Understanding the cell biology of Lokiarchaeum will be key to understanding the morphological transitions that characterized the evolution of eukaryotic cellular architecture, but Loki has not yet been cultured or seen.

Many models for the evolution of eukaryotes invoke an archaeal ancestor that is capable of phagocytosis to explain the entry of the future mitochondrion into the host cell.

Lokiarchaeum is the first prokaryote found to encode small GTPases, gelsolin, BAR domains, and longin domains, leading many to suggest that it might be compartmentalized and be capable of membrane trafficking.

Eukaryotes are thought to be a product of symbiosis between archaea and bacteria. The recently discovered Lokiarchaeum (‘Loki’) encodes more Eukaryotic Signature Proteins (ESPs) than any other archaeon, making it the closest living relative to the putative ancestor of eukaryotes.

If eukaryotes arose through a merger between archaea and bacteria, what did the first true eukaryotic cell look like? A major step toward an answer came with the discovery of Lokiarchaeum, an archaeon whose genome encodes small GTPases related to those used by eukaryotes to regulate membrane traffic. Although ‘Loki’ cells have yet to be seen, their existence has prompted the suggestion that the archaeal ancestor of eukaryotes engulfed the future mitochondrion by phagocytosis. We propose instead that the archaeal ancestor was a relatively simple cell, and that eukaryotic cellular organization arose as the result of a gradual transfer of bacterial genes and membranes driven by an ever-closer symbiotic partnership between a bacterium and an archaeon.

Interestingly, Loki is also the first bacterial or archaeal genome found to encode large numbers of proteins with clear homology to eukaryotic small GTPases. This has led to a great deal of excitement in the field because, in eukaryotes, these small GTPases plays key functions in the regulation of the cytoskeleton, cell motility, compartment identity, and intracellular trafficking. Moreover, the molecular identity of intracellular trafficking compartments and the specificity of their interactions are tightly coupled to the variety of nonredundant Rab- and Arf-type small GTPases []. In eukaryotes, the expansion of specific GTPase families through serial gene-duplication events has also been linked to an increase in compartment diversity over evolutionary time [].

Debates about the cellular nature of the last eukaryotic common ancestor (LECA) and the genetic composition of pre-LECA lineages have raged for decades. It is now widely accepted that eukaryotes represent the fruit of a symbiosis between an archaeal host [] and at least one bacterial lineage [], the former likely giving rise to the cell proper and the latter giving rise to mitochondria []. However, the lack of intermediates that bridge the gap in size and complexity between prokaryotic precursors and eukaryotes has ensured that eukaryogenesis remains one of the most enduring mysteries in modern biology. Recently, however, the falling costs of sequencing have enabled improved metagenomic sampling of diverse environments, leading to a large increase in the diversity of sequenced archaeal genomes. Remarkably, many of these contain sequences homologous to genes that play critical roles in the organization of eukaryotic cells as they grow and divide, which were previously thought to be unique to eukaryotes. These include the replication initiation complex, ubiquitin, and histones, and many of the proteins thought to underpin the dynamic architecture of eukaryotic cells, including actin, tubulin, and ESCRTIII []. It now seems clear that the bulk of the machinery governing eukaryotic intracellular architecture derives from proteins present in members of the so-called TACK superphylum of archaea []. The discovery of Lokiarchaeum (‘Loki’), a novel TACK archaeon named for the deep-sea vent near where it was identified through metagenomic sampling [], has provided strong support for this idea. The Loki composite genome encodes more homologs of Eukaryotic Signature Proteins (ESPs) than any other prokaryotic genome to date, making it an excellent candidate for a representative of the lineage that gave rise to eukaryotes ( Box 1 ).

Lokiarchaeum was discovered through a metagenomic analysis of marine sediment sampled some distance from an active vent system named Loki's Castle [] in the Arctic Mid-Ocean Ridge. 16S rRNA sequencing led to the identification of previously unidentified sequences belonging to a deep-branching TACK clade and further refinement produced a composite genome encoding 5381 putative genes given the name ‘Lokiarchaeum’. The first analysis of this remarkable ensemble genome identified 92 putative small Ras-like superfamily GTPases, six actin genes, a strikingly eukaryote-like ribosome, and clearly detectable ESCRTIII, ESCRTI, and ESCRT0 complexes. Intriguingly, longin-like proteins and a putative BAR domain protein, whose homologs play important roles in the regulation of eukaryotic cell shape and membrane organization [], were also identified. A recent sensitive reexamination of the Lokiarchaeum genome [] has identified a further 17 small GTPases, bringing the total to 109 (including some with homology to eukaryotic Rag GTPases). This analysis also revealed the presence of 38 Roadblock domains, a subset of which appears fused to Ras-like and Rag-like small GTPases, and a RLC7 dynein homolog. Finally, additional longin/longin-like domains were identified, so 41 have now been identified in total. Again, intriguingly, five of these were found fused to lokiarchaeal Arf-like small GTPases. Collectively these data support the idea that, despite the intervening events, which include the acquisition of mitochondria, a member of this or a closely related archaeal lineage gave rise to the eukaryotic cell through sequential rounds of growth and division.

