PERSPECTIVES

OPINION

Cell evolution and the problem of membrane topology

Gareth Griffiths

Abstract | Cells somehow evolved from primordial chemistry and their emergence depended on the co-evolution of the cytoplasm, a genetic system and the cell membrane. It is widely believed that the cytoplasm evolved inside a primordial lipid vesicle, but here I argue that the earliest cytoplasm could have co-evolved to high complexity outside a vesicle on the membrane surface. An invagination of the membrane, aided by an early cytoskeletal system, may have formed the first cells initially within primordial vesicles.
Genomic analysis leaves no doubt that the three main kingdoms of life eubacteria, archaea and eukaryotes evolved from a common ancestor1. The idea that all life evolved from one source is perhaps the most profound consequence of the DarwinWallace model of evolution. With increasing amounts of DNA sequence information available, the search is on to find the minimum set of genes that existed in the last common ancestor (LCA) the precursor cell(s) from which all living organisms subsequently evolved. The LCA must have been a rather sophisticated cell because it contained all the cellular machinery that is common to all present living forms, including a DNA-based information system2,3. From sequence analysis, the LCA is suggested to contain 250600 genes4,5. Mycoplasma genitalium, the simplest known prokaryote cell (which is nevertheless dependent on host-cell parasitism), requires 270 of its 380 genes for normal function, as revealed when all genes were individually knocked out6. So, the minimum conceivable cell needs a lot of genetic information. The Earth is ~4.56 billion years (Gyr) old, and the best current estimates argue that the first cells appeared by 3.03.3 Gyr at the latest7. How these first cells emerged is one of the biggest unsolved problems in biology. It seems undeniable that the LCA must have been preceded by a spontaneous generation of cells from abiotic precursor
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molecules. Even Darwin in his theory of evolution was obliged to accept the necessity of some kind of spontaneous generation. He brilliantly speculated (in a letter to Hooker) that in a warm little pond a particular chemistry involving all sorts of
Acidic Glu Asp 1 RNA Mineral surface chemistry Asn Gln 2 3 Polar uncharged Ser Pro Val Ala 4 Gly Cys 5

ammonia and phosphoric salts, light, heat, electricity etc. was present so that a protein compound was chemically formed ready to undergo more complex changes on the path towards life8. It seems likely that his (unpublished) hypothesis was not far from the mark. Modern theories were initiated by Oparin in 1924 (REF. 9) and by Haldane10, who first discussed the origin of membranes and speculated that an oily film on the surface of sea water evolved into the lipid-rich cell membrane. Here, I attempt to build up a plausible scenario for how cells may have evolved with an emphasis on the more difficult question of the origin of the cell membrane. A brief discussion of the (more actively pursued) question of the evolution of cytoplasm, including the nucleic acids, sets the stage for the main hypothesis.
The evolution of the cytoplasm Cells are made up of nucleic acids, proteins, carbohydrates, lipids and many small molecules suspended in a particular ionic
Basic Phe Tyr Aromatic His Trp

Non-polar hydrophobic Thr Ile Met Leu 7 8 9

Lys Arg 10

11

12

13

14 DNA

Evolutionary time Vesicles

RNA-DNA Phospholipids

Self-synthesizing membrane

Figure 1 | Biosynthetic pathways for amino acids, phospholipids and central metabolism. Nature Reviews | Molecular Cell Biology It has been proposed by Davis22,26 and many others that primitive biochemical pathways existed before a genetic-based system. These include the reductive carbon cycle (the equivalent of the tricarboxylic acid (TCA) cycle working in reverse), the reductive pentose pathway and the central trunk (proposed to be a remnant of the formose cycle), which together make up the central biochemical pathway (CBP). Davis proposes that the emergence of genetically encoded amino acids correlates with the number of chemical reactions from the CBP that are required to generate each amino acid (evolutionary steps 114). Acidic amino acids (Glu and Asp) are close to the CBP (12 reactions), whereas aromatic residues (such as Trp) require up to 14 steps to be synthesized and may have appeared later. The hydrophobic amino acids that could associate with membranes required four steps. The synthesis of phospholipids requires 10 steps and, therefore, self-synthesizing membranes might only have arisen after this point. Davis provides detailed analysis to argue that the evolutionary appearance of the different amino acids correlates well with the emergence of their corresponding triplet codes; this implies co-evolution of biochemistry and the genetic code, an idea extensively championed by Wong60. Davis also identified an 11-amino acid sequence in the FtsZtubulin family that he mapped to his evolutionary stage 7.5, which could support a role for this protein in cellularization. Gly, Cys and Pro cannot easily be placed into any of the five categories shown.

