in its key functions as modern mitochondria; impor- within the eukaryotic , comprehensive proteomic analy- tantly, for example, it possessed an essentially complete protein- ses of mitochondria are still relatively few (24, 28–30). import apparatus (45). Indeed, molecular reconstructions dem- Nevertheless, with the increasing availability of substantially onstrate that LECA itself was a structurally complex cell with a complete eukaryotic-genome sequences, valuable comparative sophisticated , already containing the hallmarks of a information about mitochondrial proteomes can be gleaned by in typical eukaryotic cell (46). Evidently, basic endosymbiont-to- silico data-mining methods alone. mitochondrion retailoring was essentially complete before the Although our knowledge of complete mitochondrial pro- divergence of the main eukaryotic lineages, indicating that the teomes is patchy at present, a great deal of compositional het- initial endosymbiotic event must have occurred at an earlier erogeneity is already evident, both in the total number of point in eukaryogenesis. proteins that constitute the organelle in a given organism and in their range of functional activities (31, 32). Multicellular animals Proto-Mitochondrion: An α-Proteobacterial Perspective and plants seem to have the largest repertoire of mitochondrial Comparisons of the experimentally determined or inferred mi- proteins (>1,000) although, as demonstrated in recent studies, tochondrial proteomes of extant eukaryotes allow the recon- the compositional complexity of certain unicellular protists may struction of a minimal proteome of the LMCA. Conversely, by rival that of their multicellular eukaryotic relatives (24, 30). As exploiting whole-genome sequence searches to extract eukary- detailed below, much of the variability in mitochondrial-pro- otic proteins having a close evolutionary relationship with teome composition can be understood as lineage-specific addi- α-proteobacterial homologs, and assuming that such proteins tions to a basic protein core that is conserved among eukaryotes, were contributed by the α-proteobacterial ancestor of mito- although lineage-specific losses also come into play in shaping chondria, one can gain insight into the composition of the an- the mitochondrial proteome. cestral (proto-) mitochondrial proteome. Within the set of re- Comparative mitochondrial proteomics (comprehensive com- trieved α--like proteins will be ones that are bona parison of mitochondrial-proteome data across the range of fide components of the contemporary mitochondrial proteome eukaryotes) has helped to define the composition of what we (including proteins encoded by mtDNA and nucleus-encoded may term the “last mitochondrial common ancestor” (LMCA): proteins targeted to and imported into mitochondria), as well as effectively, the mitochondrion of the LECA. In particular, α-proteobacteria–like proteins that are localized elsewhere comparative analysis of individual mitochondrial-protein com- in the cell. In fact, the latter group comprises the largest pro- plexes has highlighted the evolutionary process by which a basic portion of proteins assumed to have been transferred from the α-proteobacterial core has been modified (typically by acquisi- α-proteobacterial endosymbiont to the nuclear genome via tion of additional components) during eukaryogenesis. For EGT, effectively having been recruited to function outside the example, ETC CI (NADH: ubiquinone ) has evolving mitochondrion. expanded from 17 subunits in an α-proteobacterium, Paracoccus In an initial study along these lines, Gabaldón and Huynen denitrificans (33), to as many as 45 subunits in animals (34). (47) used a database of 77 genome sequences, including 6 Although early studies had suggested a lineage-specific distri- α-proteobacterial and 9 eukaryotic, and identified 630 clusters of bution of novel CI subunits (34, 35), with 13 of them emerging orthologous groups (COGs) that displayed a close α-proteo- specifically within metazoan animals, a broader survey of eu- bacterial/eukaryotic evolutionary relationship. This number rep- karyotic CI using enhanced bioinformatics search techniques has resents a minimal estimate of the proteins that were likely pre- revealed that virtually all of the -specific CI subunits sent in the proto-mitochondrion (this approach obviously fails to were already present in the LMCA (36, 37). ETC CV (ATP account for proteins present in the proto-mitochondrion but synthase) presents a somewhat different picture, with evidence in subsequently lost from all of the α-proteobacterial or all of the this case of species-specific subunits in addition to a eukaryote- eukaryotic genomes under consideration, as well as proteins that specific core (38–40). do not have a sufficiently strong phylogenetic signal to be Mitochondrial exhibit a similar pattern, with the detected). In a subsequent phylogenetic analysis of an expanded mitoribosome of the LCMA inferred to have had an α-proteo- database (144 genomes, including 16 eukaryotic and 11 α-pro- bacterial–contributed core consisting of 20 small-subunit (SSU) teobacterial), Gabaldón and Huynen (32) identified 842 α-pro- and 33 large-subunit (LSU) proteins supplemented, respectively, teobacteria–like eukaryotic proteins. Of this set, only 74 (8.8%) by 10 and 9 additional, eukaryote-specific subunits (41, 42). As in and 130 (15.4%) could be identified, respectively, in the yeast the case of ETC CI, retailoring of the endosymbiont ribosome and human mitochondrial proteome sets available at the time.

