Author response: Membranes, energetics, and evolution across the prokaryote-eukaryote divide

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix References Decision letter Author response Article and author information Metrics Abstract The evolution of the eukaryotic cell marked a profound moment in Earth's history, with most of the visible biota coming to rely on intracellular membrane-bound organelles. It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells. However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes. Thus, although the origin of the mitochondrion was a key event in evolutionary history, there is no reason to think membrane bioenergetics played a direct, causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity. https://doi.org/10.7554/eLife.20437.001 eLife digest Over time, life on Earth has evolved into three large groups: archaea, bacteria, and eukaryotes. The most familiar forms of life – such as fungi, plants and animals – all belong to the eukaryotes. Bacteria and archaea are simpler, single-celled organisms and are collectively referred to as prokaryotes. The hallmark feature that distinguishes eukaryotes from prokaryotes is that eukaryotic cells contain compartments called organelles that are surrounded by membranes. Each organelle supports different activities in the cell. Mitochondria, for example, are organelles that provide eukaryotes with most of their energy by producing energy-rich molecules called ATP. Prokaryotes lack mitochondria and instead produce their ATP on their cell surface membrane. Some researchers have suggested that mitochondria might actually be one of the reasons that eukaryotic cells are typically larger than prokaryotes and more varied in their shape and structure. The thinking is that producing ATP on dedicated membranes inside the cell, rather than on the cell surface, boosted the amount of energy available to eukaryotic cells and allowed them to diversify more. However, other researchers are not convinced by this view. Moreover, some recent evidence suggested that eukaryotes are no more efficient in producing energy than prokaryotes. Lynch and Marinov have now used computational and comparative analysis to compare the energy efficiency of different organisms including prokaryotes and eukaryotes grown under defined conditions. To do the comparison, the results were scaled based on cell volume and the total surface area deployed in energy production. From their findings, Lynch and Marinov concluded that mitochondria did not enhance how much energy eukaryotes could produce per unit of cell volume in any substantial way. Although the origin of mitochondria was certainly a key event in evolutionary history, it is unlikely to have been responsible for the diversity and complexity of today's life forms. https://doi.org/10.7554/eLife.20437.002 Introduction The hallmark feature distinguishing eukaryotes from prokaryotes (bacteria and archaea) is the universal presence in the former of discrete cellular organelles enveloped within lipid bilayers (e.g. the nucleus, mitochondria, endoplasmic reticulum, golgi, vacuoles, vesicles, etc.). Under a eukaryocentric view of life, these types of cellular features promoted the secondary origin of genomic modifications that ultimately led to the adaptive emergence of fundamentally superior life forms (Martin and Koonin, 2006; Lane and Martin, 2010). Most notably, it has been proposed that the establishment of the mitochondrion provided an energetic boost that fueled an evolutionary revolution responsible for all things eukaryotic, including novel protein folds, membrane-bound organelles, sexual reproduction, multicellularity, and complex behavior (Lane, 2002, 2015). However, despite having more than two billion years to impose their presumed superiority, eukaryotes have not driven prokaryotes extinct. Prokaryotes dominate eukaryotes both on a numerical and biomass basis (Whitman et al., 1998; Lynch, 2007), and harbor most of the biosphere's metabolic diversity. Although there is no logical basis for proclaiming the evolutionary inferiority of prokaryotes, one central issue can be addressed objectively – the degree to which the establishment of eukaryotic-specific morphology altered energetic efficiency at the cellular level. Drawing on observations from biochemistry, physiology, and cell biology, we present a quantitative summary of the relative bioenergetic costs and benefits of the modified architecture of the eukaryotic cell. The data indicate that once cell-size scaling is taken into account, the bioenergetic features of eukaryotic cells are consistent with those in bacteria. This implies that the mitochondrion-host cell consortium that became the primordial eukaryote did not precipitate a bioenergetics revolution. Results The energetic costs of building and maintaining a cell The starting point is a recap of recent findings on the scaling properties of the lifetime energetic expenditures of single cells. All energy utilized by cells can be