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Protocytoplasm Lipid vesicle P + P + P P + + + P + P Obcell Protocell Mineral surface Mineral + surface

b Cytoplasm inside
vesicle Gel

Cytoplasm outside vesicle

P + +

Unstable permeable bilayer

Transient breaks in the membrane RNA

Less permeable bilayer

Stable bilayer

Liposome

Ribosome Protein

Autonomous cell divides

Pore/channel Single acyl chain lipids Monoglycerides Fatty acids Carboxylic acids Two acyl chain phospholipids Impermeable membrane Electric potential Proton gradient

Figure 2 | Surface interactions and the membrane problem. a | A pure lipid vesicle in contact with a protocytoplasmic milieu. The system provides two catalytic surfaces, the positively charged mineral surface and the negatively charged, hydrophobic lipid vesicle. b | Two proposed membrane scenarios for cellularization during evolution. In the inside-out or leaky liposome model (left) the cytoplasm evolved within the vesicle and the topology of the membrane (outer leaflet outside) remains in place during the evolutionary process. In the cytoplasm outside model, the cytoplasm co-evolves with the membrane by associating with the outer membrane surface (which will later become the cytoplasmic surface). A gel-like filamentous material may prevent the diffusion of protocytoplasmic components36. c | The cytoplasm inside the vesicle hypothesis requires that the molecular precursors of life must have found a way to pass selectively through the liposome barrier. Deamer proposed that the earliest vesicles

contained bilayer membranes made up of single acyl chain lipids that are Nature Reviews | Molecular Cell Biology more permeable to many molecules, including ions. As the system evolved to use the more complex two acyl chain phospholipids, the membrane became more impermeable. Additional proposed mechanisms that overcome the permeability barrier include osmotic forces, transient breaks in the membrane caused by polymerized amino acids such as polyleucine, and transient openings in the bilayer caused by temperature changes or freeze-thaw cycles20. d | The obcell model of the outside-in hypothesis of cellularization, as proposed by Blobel38 and Cavalier-Smith39. The early liposome system is postulated to induce cisternae to fuse with themselves to form double-membrane obcells (inside-out cells)39. The model necessitates the loss of the outer of the two membranes to release a protocell that has the correct topology (with the luminal domains of the membrane proteins facing outwards).

environment. Before cells emerged, how were sufficient amounts of the necessary starting materials generated? Following the pioneering experiments of Miller9, extensive studies described many chemical reactions that plausibly occurred under the presumed conditions of the early Earth. These reactions could synthesize many (but not all) amino acids and other key precursors, such as purines, and precursors of liposomes such as carboxylic acids1113. The key precursors of life could have been made on Earth or in outer space and carried to Earth via meteorites, such as the Murchison meteorite that landed in Australia in 1969 (REF. 14). Meteorites, as well as interplanetary dust particles, bring an extraordinary selection and amount of chemicals and biochemical precursors of life from space, including >90 different amino acids (of which 19 are found in living organisms) and, as discussed below, bilayer-forming lipids15,16.
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The cytoplasms of most modern cells have a similar chemical composition with a reducing environment, neutral pH and an ionic composition that is rich in K+, Cl and Mg2+ but low in Ca2+ and Na+. Perhaps this universal composition reflects the environment where the first cells evolved; if so, the environment where the earliest chemistry that preceded life occurred is likely to have been in fresh water17,18 rather than the high-salt ocean environment proposed by others10,19. The latter is also difficult to reconcile with the need for primordial lipid vesicles, which are unstable at high salt concentrations20. For a protocytoplasm to emerge, many schemes have been postulated that involve the occurrence of chemical catalysis and autocatalytic reactions on the surface of positively charged minerals, such as clay or iron pyrites2125. A useful yardstick for timing the main events leading to cellularization is a scheme proposed by