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In a more recent analysis of this type, Wang and Wu (8) used a COLLOQUIUM greatly expanded genome database (1,613 total, 30 eukaryotic, 171 α-proteobacterial), retrieving 394 COGs that they consider to represent mitochondrion-derived nuclear genes: a substan- tially lower number than the 842 reported by Gabaldón and Huynen (32). Wu and Wang (8) claim that their analysis is more specific and more sensitive than that of Gabaldón and Huynen (32), resulting in a “leaner” reconstructed metabolism in the proto-mitochondrion, but one in which a number of gaps in the latter study are filled by the identification of additional compo- nents of various pathways. In their reconstruction, Gabaldón and Huynen (32) included as mitochondrial proteins ones that clus- tered with β- and γ-proteobacteria, a concession necessary to recover the bulk of Reclinomonas americana mtDNA-encoded ribosomal proteins. However, this problem proved not to be an issue with the increased organismal sampling in the Wu and Wang (8) study. In fact, Gabaldón and Huynen (32) noted that the number of COGs they recovered varied substantially, depending on the parameters they selected for phylogenetic analysis, and could be reduced substantially by application of more selective criteria (e.g., by setting more stringent cutoffs). The results of these reconstructions are obviously highly influ- enced by the database used (both number and phylogenetic Fig. 1. Representation of the evolutionary ancestry of the mitochondrial coverage of the genome sequences examined) as well as the proteome, deduced from phylogenetic-tree reconstructions. Proportions of the four categories vary depending on the organism in question, but the methods used and parameters chosen for tree construction. α-proteobacterial component typically constitutes ∼10–20% of the total Despite these qualifications, these studies do provide a mini- mitochondrial proteome (8, 32).

malist picture of the proto-mitochondrial metabolism, retrieving EVOLUTION virtually complete pathways for pyruvate metabolism, TCA cycle, coupled electron transport-oxidative phosphorylation, fatty-acid The mosaic character of the mitochondrial proteome raises , β-oxidation, branched-chain degrada- a number of questions about the acquisition of the individual tion, and biosynthesis of ubiquinone, biotin, 1-carbon units, and proteins. As detailed above, we can safely infer that the α-pro- iron-sulfur clusters, most of which metabolism is localized in teobacterial component (APC) of the mitochondrial proteome contemporary mitochondria. Proteins involved in mitochondrial- was introduced into the evolving eukaryotic cell by the endo- ribosome biogenesis and translation are prominent in these re- symbiont that also brought in the mitochondrial genome, with constructions, as are metabolite transporters, but replication and many endosymbiont genes subsequently either lost or under- proteins are virtually absent, with heterotrophic going EGT to the nucleus. The origin of the non–α-proteo- carbohydrate metabolism (e.g., ) entirely missing. No- bacterial component (NPC) of the mitochondrial proteome is table α-proteobacterial contributions to nonmitochondrial me- less clear. With some exceptions, NPC proteins were most likely tabolism include several ER-localized involved in acquired subsequently by the evolving mitochondrion and pre- () metabolism, as well as enzymes participating in purine sumably might have had one of three origins: (i) acquisition from and UDP-sugar biosynthesis (8). the host of preexisting proteins (i.e., proteins already present in The overall impression from these studies is that of a (facul- the host); (ii) elaboration de novo within the eukaryotic cell; or tatively) aerobic endosymbiont exhibiting a streamlined metab- (iii) lateral gene transfer (LGT) from diverse prokaryotes (bac- olism with a bias toward conversion, amino acid bio- teria other than α-proteobacteria or ) or . Acqui- synthesis, and protein synthesis. This α-proteobacteria–derived sition of proteins via LGT must have occurred during the period portion of the mitochondrial proteome constitutes much of the of eukaryogenesis leading up to LECA (after the establishment functional core that is conserved throughout eukaryotes. of the proto-mitochondrion) although examples of lineage-spe- cific, post-LECA LGT are certainly evident. Contending