partitioned into two basic categories: that employed in cell maintenance and that directly invested in building the physical infrastructure that comprises a daughter cell. Maintenance costs involve a diversity of cellular functions, ranging from turnover of biomolecules, intracellular transport, control of osmotic balance and membrane potential, nutrient uptake, information processing, and motility. Cell growth represents a one-time investment in the production of the minimum set of parts required for a progeny cell, whereas cell maintenance costs scale with cell-division time. The common usage of metabolic rate as a measure of power production is uninformative from an evolutionary perspective, as it fails to distinguish the investment in cellular reproduction from that associated with non-growth-related processes. To make progress in this area, a common currency of energy is required. The number of ATP→ADP turnovers meets this need, as such transformations are universally deployed in most cellular processes of all organisms, and where other cofactors are involved, these can usually be converted into ATP equivalents (Atkinson, 1970). When cells are grown on a defined medium for which the conversion rate from carbon source to ATP is known (from principles of biochemistry), the two categories of energy allocation can be quantified from the regression of rates of resource consumption per cell on rates of cell division (Bauchop and Elsden, 1960; Pirt, 1982; Tempest and Neijssel, 1984). A summary of results derived from this method reveals two universal scaling relationships that transcend phylogenetic boundaries (Lynch and Marinov, 2015). First, basal maintenance costs (extrapolated to zero-growth rate, in units of 109 molecules of ATP/cell/hour, and normalized to a constant temperature of 20∘C for all species) scale with cell volume as a power-law relationship (1a) CM=0.39V0.88, where cell volume V is in units of μm3. Second, the growth requirements per cell (in units of 109 molecules of ATP/cell) scale as (1b) CG=27V0.97. The total cost of building a cell is (1c) CT=CG+tCM, where t is the cell-division time in hours. Derived from cells ranging over four orders of magnitude in volume, neither of the preceding scaling relationships is significantly different from expectations under isometry (with exponent 1.0), as the standard errors of the exponents in Equations (1a,b) are 0.07 and 0.04, respectively. Moreover, as there is no discontinuity in scaling between prokaryotes and eukaryotes, these results suggest that a shift of bioenergetics from the cell membrane in prokaryotes to the mitochondria of eukaryotes conferred no directly favorable energetic effects. In fact, the effect appears to be negative. Taking into account the interspecific relationships between cell-division time and cell volume (Lynch and Marinov, 2015) and using Equation (1b), one can compute the scaling of the rate of incorporation of energy into biomass, CG/t. For bacteria, cell-division times decline with increasing cell volume as ∼V-0.17, albeit weakly (the SE of the exponent being 0.11), implying that the rate of biomass accumulation scales as ∼V0.97+0.17=V1.14 on a per-cell basis and as ∼V1.14-1.00=V0.14 on a cell volumetric basis (with the SEs of both exponents being 0.12). In contrast, in most eukaryotic groups, cell-division times increase with cell volume, on average scaling as ∼V0.13, implying a scaling of ∼V0.84 for the rate of biomass accumulation per cell and ∼V-0.16 on a volumetric basis (with SEs equal to 0.06 for the exponents). Thus, in terms of biomass production, the bioenergetic efficiency of eukaryotic cells declines with cell volume, whereas that of bacterial cells does not. The pattern observed in bacteria is inconsistent with the view that surface area limits the rate of energy production, as this leads to an expected scaling of ∼V2/3 on a per-cell basis. Energy production and the mitochondrion The argument that mitochondria endow eukaryotic cells with exceptionally high energy provisioning derives from the idea that large internal populations of small mitochondria with high surface area-to-volume ratios provide a dramatic increase in bioenergetic-membrane capacity (Lane and Martin, 2010). In prokaryotes, the F0F1 ATP synthase (the molecular machine that transforms ADP to ATP in the process of chemiosmosis) and the electron transport chain (ETC) components (which create the chemiosmotic proton gradient) are restricted to the cell membrane, but in eukaryotes, they are confined to inner mitochondrial membranes. A key question is whether the bioenergetic capacity of cells is, in fact, limited by membrane surface area. Although the situation at the time of first colonization of the mitochondrion is unknown, the iconic view of mitochondria being tiny, bean-shaped cellular inclusions is not entirely generalizable. For example, many unicellular eukaryotes harbor just a single mitochondrion or one that developmentally moves among alternative reticulate states (e.g. Rosen et al., 1974; Osafune et al., 1975; Biswas et al., 2003; Yamaguchi et al., 2011). Such geometries necessarily have lower total surface areas than a collection of spheroids with similar total volumes. For the range of species that have been examined, many of which do have small individualized mitochondria, the total outer surface area of mitochondria per cell is generally on the order of the total area of the plasma membrane, with no observed ratio exceeding 5:1, and many being considerably smaller than 1:1 (Figure 1a). It may be argued that the outer surface area of the mitochondrion is of less relevance than that of the inner membrane (where the ATP synthase complex sits), but the ratios of inner (including the internal cristae) to outer membrane areas for mitochondria in mammals, the green alga Ochromonas, the plant Rhus toxicodendron, and the ciliate Tetrahymena are 5.0 (SE = 1.1), 2.4, 2.5, and 5.2, respectively (Supplementary material). Thus, the data are inconsistent with the idea that the mitochondrion engendered a massive expansion in the surface area of bioenergetic membranes in eukaryotes. Figure 1 Download asset Open asset Scaling features of membrane properties with cell size. (a) Relationship between the total outer surface area of mitochondria and that of the plasma membrane for all species with available data. Diagonal lines denote three idealized ratios of the two. (b) The number of ATP synthase complexes per cell scales with cell surface area (S, in μm2) as 113⁢S1.26 (r2=0.99). (c) Relationship between the total (inner + outer) surface area of mitochondria and cell volume for all species with available data. Open points are extrapolations for species with only outer membrane measures, derived by assuming an inner:outer ratio of 4.6, the average of observations in other species. References to individual data points are provided in Appendix 1–tables 1 and 2. https://doi.org/10.7554/eLife.20437.003 Three additional observations raise questions as to whether membrane surface area is a limiting factor in ATP synthesis. First, the localization of mitochondrial ATP synthase complexes is restricted to two rows on the narrow edges of the inner cristae (Kühlbrandt, 2015). Because this confined region comprises <<10% of the total internal membrane area, the surface area of mitochondrial membranes allocated to ATP synthase appears to be less than the surface area of the cell itself. Second, only a fraction of bacterial membranes appears to be allocated to bioenergetic functions (Magalon and Alberge, 2016), again shedding doubt on whether membrane area is a limiting factor for energy production. Third, in every bacterial species for which data are available, growth in cell volume is close to exponential, that is, the growth rate of a cell increases as its cell volume increases despite the reduction in the surface area:volume ratio (Voorn and Koppes, 1998; Godin et al., 2010; Santi et al., 2013; Iyer-Biswas et al., 2014; Osella et al., 2014; Campos et al., 2014). Further insight into this issue can be achieved by considering the average packing density of ATP synthase for the few species with proteomic data sufficient for single-cell counts of individual proteins. By accounting for the stoichiometry of the various subunits in the complex, it is possible to obtain several independent estimates of the total number of complexes per cell under the assumption that all the proteins are assembled (Supplementary material). For example, the estimated number of complexes in E. coli is 3018, and the surface area of the cell is ~15.8 μm2. Based on the largest diameter of the molecule (the F1 subcomplex), a single ATP synthase in this species occupies ~64 nm2 (Lücken et al., 1990) of surface area, so the total set of complexes occupies ~1.8% of the cell membrane. Four other diverse bacterial species for which these analyses can be performed yield occupancies ranging from 0.6% to 1.5%, for an overall average of 1.1% for bacteria. This will be an overestimate if only a fraction of proteins are properly assembled and embedded in the cell membrane. This kind of analysis can be extended to eukaryotes, noting that eukaryotic ATP synthases are slightly larger, with maximum surface area of ~110 nm2 (Abrahams et al., 1994; Stock et al., 1999). Although ATP synthase resides in mitochondria in eukaryotes, it is relevant to evaluate the fractional area that would be occupied were they to be located in the cell membrane. Such hypothetical packing densities are 5.0% and 6.6%, respectively, for the yeasts S. cerevisiae and S. pombe, and 6.6% and 6.8% for mouse fibroblasts and human HeLa cells. Although these observations suggest a ~5 fold increase in ATP synthase abundance with cell surface area in eukaryotes, the data conform to a continuous allometric function with no dichotomous break between the bacteria and eukaryotes (Figure 1b). Similar conclusions can be reached regarding the ETCs, although direct comparisons are more difficult due to the diversity