Davis22,26 (FIG. 1; see below). The evolution of the cytoplasm is often proposed to have occurred in parallel with the emergence of a liposome system, which eventually became the delimiting membrane of the cell.
The emergence of membranes In a water-based system, many lipids can self-assemble into bilayer-containing vesicles. Phospholipids are the main bilayerforming lipids in bacteria and eukaryotes (assuming that the ether-lipid-based membranes in archaea are a later adaptation to extreme environments22). However, the simplest bilayer-forming lipids are long-chain (>C9) fatty acids, carboxylic acids and monoglycerides18,20. When such lipids were extracted with solvents from the Murchison meteorite, they formed bilayered vesicles in aqueous solution16. Deamer and colleagues proposed that vesicles containing lipids made up of single acyl chains formed the first template for the
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Box 1 | Double membrane compartments in modern cells

a Vaccinia virus
TGN

Golgi complex Plasma membrane

IMV

(Microtubule)

(Actin)

EEV

b Budding yeast

Golgi Spore

Spindle pole body

Nucleus

c Autophagy

lipid membranes are more permeable to uncharged and charged molecules, their overall permeability is several orders of magnitude lower than phospholipid bilayers. Significantly, all bilayers are highly impermeable to ions such as K+ (REFS 18,20). As membrane complexity increases, the bilayer becomes more impermeable. In this cytoplasm within the vesicle scenario, the emerging ribosomes inside the vesicle would eventually evolve the capacity to insert membrane proteins from the inside with their extracellular domains facing outwards, as in modern cells (FIG. 2b,c). Many elegant experiments have been conducted to try and reconstitute some aspects of this hypothesis. For example, investigators trapped enzymes and whole transcription translation systems within vesicles and found that these became functionally active 20,3335 (FIG. 2c). Such analyses are still a long way from reconstituting life in such vesicles, as conceded even by some of the strongest proponents of this hypothesis18. The outside the vesicle scenario. Given the difficulties with the above hypothesis, let us consider another scenario in which the cytoplasm evolved to a high degree of complexity outside the vesicles. A mineral surface could provide an environment on which lipid vesicles become attached, and these surfaces could provide the environment where some rudimentary components of a complex protocytoplasm for example, perhaps ATP, GTP, proteins, RNA and maybe even DNA could evolve. Given the problem of these key precursors diffusing away from the site of action, it is attractive to consider the emergence of a polymer in the space around the vesicles that can form a hydrophilic gel (REF. 36). This could initially be a simple polymer such as polysugars or polyglutamic acid that might later be replaced by a cytoskeletal protein polymer such as actin (see below). Such systems of filaments could serve to attach the vesicles to the mineral surface, capture precursor molecules within their gel-like matrix or provide a scaffold for protection as well as an additional catalytic surface for emerging biochemistry. Under these conditions, there would be three catalytic surfaces the positively charged mineral surface24,25, the surface of the gel36 and the negatively charged lipid vesicle surface that could provide a rich surface for many reactions37. The mineral surface could itself catalyse the assembly of lipid vesicles25. In this microenvironment, sandwiched between these surfaces, a sophisticated protocytoplasm
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Mitochondrion