The internal architecture of all eukaryotic cells is drastically different from that of their distant relatives bacteria and archaea. Most obviously, they differ in size: eukaryotes are thought to have arisen from prokaryotic ancestors, but eukaryotic cells tend to be one to two orders of magnitude larger in mass than prokaryotes. Further, while the cytoplasm of most prokaryotes is bounded by one or two [] simple membranes, a series of internal membranes divides the cytoplasm of all eukaryotic cells into numerous internal compartments. The dynamic organization of these compartments is regulated by a startling array of regulatory and structural proteins [], with many layers of molecular machinery working to ensure the controlled distribution of compartments between daughter cells at cell division [].

Unfortunately, at present, members of the Lokiarchaeota and their relatives have yet to be isolated, imaged, or cultured. All that is available is a genome sequence. This forces us to ask an age-old question in biology: is it possible to predict phenotype from genotype? Inferring the form and behavior of an organism from genomic information alone is difficult, especially when the gene families of relevance are ancient and their relationships uncertain. This problem is well illustrated for proteins like actin, where a clear correspondence between the six actin homologs in Loki and the actin genes in eukaryotes remains to be established. Additionally, the problem of inferring cell morphology from sequence data is confounded by the nonlinear relationship between genotypic and phenotypic information. For example, small variations in the structure of a monomer of a cytoskeletal protein can lead to dramatic changes in the behavior of the filament polymer and the resulting cellular phenotypes []. Nevertheless, despite these challenges, some insights about the appearance of Loki can be gained using phylogenetics, bioinformatics, and cell biology as a guide. Following this line of reasoning we argue that Loki is likely to be a structurally simple cell and that the origins of eukaryotic complexity lie elsewhere. We suggest that a partnership between an ancient Loki-like archaeon and a pre-mitochondrial bacterium allowed lokiarchaeal GTPases to combine with bacterial lipid synthesis, enabling the subsequent evolution of quintessentially eukaryotic membrane-bound compartments.

As a potential living model for the protoeukaryotic cell, we might wonder what Loki looks like. Does it have a rudimentary trafficking system and primitive organelles, as suggested by recent commentaries [], or might it be a small, structurally simple archaeon with a large complement of regulatory genes? In the first scenario, a Loki-type cell with complex internal organization may have engulfed a bacterial cell leading to late acquisition of mitochondria. Alternatively, in the second scenario, eukaryotic cellular architecture may have emerged gradually through the influx of lipids and lipid metabolic genes from a bacterial partner [] during a long period of increasing intimacy. In the latter case, eukaryogenesis was a true collaborative venture that relied on structural and information-processing genes from archaea and on lipid metabolism from bacteria. These are drastically different ways of viewing the origins of eukaryote cell architecture, and Loki holds the key to distinguishing between them.

Loki GTPases and Membranes

23 Klinger C.M.

et al. Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks. 23 Klinger C.M.

et al. Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks. 17 Ball S.G.

et al. Pathogen to powerhouse. 18 Archibald J.M. Endosymbiosis and eukaryotic cell evolution. 19 Embley T.M.

Williams T.A. Evolution: steps on the road to eukaryotes. In search of clues to resolve this question, we look at the lessons that can be learned from small GTPases. The identification of numerous ‘Ras-like’ (Ras/Rho/Rab/Ran) and ‘Arf-like’ (Arf/Sar) small GTPases, as well as homologs of the atypical vacuolar/lysosomal Rag GTPases [], was one of the major surprises of the Loki genome. Although the phylogenetic analyses performed thus far provide only modest support for an archaeal origin of the Ras-like, Arf-like, and Rag-like small GTPase subgroups [], this information has been used to support the argument that Loki is likely to possess intracellular compartments and, perhaps, a primitive form of phagocytosis []. If true, this finding would be significant because it provides a mechanism by which a Loki-like cell could have engulfed the future mitochondrion. There are, however, problems with this reading of the data. The presence of large numbers of small GTPases in the Loki genome provides strong evidence of ancestry and the capacity for regulatory complexity but does not by itself imply conservation of function. What, then, is the evidence that, like their eukaryotic counterparts, Loki small GTPases regulate membrane dynamics and compartment identity?

12 Pfeffer S.R. Rab GTPase regulation of membrane identity. 13 Barr F.A. Review series: Rab GTPases and membrane identity: causal or inconsequential?. 24 Leung K.F.

et al. Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases. 25 ten Klooster J.P.