models Origin and Evolution of the Mitochondrial Proteome for the origin of mitochondria have different implications for the Acceptance that the mitochondrial genome is of α-proteobac- origin of the mitochondrial NPC, in particular the prokaryotic terial origin naturally prompted the question: How much of the cohort that does not cluster with α-proteobacteria. total mitochondrial proteome is also of α-proteobacterial an- cestry? Initial phylogenetic analyses provided a surprising an- Phagotrophic vs. Syntrophic: Two Contending Scenarios swer: relatively less than might have been anticipated (47–49). Although many specific symbiotic models for the origin of mi- These studies revealed that mitochondrial proteins can largely be tochondria have been proposed over the past five decades (see, binned into four categories: category I, strong relationship with e.g., ref. 50), these hypotheses can be broadly grouped into one α-proteobacteria; category II, non–α-proteobacterial homologs of two scenarios: phagotrophic, in which an α-proteobacterial in prokaryotes, often without a strong affiliation to any particular endosymbiont is taken up by a host cell that already has some bacterial or archaeal group; category III, homologs exclusively in degree of eukaryotic character, in particular the ability to carry other eukaryotes; and category IV, proteins unique to the or- out phagocytosis (ingestion of other as a food source); ganism in question, lacking evident homologs in other eukary- and syntrophic, where intimate metabolic interdependence be- otes (Fig. 1). This pattern, initially deduced for yeast, S. cerevisiae tween two different kinds of prokaryotic cell leads eventually to (48, 49), has been observed for the mitochondrial proteome of one cell (the host, assumed to be an archaeon) subsuming the other organisms, although with variation in the proportions of the second cell (the symbiont, assumed to be a eubacterium) (see, different categories (e.g., ref. 28). Consistently, only ∼10–20% of e.g., refs. 3, 4, 14, 46, 51, and 52 for recent synopses and cri- the mitochondrial proteome in diverse eukaryotes exhibits a tiques). [Although this categorization is useful for discussion strong α-proteobacterial phylogenetic signal (8, 32). purposes, it should be emphasized that phagotrophy and syntrophy,

Gray PNAS | August 18, 2015 | vol. 112 | no. 33 | 10135 Downloaded by guest on September 27, 2021 as biological processes, are not mutually exclusive: It is conceivable, ancient origin. The idea here of “ancient origin” is an origin that for example, that a syntrophic association might have occurred in- considerably predates that of the mitochondrial endosymbiosis: volving a host cell that already had some eukaryotic characteristics, i.e., proteins that had existed for a long time in the host since rather than an archaeon per se (see ref. 4 for a pertinent discussion their acquisition (e.g., by LGT) from a prokaryotic source, or of this point).] that were present in LUCA and inherited vertically, before the In perhaps the best-known and most widely discussed syntro- advent of the mitochondrial endosymbiosis. Thus, lack of affili- phic model, the hydrogen hypothesis of Martin and Müller (53), ation with a specific bacterial or archaeal group could simply the host is a strictly anaerobic methanogenic archaeon, which is reflect progressive sequence divergence over a long evolutionary dependent on hydrogen produced by the symbiont (a faculta- history. A similar issue arises with the : In a phylo- tively aerobic α-proteobacterium). Integration of the α-proteo- genetic investigation of the evolutionary origin of peroxisomal bacterial partner into the archaeal partner followed by massive proteins, Gabaldón et al. (60) found a substantial number of gene transfer from symbiont to host genome effectively initiates cases of unresolved ancestry that “imply the existence of ho- the process of eukaryogenesis, with the symbiont undergoing mology to prokaryotic sequences without a tree that specifically subsequent conversion into the mitochondrion. In syntrophic supports a bacterial or archaeal origin.” models, the origin of the mitochondrion is coincident with the In their analysis of the yeast mitochondrial proteome (a set of origin of the eukaryotic cell, per se, and is the conditio sine qua 423 proteins), Karlberg et al. (48) found that about half had non for eukaryogenesis. homologs