Fusion with lysosome

There are at least three examples in modern cells that are topologically similar to the life outside the vesicle model (see figure). a | During the cellular release of vaccinia virus, the|trans GolgiCell Biology Nature Reviews Molecular network (TGN) cisternal domain engulfs the intracellular mature virus (IMV)56. This intracellular enveloped virus is transported along microtubules and fuses with the plasma membrane. During fusion with the plasma membrane, or shortly thereafter, the local polymerization of actin around the TGN-derived membrane facilitates virus release into the extracellular space (extracellular enveloped virus (EEV))57. As proposed in the evolutionary schemes presented in FIG. 3, actin and microtubules also interact here with membranes. b | During sporulation in the budding yeast Saccharomyces cerevisiae, a double-membrane prospore cisterna is formed that originates from post-Golgi vesicles that fuse around the forming (haploid) spore (only one spore is shown but up to four may be made). The Golgi vesicles aggregate on the surface of the nucleus (pink) at the spindle pole body to enclose the spore within two membranes58. c | The formation of autophagic vacuoles is proposed to occur by the formation of a double-membrane structure59. A cytoplasmic cisternal structure (of unknown origin but suspected to originate from the endoplasmic reticulum) wraps around cytoplasmic components such as a mitochondrion to form a double-membrane vesicle. The outer membrane fuses with a late endocytic compartment, whereas the inner membrane is expected to be lysed by the hydrolytic conditions of the lysosomal lumen. In parts a and c, the purple ball and stick structure represents a membrane-spanning protein, with the ball representing the luminal domain.

cell membrane and that more complex (two acyl chain) phospholipids emerged later20 (FIG. 2). Phospholipids can be synthesized without enzymes under plausible abiotic conditions12,13,2729, but it seems likely that these lipids became more important when RNA ribozymes or ribosomes and protein enzymes emerged that could synthesize them (FIG. 1).
The evolution of cells If we assume that an increasingly complex cytoplasm with protein-synthesizing ribosomes, nucleotides, RNA and even DNA
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emerged in contact with a system of liposomes, we face a crucial unresolved issue in understanding the origin of cells and the cell membrane: on which side of the liposomes did the first key reactions occur? The scenario of life within the vesicle. Most specialists think that such a system could only have evolved within the primordial vesicle20,3034 (FIG. 2b). How then could the precursors of the key molecules of life have crossed a bilayer that presents a significant barrier to charged molecules? Although vesicles comprised of single acyl chain

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Box 2 | Interactions of actin and microtubules with membranes in modern cells
Filopodia Myosin +

Lamellipodia Cell motility

Exocytosis

Comet Endocytosis Cytokinesis Membrane nucleation of actin +

of an obcell or inside-out cell (FIG. 2d). Whereas the life within the vesicle model (FIG. 2c) has no obvious mechanistic precedents in modern cells, there are examples from present-day cells in which a cisternal wrapping process occurs that is topologically similar to the cytoplasm outside the vesicle model (BOX 1). Below, I argue that this (admittedly complex) inversion occurred by numerous steps and depended on the earliest cytoskeletal filaments.