of electron transport chain complexes in prokaryotes (Price and Driessen, 2010). The number of ETC complexes is comparable to that of ATP synthases in both bacteria and eukaryotes (Supplementary Material), and the physical footprint of the ETC is ~5× that of F0F1 (~570 nm2; Dudkina et al., 2011), implying that an average of ~5.5% of bacterial cell membranes is dedicated to the ETC and that the corresponding hypothetical packing density for eukaryotes would be ~30% (if in the cell membrane). There are a number of uncertainties in these packing-density estimates, and more direct estimates are desirable. The optimum and maximum-possible packing densities for ATP synthase also remain unclear. Nonetheless, the fact remains that any packing problems that exist for the cell membrane are also relevant to mitochondrial membranes, which have additional protein components (such as those involved in internal-membrane folding and transport into and out of the mitochondrion). The biosynthetic cost of lipids Any attempt to determine the implications of membranes for cellular evolution must account for the high biosynthetic costs of lipid molecules. There are two ways to quantify such a cost. First, from an evolutionary perspective, the cost of synthesizing a molecule is taken to be the sum of the direct use of ATP in the biosynthetic pathway plus the indirect loss of ATP resulting from the use of metabolic precursors that would otherwise be converted to ATP and available for alternative cellular functions (Akashi and Gojobori, 2002; Lynch and Marinov, 2015). Second, to simply quantify the direct contribution to a cell's total ATP requirement, the costs of diverting metabolic precursors are ignored. By summing the total costs of all molecules underlying a cellular feature and scaling by the lifetime energy expenditure of the cell, one obtains a measure of the relative drain on the cell's energy budget associated with building and maintaining the trait. This measurement, sc, can then be viewed as the fractional increase in the cell's energy budget that could be allocated to growth, reproduction, and survival in the absence of such an investment, ignoring the direct fitness benefits of expressing the trait, sa. For selection to be effective, the net selective advantage of the trait, sn=sa-sc, must exceed the power of random genetic drift, 1/Ne in a haploid species and 1/(2⁢Ne) in a diploid, where Ne is the effective population size. Most cellular membranes are predominantly comprised of glycerophospholipids, which despite containing a variety of head groups (e.g. glycerol, choline, serine, and inositol), all have total biosynthetic costs per molecule (in units of ATP hydrolyses, and including the cost of diverting intermediate metabolites) of (2a)cL≃320+[38⋅(NL−16)]+(6⋅NU),(2b)cL≃340+[40⋅(NL−16)]+(6⋅NU), in bacteria and eukaryotes, respectively, where NL is the mean fatty-acid chain length, and NU is the mean number of unsaturated carbons per fatty-acid chain (Supplementary material). Although variants on glycerophospholipids are utilized in a variety of species (Guschina and Harwood, 2006; Geiger et al., 2010), these are structurally similar enough that the preceding expressions should provide The which the loss of from the of metabolic is in bacteria and eukaryotes, respectively. From the of a cell's total energy the evolutionary cost of a lipid molecule is For most lipids in membranes, and so the cost per lipid molecule is generally in the range of to although the average over all lipids deployed in membranes is much which more than of membrane lipids is having an evolutionary cost of a cost of To these expenditures into perspective, the evolutionary biosynthetic costs of of the four is ATP per molecule (Lynch and Marinov, whereas the average cost of an is ATP (Atkinson, and Gojobori, of the preceding expressions to the known membrane of cells that the biosynthetic costs of eukaryotic lipids are than those in bacteria (Supplementary For example, for a diversity of bacterial species the average direct cost per lipid molecule in the plasma membrane is (SE = whereas that for eukaryotes is The is to the mean for eukaryotic but the cost of mitochondrial lipids is in eukaryotes are of the cost of mitochondrial of to and the for eukaryotic lipids to have containing more To the total bioenergetic cost associated with membranes, we information on the of lipid molecules required for membrane which is to the total surface area of the membrane by the number of lipid surface area, and by two to account for the lipid of the areas of membrane lipids are within of an average of et al., et al., 2011), so the cost of a membrane (in units of and ignoring lipid turnover and the occupied by is where A is the membrane surface area in units of and is the average cost of a information is available on the total investment in mitochondrial membranes that a can be Over the eukaryotic the total surface area of mitochondria (inner plus outer membranes, over all mitochondria, in