Hordijk P.L. Targeting and localized signalling by small GTPases. 24 Leung K.F.

et al. Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases. 26 Resh M.D. Covalent lipid modifications of proteins. In eukaryotes, many small GTPases are physically associated with membranes and this membrane anchoring plays a fundamental role in linking the GTP–GDP cycle to membrane identity, dynamics, and compartmentalization []. Small GTPases are recruited to membranes through multiple targeting mechanisms. Most commonly this relies on polybasic sequences that provide an electrostatic interaction with the membrane surface, together with the cotranslational or post-translational addition of one or more lipid tails []. These lipid modifications include N-myristoylation (Arf GTPases), palmitoylation (H-Ras), farnesylation (Ras), and geranylgeranylation (Rab and Rho). Farnesylation and geranylgeranylation (collectively known as prenylation) usually rely on the presence of a ‘CAAX’ box (Cys-aliphatic-aliphatic-X) at the carboxyl terminus of target proteins, where the C-terminal amino acid (X) determines whether the protein will be modified by the closely related enzyme farnesyl transferase (FTase) or geranylgeranyltransferase I (GGTase I). Rab proteins are geranylgeranylated at two C-terminal cysteines by GGTase II, with the aid of a Rab escort protein (REP), which provides specificity []. While a single geranylgeranyl tag can ensure the stable association of a protein with a membrane, proteins that are farnesylated often require a second signal (e.g., palmitate tag, polybasic charged residue cluster) for membrane binding. Importantly, the enzymes responsible for these key post-translational lipid modifications are encoded by highly conserved, essential genes ubiquitous across eukaryotes []; homologs have yet to be identified in prokaryotes.

23 Klinger C.M.

et al. Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks. 27 DerMardirossian C.

Bokoch G.M. GDIs: central regulatory molecules in Rho GTPase activation. Table 1 a a ‘Present’ indicates that a putative or confirmed protein ortholog (or orthologous group/orthologous domain) has been identified in one or more representative species within each column. Phylogenetic Distribution of Membrane-Trafficking Building Blocks Bacteria TACK Archaea Lokiarchaeum Eukaryotes Refs Small GTPases − − Present Present 10 Spang A.

et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. 23 Klinger C.M.

et al. Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks. CAAX domains − − − Present Prenyltransferases − − − Present Fatty acid transferases − − − Present GDI/GDF/REP/accessory − − − Present Longin/Roadblock Present Present Present Present 23 Klinger C.M.

et al. Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks. 60 De Franceschi N.

et al. Longin and GAF domains: structural evolution and adaptation to the subcellular trafficking machinery. SNARE − − − Present Coat proteins − − − Present Dynamins Present − − Present 57 Low H.H.

Löwe J. A bacterial dynamin-like protein. Actin/actin-like proteins Present Present Present Present 10 Spang A.

et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. 22 Michie K.A.

Löwe J. Dynamic filaments of the bacterial cytoskeleton. 72 Izoré T.

et al. Crenactin from Pyrobaculum calidifontis is closely related to actin in structure and forms steep helical filaments. Until the discovery of Loki, it seemed clear that small GTPases and their ubiquitous lipid-modifying enzymes coevolved. This is no longer the case. Loki has no detectable orthologs of any of the lipid modification enzymes or accessory proteins discussed above ( Table 1 ). An analysis of all 109 putative Loki small GTPase sequences [] (NCBI) shows that none has a C-terminal CAAX domain, although putative C-terminal interaction sites for GGTase II were identified in two Loki GTPases (Table S1 in the supplemental information online). In addition, the Loki genome ensemble appears to lack homologs of RhoGDI and RhoGDF, the proteins in eukaryotes that act to regulate the association of lipid-modified small GTPases with membranes (GDI masks the lipid moiety enabling it to maintain small GTPases in the cytosol until they are displaced through the action of GDF []), again arguing against Loki GTPases being subject to eukaryote-like lipid modifications. Of course, this does not preclude the presence of an alternative mode of lipid modification in Loki. Since protein–lipid and lipid–lipid interactions are strongly dependent on environmental pressure, temperature, and chemical conditions, it is possible that, for example, the tethering of GTPases to archaeal-type membranes present in Loki at 4°C necessitates a different type of chemical modification. The identification of high temperature and/or mesophilic Lokiarchaeota will help to make the role of the environment clearer.