Functions of the cytoskeleton Not so long ago, textbooks dogmatically stated that only eukaryotes contain actin filaFusion + ments and microtubules. Recently, this idea Microtubule has been overturned with the realization that Phagocytosis the actin- and microtubule-family proteins Macropinocytosis are universally expressed and interact with + Motor + Motor membranes. The prokaryotic GTPase FtsZ, MreB (actin homologue) a homologue of tubulin, was shown to form filaments and to interact with the membrane during bacterial cell division40. FtsZ (tubulin homologue) Actin homologues have been discovered in eubacteria and archaea. It had been long appreciated that hexokinase and chaperones of the heat shock protein-70 (HSP70) family were homologous to actin41. Recently, new FtsA (actin homologue) Cytokinesis members of this family, the MreB and In eukaryotes, the actin family is involved in many membrane-dependent processes such as exocytosis, endocytosis, phagocytosis, cell motility (for example, Nature Reviews | Molecular Cell Biology Mb1 subfamilies, were shown to be true using lamellipodia), cytokinesis and actin homologues that can form actin-like cell polarity. Actin (red) and its prokaryotic homologues have many intimate connections with membranes in eukaryotes (see figure; top panel) and prokaryotes (bottom panel). Membranes can filaments in vitro and in bacteria42,43. As in nucleate the assembly of actin and, in all known cases, the topology is such that the fast-growing end eukaryotic cells, these filaments polymerize (barbed end or plus end (+)) is localized adjacent to the membrane this means that the insertion of while being attached to the membrane new monomers occurs at this site. Of the different mechanisms and processes shown, only two can be surface, providing a force that contributes to considered to be well understood. First, myosin motors attached to membrane organelles can walk prokaryote shape44,45. When these proteins along actin; most myosins move towards the plus ends. Second, actin comets, which were first are knocked out in rod-shaped bacteria, described in the intracellular transport of Listeria monocytogenes and other cytoplasmic pathogens, for example, the cells are converted into transport vesicles such as endocytic vesicles and phagosomes. For the most part, however, the spheres44. One actin homologue, FtsA, is mechanistic details of how actin or its homologues in prokaryotes interact with membranes is poorly also involved in bacterial cell division and understood49,52. Even less is known about the interactions of microtubules with membranes, with the has recently been shown to interact directly exception of motor proteins that can be bound to membrane organelles; kinesin transports cargo towards the plus end of the microtubule, whereas dynein transports cargo in the opposite direction. with FtsZ; thus, the actin- and tubulin-based In prokaryotes, actin homologues interact with the cell membrane, whereas the tubulin homologue systems work together to drive bacterial FtsZ, which also assembles into filaments (green), interacts with the membrane at the site of cell cytokinesis46 (BOX 2). division (it forms a ring structure at the septum known as the Z ring) and is essential to carry out In eukaryotes, the actin family is involved cytokinesis (bottom panel). There are no comprehensive models to predict how FtsZmembrane in many membrane-dependent processes interactions operate mechanistically in this process but, recently, the actin homologue FtsA has such as exocytosis, endocytosis, phagobeen shown to interact directly with FtsZ46. A similar lack of understanding pertains to the role of cytosis, cell motility, cytokinesis and cell actin in the much more complex process of cytokinesis in eukaryotic cells; microtubules and polarity47,48. In many fusion processes, actin microtubule-associated proteins are also implicated in this process. polymerizes on membranes and somehow provides a force to pull membranes together might have emerged that interacted first with their extracellular domains facing the and/or push them or keep them apart4952 with the outer surface of the vesicles. Small inside of the vesicle38. To develop normal (BOX 2). Recent data have shown direct molecules may indeed have crossed sponcell-membrane topology, the vesicle would interactions between membrane nucleation taneously into the vesicle lumen and I will have to invert upon itself and fuse to form a mechanisms for actin assembly (N-WASP) revisit this issue below. double-layered vesicle (FIG. 2d). The outer of and machinery for non-clathrin-mediated If ribosomes could make hydrophobic the two vesicles would then need to lyse to endocytic vesicle formation and for polypeptides, these would interact with the dorsal cell ruffling. In these processes, release the cell. This clever idea by Blobel38 vesicle outer surface and, in time, the system is rarely cited by origin-of-life specialists sorting nexin-9 (SNX9) was identified as a would evolve the capacity to insert membut has been extensively championed by membrane scaffold protein that stimulates brane proteins that spanned the membrane, Cavalier-Smith39, who conceived the idea N-WASP and ARP2/3-dependent actin
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a
Pre-cytoplasmic environment Ribosome Lumen Actin Microtubule/FtsZ + + + + + + + + +

External milieu Extracellular space

Protocell Channel

Cytokinesis

Extracellular space Luminal fusion Autonomous cells Cytoplasmic fusion Inverted vesicle

Figure 3 | The outside-in model of cellularization. Shown is a possible evolutionary mechanism by which the outside-in model (FIG. 2d) may have occurred. a | An evolutionary stage exists in which a complex protocytoplasm has a genetic code that is RNA- or even DNA-based, ribosomes for synthesizing proteins and a relatively advanced biochemistry. The system has evolved the capacity to insert membrane proteins such that the future extracellular or luminal domains are inside the liposome. In parallel, an actin- and tubulinbased cytoskeleton evolved the capacity to interact with the liposome surface. b | The emergence of the cytoplasmic fusion machinery (orange) allows liposomes to fuse together and bend membranes. c | The fusion machinery could collaborate with the cytoskeleton to form an inwards-budding vesicle. This budding process, which is similar to modern endocytosis but has the opposite