in prokaryotes and, of this portion, 108 (25% of the In phagotrophic models, the origin of the mitochondrion is total) were represented in bacteria and archaea as well as eu- placed later in eukaryogenesis and may in fact be one of the last karyotes. The authors suggested that these 108 proteins might major eukaryotic features to be elaborated. Cavalier-Smith, in have been present in the last universal common ancestor particular, has long championed a phagotrophic model of mito- (LUCA), and therefore conceivably some could already have chondrial origin (11, 54–57), with detailed schema of eukary- been resident in the host cell before uptake of the α-proteo- ogenesis developed particularly from a cell-biological perspective. bacterial endosymbiont. Other prokaryotic genes might have In both scenarios, the emergence of the mitochondrion is a been contributed by a “slow-drip” process in which successions of singular event. In the syntrophic scenario, it is seen as a radical LGT over a long period seeded the evolving eukaryotic genome innovation resulting from an extremely rare, even unlikely, event; with prokaryotic genes from diverse lineages (61). The results in the phagotrophic scenario, it is regarded as a graduated in- of an analysis of mitochondrial aminoacyl-tRNA synthetases novation resulting as an almost inevitable consequence of a (aaRSs), which indicated that “a majority of the mitochondrial process (phagocytosis) that is now common among microbial eu- aaRSs originate from within the bacterial domain, but none karyotes. [It is interesting to consider, as O’Malley (3) has pointed specifically from the α-Proteobacteria” (62), would seem con- out, the way in which proponents of syntrophic and phagotrophic sistent with this view. The point is that, within a phagotrophic scenarios use the monophyletic origin of mitochondria to bolster model of mitochondrial origin, much of the mitochondrial NPC, their particular viewpoint. Syntrophic proponents ask why, if phago- at least that portion that is prokaryotic but not specifically cytosis is so common, mitochondria emerged only once whereas α-proteobacterial, might well have been present in the proto- phagotrophic proponents ask why, if a syntrophic scenario is so eukaryotic host organism that assimilated the α-proteobacterial “unspeakably rare” (11), it happened at all.] symbiont. As early as 1987, Cavalier-Smith (55) asserted that the Within these two scenarios, how can we account for those host “was probably a facultative aerobe” and, as such, “it would mitochondrial proteins that seem to have a prokaryotic but not probably already have most of the proteins now present in the specifically α-proteobacterial ancestry (referred to below as mitochondrion, so they could be transferred into it and allow “prokaryotic NPC proteins”)? Trivially, some mitochondrial deletion of the homologous symbiont genes without any gene proteins that score as NPC in phylogenetic reconstructions may transfer being necessary”—an overreach, perhaps, in light of have diverged too far to be readily recognizable as α-proteo- current knowledge about the composition of the mitochondrial bacterial. However, this possibility is unlikely to account for the proteome, but nevertheless an early explicit statement of the entirety of this cohort. Martin and coworkers (58, 59) have ar- idea of a substantial host contribution to the mitochondrion of gued for a fluid prokaryotic- model in which LGT preendosymbiont proteins. among free-living prokaryotes preceding and subsequent to the If we allow that certain proteins destined to be incorporated origin of mitochondria could explain the observed pattern. into the mitochondrion preexisted in the host, what might they However, although contemporary α-proteobacterial (and other have been doing there? Recently, in what I dubbed the “pre- prokaryotic) genomes almost certainly differ in gene content from endosymbiont hypothesis,” I raised the possibility that a mem- their ancestral genomes existing at the time of the mitochondrial brane-bound organelle (“premitochondrion”) might have com- endosymbiosis, and accepting that LGT has played a major role in partmentalized some of the metabolic activities that were eventually prokaryotic genome evolution, this explanation would seem to relocated to the mitochondrion (5). In effect, such an endogenously demand that the genes in question were preferentially lost