28 Lawrence J.G.

Roth J.R. Selfish operons: horizontal transfer may drive the evolution of gene clusters. 29 Koonin E.V.

et al. Horizontal gene transfer in prokaryotes: quantification and classification. 30 Sharpe H.J.

et al. A comprehensive comparison of transmembrane domains reveals organelle-specific properties. 31 Yutin N.

et al. The origins of phagocytosis and eukaryogenesis. 32 Samson R.Y.

et al. A role for the ESCRT system in cell division in archaea. Thus, either Loki has GTPase-regulated compartments but utilizes a currently unknown mode of membrane association or Loki carries a large complement of GTPases not physically associated with membranes that perform diverse regulatory functions like those played by kinases in modern eukaryotes. In this case, small GTPases emerged as a diversified family of non-membrane-associated regulators in archaea that became associated with membranes during the subsequent process of eukaryogenesis. Although we do not currently have access to cell biological data for Loki, there may be ways to test these two ideas. While functional studies in Loki remain a distant dream, clues can be found in the organization of the genome. This is because many genes in bacteria and archaea – including those of Loki – are assembled into operons. These coregulatory units facilitate the coexpression and coinheritance [via horizontal gene transfer (HGT)] of functionally related genes []. The identification of proteins that lie alongside each of the different GTPases in Loki operons will therefore provide a clue to their subcellular localization and function (e.g., lipid modification enzymes, kinases, actin homologs, membrane proteins). In addition, it may be possible to determine whether Loki cells are likely to possess physically distinct membrane domains like those that characterize eukaryotic compartments, by looking for patterns of amino acid use and hydrophobicity within transmembrane regions of proteins encoded in the composite genome []. At present, without such data, it is hard to argue that Loki has a capacity for intracellular trafficking or phagocytosis as seen in eukaryotes. Indeed, specialized phagocytic machinery in eukaryotes does not appear to be ancestral []. Note that this does not preclude there being proteins present in the Loki genome that have the capacity to bend, push, or invaginate membranes, since such proteins are a prerequisite for cell division [] in both archaea and bacteria.

33 Hetzer M.

et al. The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly. 34 Clarke P.R.

Zhang C. Spatial and temporal coordination of mitosis by Ran GTPase. 35 Jékely G. Origin of the nucleus and Ran-dependent transport to safeguard ribosome biogenesis in a chimeric cell. 36 Jékely G. Small GTPases and the evolution of the eukaryotic cell. 37 Baum D.A.

Baum B. An inside-out origin for the eukaryotic cell. 38 de Boor S.

et al. Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation. 39 Cavazza T.

Vernos I. The RanGTP pathway: from nucleo-cytoplasmic transport to spindle assembly and beyond. 40 Kiyomitsu T.

Cheeseman I.M. Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation. 41 Fan S.

Margolis B. The Ran importin system in cilia trafficking. 42 Dishinger J.F.

et al. Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-β2 and RanGTP. What can be concluded if these investigations fail to support any membrane-associated role of Loki's small GTPases? Perhaps the small GTPases found in the Loki genome function more like the GTPase Ran []. Ran has been suggested to be the primordial eukaryotic small GTPase, in part because it is highly conserved and is present in a single copy in all eukaryotes known to date []. Ran GTPase is not known to insert or associate with membranes (although it is lysine acetylated []). Intriguingly, Ran controls traffic across compartments that are separated not by a continuous membrane but by large, semipermeable aqueous channels such as the nuclear pore complex and the ciliary base. This is achieved through the establishment of gradients of Ran GTP activity driven by the spatial separation of its activators and inhibitors. For example, the binding of a Ran GEF to chromatin is used to control the shuttling of proteins between the nucleoplasm and cytoplasm [] and for spindle-pole positioning []. Ran is thought to function a similar way to aid the selective accumulation of proteins within cilia [].

43 Bielli A.

et al. Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission. 44 Fransson S.

et al. The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking. 45 Fransson A.

et al. Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis. 46 Embley T.M.

Martin W. Eukaryotic evolution, changes and challenges. There is a second set of small GTPases that are not subject to lipid modification that is exemplified by the GTPase Sar1 and the atypical GTPases Miro1/2 (together with Rit and RhoBTB). In eukaryotes, these small GTPases carry membrane-insertion domains. In the case of Sar1, which is present in a single copy in most eukaryotic genomes, this serves to induce the budding of membrane from the endoplasmic reticulum (ER) []; in the case of Miro1/2, this hydrophobic domain tethers the GTPases to the outer mitochondrial membrane [], where they regulate mitochondrial activity and dynamics. These further exceptions to the rule in eukaryotes are interesting in that they represent small GTPases that associate with stable organelles, the ER, the nuclear envelope, and mitochondria rather than self-organizing, dynamic cellular compartments like those regulated by Arf and Rabs. Moreover, it has been argued that the acquisition of these ubiquitous eukaryotic compartments – the continuous nuclear envelope and ER and mitochondria – is likely to represent two key steps in eukaryogenesis []. Interestingly, two small GTPases in the Loki genome have hydrophobic alpha helices: KKK46087 and KKK46086 (Table S1), suggesting that they may associate with membranes in this way.