topology, allows the genetic material (not shown) and the protocytoplasm to Nature Reviews | Molecular Cell Biology enter into a vesicle within a larger vesicle the protocell. Channels and transporters that allow ions to cross the membrane would be important for maintaining the ionic homeostasis of the protocytoplasm inside the protocells and in the extracellular space. d | The emergence of luminal fusion mechanisms allows the process of fission out of the parental vesicle to occur. This machinery also allows the protocells to fuse together within the extracellular space. e | The cytoplasmic fusion machinery evolves into the process of cytokinesis, which allows protocells to divide in a regulated fashion such that each daughter cell contains everything it needs to metabolize and replicate. The protocells are eventually released when the outer membrane lyses. These are now independent living forms that are capable of self-replication.

assembly, especially when it is allowed to oligomerize on phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2)-enriched membrane domains in vitro. SNX9 contains a BAR domain that is known to facilitate membrane bending53. Such studies start to resolve the long-elusive molecular links between actin, its assembly and membrane functions. Besides its role in cytoplasmic fusion processes (where the cytoplasmic leaflets of membranes first interact), a recent study shows that N-WASP-based actin polymerization is also essential for two apposing plasma membrane luminal domains to fuse completely during Drosophila melanogaster myoblast fusion54. Thus, actin facilitates both cytoplasmic and luminal fusion events (FIG. 3). Homologues of actin and tubulin have therefore been identified in all kingdoms of life. The ATPase actin and the GTPase tubulinFtsZ protein family may have appeared before the cell became surrounded
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by a membrane and, if so, could have functioned in cellularization. These suggestions are supported by an analysis by Davis, who argued that the order in which coded synthesis of the different amino acids and lipids emerged during evolution correlates with the number of reactions needed for their synthesis from an already evolved biochemical system, which included the tricarboxylic acid (TCA) cycle and the pentose pathway22,26 (FIG. 1). These reaction systems are universally involved in the synthesis of all 20 common amino acids used by modern cells. Davis26 identified a conserved 11-residue sequence in the FtsZtubulin family that he mapped to his evolutionary stage 7.5, a stage he classified as occurring before the system could self-synthesize membrane phospholipids (stage 10; FIG. 1). Because microtubules and FtsZ, and especially actin, have intricate interactions with modern membranes, I propose a speculative model for the role of their precursors in cellularization.