from evolved host organelle could have provided an immediately the α-proteobacterial genomes under consideration, or were so available source of proteins for the endosymbiont-to-organelle widely transferred among non–α-proteobacterial genomes that transformation, with duplicate pathways being rationalized via evidence of their α-proteobacterial provenance has effectively preferential loss/transfer of endosymbiont genes and consequent been erased. Although such a scenario might apply in isolated marked reduction in the gene content of what would become the cases, it is unlikely, in my view, that it could rationalize the entirety mitochondrial genome (Fig. 2). of prokaryotic NPC mitochondrial proteins. Considering that the In some respects, in contemporary eukaryotic cells, the per- mitochondrial APC is relatively constant, whereas the NPC sub- oxisome encapsulates some of the features ascribed to the pre- sets are quite diverse, one would have to imagine a very large mitochondrion: an endogenously evolved (63), membrane-bound proto-mitochondrial genome if the bulk of the NPC were assumed organelle compartmentalizing (in different eukaryotes) various to have been present in, and contributed by, an α-proteobacte- activities involved in the oxidative metabolism of carbohydrates, rial genome in a syntrophic scenario. Lang and Burger (14, 52) , amino acids, and purines, and the biosynthesis of certain have presented other arguments in strongly argued critiques of a lipids (56, 64, 65). Mitochondria and are known to syntrophic scenario. be linked metabolically (sharing pathways such as β-oxidation of An alternative, straightforward explanation for lack of phylo- very long-chain fatty acids, with metabolites shuttling between genetic signal in these cases is that the proteins in question are of the two organelles) as well as evolutionarily, with a significant

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A recently proposed model of eukaryogenesis [“phagocytosing COLLOQUIUM ATP pre-mitochondrion archaeon theory” (PhAT)] envisages an emerging amitochondriate (autogenous) host cell in which rampant LGT (a consequence of phagocytotic NPC ATP acquisition of genomic DNA from various sources) leads to “a highly mosaic host genome” (74). This theory is compatible with ancestral the view (5) that many of the proteins acquired later by the mitochondrion evolving mitochondrion (part of the NPC) could already have existed in the host cell (Fig. 2); it is also consistent with the idea APC that much of the evolution of eukaryotic cellular complexity α-proteobacterial occurred before the acquisition of an α-proteobacterium and its endosymbiont transformation into the mitochondrion (11, 57). (exogenous) Concluding Remarks Despite considerable progress over the past 15 y in elucidating Fig. 2. A schematic view of the preendosymbiont hypothesis. The pre- the composition of the mitochondrial proteome in a variety of mitochondrion is seen as a membrane-bound entity endowed with a pro- eukaryotes, we still cannot infer with any certainty the precise tein-import system and various /small-molecule transporters, compart- evolutionary origin of many of the mitochondrial proteins, par- mentalizing many of the metabolic functions of the mitochondrion. The ticularly the prokaryotic portion of the NPC. Nor can we assert premitochondrion is assumed to have evolved endogenously within the with confidence at what time point any individual protein was preeukaryote cell and to contain proteins that would later contribute to introduced into the evolving eukaryotic cell to become associated the NPC (non–α-proteobacterial component) of the contemporary mito- chondrial proteome. An α-proteobacteria–like endosymbiont is converted to with the mitochondrion. At present, phagotrophic and syn- the ancestral mitochondrion, effectively “capturing” protein components throphic models of mitochondrial origin account in quite dif- and functions of the premitochondrion. The endosymbiont contributes the ferent ways for the NPC component of the mitochondrial α-proteobacterial component (APC) of the mitochondrial proteome, which is proteome. What we still lack is compelling evidence, of the sort largely directed toward specification of energy-generating capacity in the that convincingly demonstrated the α-proteobacterial nature of form of coupled electron transport/oxidative phosphorylation; as well, the the mitochondrial genome, that will robustly turn the tide in favor mitochondrial inner and outer membranes are assumed to be endosymbi-