μm2) scales with cell volume in units of as (Figure SEs of and on are and this to Equation with the average total cost of mitochondrial lipids Appendix and using the for the total growth requirements of a cell, Equation (1b), the relative cost of mitochondrial membrane lipids is or of the total growth budget of a eukaryotic cell, and independent of cell within the range typically in eukaryotes (SE of the exponent is The direct contribution of mitochondrial membrane lipids to a cell's growth budget is of this total cost. costs of mitochondrial membranes a not by prokaryotes, associated with bioenergetics to the of eukaryotic that is, the additional costs of maintenance of mitochondrial lipids is unknown, but for the of a cell's energy budget is allocated to growth (Lynch and Marinov, so the costs should as for the costs will be or lower on whether the cost of maintenance is or that for total cellular do not of membranes, so accounting for this would the preceding results by a factor For cells internal membranes, the relative contribution of the cell membrane to a cell's total energy budget is expected to decline with increasing cell to the decline in the surface area to volume For the cells of and of and and of a cell's growth budget must be allocated to the plasma membrane, but for the larger and coli and these to and and they would be expected to to decline with increases in cell scaling with the of the cell. In contrast, to the investment in internal membranes, the fraction of a eukaryotic cell's energy budget to membranes does not with increasing cell size. Although there are only a few eukaryotic cell types for which this issue can be the data three orders of magnitude in cell volume and suggest that to of the total growth budget is allocated to lipid and that an increasing fraction of such costs is associated with internal membranes in cells of increasing size. The alga which has a cell volume of just that of many of its energy budget to membranes, and of these costs of the total cell are associated with internal membranes. A cell a similar ~30% of its energy budget to membranes, but of these costs of the total cell are associated with internal membranes. 1 of membranes to total cellular growth the green alga the the green alga and the in Cell and total membrane areas are in units of and respectively, with the membranes associated with the in the species. The fraction of the total cell growth budget allocated to membranes is by the ratio of Equations (1b) and using the costs in 1 where available, and otherwise the for eukaryotic this total cost is then into different fractional in the of cell growth + these observations that the use of internal membranes a drain on the total energy of eukaryotic much more than would be expected in bacteria of comparable size. Moreover, the lipids associated with mitochondria to of a eukaryotic cell's investment in membranes the energetic of membrane bioenergetics to mitochondria is that the observations in Figure are derived from a diversity of with many it is considering whether the overall conclusions are consistent with the known capacity of ATP First, it noting that only a fraction of the energy invested in is derived directly from the chemiosmotic of ATP For example, of and for every ATP (Akashi and Gojobori, that of the former is to ATP hydrolyses, this implies that only of the energy invested in ATP in the the ratio of use of to ATP is more on the order of in lipid the direct investment in ATP to Thus, as the of the energetic cost of building a cell is associated with synthesis of the building of proteins and membranes, only of biosynthetic energy may be derived from ATP the known energy requirements for the maintenance and growth of a cell, the cell-division time, and the number of ATP synthase complexes per cell, it is possible to the required rate of ADP ATP per the cellular energetic data (Lynch and Marinov, 2015) and the ATP synthase in Appendix the maximum by the estimated required rates of ATP and respectively for the bacteria E. and and and for the yeasts S. cerevisiae and S. have been to the maximum turnover rates for F0F1 ATP usually in and these average in bacteria et al., and et al., 2007), in and and in S. cerevisiae et al., 2010), and in and Thus, that a substantial fraction of complexes are to be in membranes, the based estimates of the of ATP turnovers per cell to be consistent with the known capacity of ATP The cellular investment in The of a cell a of its bioenergetic to the large number of proteins required to the complex, are and the number per cell appears to be universally with cellular growth rate and Tempest et al., and and 1974; and 1974; et al., 1975; and 1975; and 1975; et al., 2010). out that the total and mean number of per scale with cell volume as and respectively, and that the are and for with no dichotomous break between prokaryotes and eukaryotes (Lynch and Marinov, 2015). with the they process and the proteins they the of per cell also to scale with cell volume, in a