The cytoskeleton in cellularization? My model of cellularization starts with interactions of the protocytoplasm with the outer surface of the initially pure lipid liposomes (FIG. 3a). In the model, I propose that proteins (made by ribosomes present in the protocytoplasm) evolved hydrophobic domains that allowed them to interact with liposomes. Later, membrane-spanning proteins, including channels and pumps, were inserted. The proton and other pumps formed chemical and electrical gradients and synthesized ATP on the cytoplasmic side of the membrane. The inside of the vesicle could then develop a different composition to the outside and might later have become the extracellular space. Thus, in this model, even sophisticated membrane functions such as proton and electrochemical gradients emerged before cellularization. Actin and/or tubulin ancestors then interacted with the outer surface of the membrane and facilitated membrane bending.
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A cytoplasmic machinery evolved that fused the vesicles together (FIG. 3b). A coordinated effort between the cytoskeleton and the cytoplasmic fusion machinery then allowed apposing membrane lipid coalescence and the inwards pinching of vesicles to form protocells (FIG. 3c). These inverted vesicles within the larger vesicle enclose the protocytoplasm and the genetic material (DNA or RNA); it is implicitly assumed that this evolving genetic material is intimately associated with the cytoplasmic surface of the vesicle (not shown in FIG. 3). The formation of these vesicles is topologically equivalent to the budding of vesicles into the lumen of specialized endocytic organelles multivesicular bodies in modern eukaryotic cells. Additional machinery must have evolved that allowed the luminal domains of the protocell membranes to fuse together. The same machinery could allow cells to bleb off a membrane vesicle by fission, a process equivalent to blebbing in modern cells. The protocells could undergo selection by content mixing driven by fusion with themselves and with newly made protocells bringing in new components from the external milieu (FIG. 3d). At a later stage, the cytoplasmic fusion machinery facilitates the separation of daughter protocells in a regulated fashion (cytokinesis) (FIG. 3e). The final stage is lysis of the first outer membrane to release independent cells. These are expected to have probably several hundred DNA-encoded genes in order for the minimal cell functions to be permitted. The extracellular compartment (FIG. 3c) would offer several advantages to the emerging cells bathing within this space. The evolution of mechanisms to transfer glycoconjugates bound to lipids or proteins onto and beyond the luminal surface of the boundary membrane of protocells could provide a reserve energy source for cells if sugar hydrolases were also secreted into this space. A high-viscosity environment here could also protect the enclosed cells from extreme environmental changes in the external environment. Ion channels in the boundary membrane and the protocell membrane could concentrate some ions, such as Ca2+ and protons, which would be stored in the extracellular buffering compartment and injected into the protocells as required. It is conceivable that many of the mechanisms proposed for permeation of the membrane in the inside the vesicle scenario (FIG. 2c) could be operational in the outside the vesicle model (FIG. 3). However, instead
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of molecules entering the future cytoplasm, in the latter model, the transport of molecules into the lumen of the vesicles would allow the future extracellular space to evolve. The stage of protocells within a vesicle might, perhaps, have existed for a relatively long time. The evolutionary split between eukaryotes and prokaryotes55 could also have developed in this system, protected by two membranes.
Conclusions and perspectives I have outlined a plausible scenario for the co-evolution of the cytoplasm and the membrane based on existing cell biological principles. I have discussed the two principal theories with respect to the evolution of the membrane and suggested that the cytoplasm outside model (FIG. 3) is more plausible than the more favoured cytoplasm inside the vesicle model. If the inside-out (or obcell) model is correct, it seems reasonable to suggest that it needed the active participation of a cytoskeleton to invert the topology of the membrane system and initiate the cellularization process. The model predicts that the actin and tubulin family preceded the LCA and that they are probably as universal as the vacuolar ATPase family. Experimentally, one could ask how a liposome system with a subset of reconstituted membrane proteins would behave with cytosolic extracts and factors such as glucose, GTP and ATP. If the cytoplasmic domain of these membrane proteins all faced outwards, some aspects of the life outside the vesicle model presented here could be tested. Alternatively, if they had the opposite orientation, they could perhaps be used to test some aspects of the life within the vesicle scenario.
Gareth Griffiths is at the European Molecular Biology Laboratory, Heidelberg, Germany. e-mail: gareth.griffiths@embl-heidelberg.de doi:10.1038/nrm2287 Published online 31 October 2007
1. 2. 3. 4. 5. 6. 7. Doolittle, R. F. Searching for the common ancestor. Res. Microbiol. 151, 8589 (2000). Poole, A., Jeffares, D. & Penny, D. Early evolution: prokaryotes, the new kids on the block. Bioessays 21, 880889 (1999). Reichard, P. The evolution of ribonucleotide reduction. Trends Biochem. Sci. 22, 8185 (1997). Gil, R., Silva, F. J., Pereto, J. & Moya, A. Determination of the core of a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68, 518537 (2004). Koonin, E. V. Comparative genomics, minimal genesets and the last universal common ancestor. Nature Rev. Microbiol. 1, 127136 (2003). Glass, J. I. et al. Essential genes of a minimal bacterium. Proc. Natl Acad. Sci. USA 103, 425430 (2006). Brasier, M., McLoughlin, N., Green, O. & Wacey, D. A fresh look at the fossil evidence for early Archaean cellular life. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 887902 (2006).

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Acknowledgements

DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/sites/ entrez?db=genomeprj Listeria monocytogenes | Mycoplasma genitalium UniProtKB: http://beta.uniprot.org/ FtsA | FtsZ | N-WASP | SNX9

FURTHER INFORMATION
Gareth Griffithss homepage: http://www-db.embl.de/jss/EmblGroupsOrg/g_79.html

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