of one or the other model. EVOLUTION ont-derived (57). The conversion of endosymbiont to ancestral mitochon- We should not, of course, exclude postendosymbiont LGT, drion is greatly facilitated by the existence of the reservoir of premito- chondrial NPC proteins in the host cell. Endosymbiont-to-nucleus gene either before or after the emergence of the LECA, as an addi- transfer (EGT), coupled with rationalization of redundant pathways, results tional source of NPC mitochondrial proteins. An important ex- in the formation of evolutionarily chimeric enzymatic pathways and protein ample is the early replacement of the original mtDNA-encoded complexes in the ancestral mitochondrion, as well as functional relocation of bacterial-type RNA polymerase (today found only in jakobid the products of some transferred α-proteobacterial genes to other cellular flagellates) with a nucleus-encoded, single-subunit, phage T3/T7- compartments. The premitochondrion is assumed to be a non–energy-gen- like RNA polymerase that now functions as the mitochondrial erating organelle that imports ATP; a key innovation in the transition to the transcriptase in all other eukaryotes (75). Convincing cases of contemporary mitochondrion is the acquisition of an ADP/ATP transporter LGT events of this sort might be recognized through compara- that reverses this flow, ultimately allowing the mitochondrion to become tive studies of particular mitochondrial metabolic pathways, but the primary site of ATP generation for cellular functions. Modified from ref. 5. such studies will likely require direct biochemical confirmation in addition to sequence comparisons. Post-LECA lineage-specific fraction of peroxisomal proteins seemingly recruited from mi- LGT from particular bacterial or archaeal groups, as well as tochondria (60). Peroxisomes have been implicated as the evo- lineage-specific evolution of novel mitochondrial proteins, must lutionary source of the mitochondrial ADP/ATP exchanger (57), certainly be invoked to account for the substantial differences in as well of the organellar division machinery (66). Thus, it is not overall composition: e.g., between the yeast and human mito- inconceivable that the primitive peroxisome might have har- chondrial proteomes (32). bored a wider variety of metabolic processes, some of which were Accordingly, there is a continuing place for comparative mi- ultimately relocated to the evolving mitochondrion. tochondrial proteomics in better defining the conserved func- tional core of the mitochondrion: that cohort of proteins that was A Phagotrophic Perspective: 2015 present in the LMCA and has been preserved throughout eu- Recent studies have reinforced an emerging view that eukaryotes karyotes, despite evident lineage-specific reworking of the mi- arose from within (rather than as a sister group to) Archaea and tochondrial proteome. Particular efforts should be made to more are specifically affiliated with the TACK superphylum, comprising solidly identify and define -derived NPC proteins that the phyla Thaumarchaeota, Aigarchaeota, Crenarchaeota, and might be part of this core. To date, few studies have examined in Korarchaeota (67–73). This inference, based largely on phylo- depth the mitochondrial proteome in early diverging eukaryotes; genetic reconstructions, draws support from the finding of what such studies may hold important additional clues to the origin were previously deemed to be eukaryotic signature proteins and evolution of the mitochondrial proteome, as they have in the (ESPs) in TACK genomes, including archaeal homologs of actin, case of the mitochondrial genome. , ESCRT proteins, and several proteins involved in eukaryotic transcription and translation (69, 73). These new in- ACKNOWLEDGMENTS. I am grateful to Tom Cavalier-Smith for comments that were most helpful in the preparation of this review, in particular for sights allow the possibility that the archaeal ancestor of the pointing out that the peroxisome might have had the preconditioning eukaryotic cell might have had more eukaryotic character than function that I have ascribed “to an evanescent separate premitochondrial previously supposed, including a primitive ability to phagocytose. compartment.”

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