Alerts User Alerts. You will receive an email whenever this article is corrected, updated, or cited in the literature. You can manage this and all other alerts in My Account. This feature is available to authenticated users only.
Get Citation Citation. Get Permissions. Aging of post-mitotic tissues is associated with a continuous decrease of the mitochondrial capacity to produce ATP by oxidative phosphorylation resulting in a general decline of vital cellular functions.
Thus, decreased ATP production and enhanced oxidative stress are major triggers of senescent dysfunction of long-lived post-mitotic cells, such as neurons, cardiac myocytes, skeletal muscle fibers and RPE.
The RPE fulfills metabolic functions that are essential for proper action and survival of retinal photoreceptors. These vital functions include maintenance of the visual cycle by continuous uptake, processing, transport, and release of vitamin A; generation of ion gradients within the photoreceptor matrix; mediation of active transport of nutrients between the choroid and the photoreceptors; formation of the outer blood brain barrier; phagocytic uptake; and degradation of the constantly shed photoreceptor outer segments.
He et al. As a consequence of electron leakage from the electron transport chain reactive oxygen species ROS , including the superoxide anion radical and hydroxyl radical, form within mitochondria during normal respiration, thereby initiating oxidative damage to mitochondrial proteins, nucleic acids, and lipids.
Thus, RPE cells are at permanent risk for oxidative damage due to their location in the highly oxygenated environment of the outer retina and their exposure to light.
Although lysosomal dysfunction causing buildup of undegradable material seems to be the initial incident, 11 — 13 the accumulation of waste material finally results in the formation of lipofuscin granules that are highly phototoxic by generating ROS upon illumination. In aging cells, oxidative damage is fostered by decreasing activities of antioxidant enzymes, including dismutases and catalases and by a decline of intracellular low molecular weight scavengers of oxidizing species.
Reduced glutathione represents the most abundant and most important intracellular antioxidant. Consequently, a high intracellular level of its reduced form is considered as a marker of cells that are well shielded from severe oxidative damage, whereas vast consumption of reduced glutathione is a hallmark of cells experiencing severe oxidative stress.
Mitochondrial dysfunction, reducing ATP generation, is linked to glutathione deficiency by the Gibbs free energy equation. Thus, age-related reduction of intracellular ATP levels should result in an equivalent decrease of reduced glutathione levels thereby pushing the aging process.
In the present study we applied atractyloside for inhibition of mitochondrial ATP synthesis to test the effects of moderate ATP depletion in cultured human RPE cells on cellular oxidative stress response, autophagy and related anti-aging mechanisms, and photoreceptor outer segment phagocytosis as an example for a vital RPE function.
Although these most abundant retinopathies occur by different multifactorial mechanisms, reduced ATP levels and the resulting adverse effects addressed in our study may be common contributors to the etiologies of both disorders. All subjects were treated in accordance with the Declaration of Helsinki. Intracellular ATP was determined by bioluminescent luciferase assay, including thermostable firefly luciferase and luciferin substrate.
Reduced and total glutathione were determined with a commercial glutathione assay kit Biovision, Mountain View, CA. Lactate in conditioned medium was assayed according to Bergmeyer. Turnover of endogenous proteins was measured in pulse-chase experiments as described previously. After removal of the radioactive medium, the cell layers were washed and chased with nonradioactive medium chase phase.
Protein degradation was determined by measurement of the low-molecular TCA-soluble radioactivity released into the medium. Therefore, the effect of this compound on intracellular protein turnover can be taken as a measure of autophagic sequestration. Autophagic protein degradation was calculated by subtracting degradation rates in the presence from the rates observed in absence of 3-methyladenine. Thus, lysosomal protein degradation ammonia-sensitive degradation was calculated by subtracting degradation rates in the presence of NH 4 Cl from the rates observed in absence of NH 4 Cl.
POS was isolated from pig eyes obtained from a local slaughter house according to the method of Schraermeyer et al. The anterior half of the eye was dissected, the vitreous and retina removed. Isolated retina were agitated in KCl-buffer 0. The supernatant containing POS was filtered through gauze and diluted with KCl-buffer and centrifuged at rpm for 7 minutes. Radiolabeling was stopped by removal of the iodination beads. RPE cultures were grown to confluence on well tissue culture plates and maintained for 4 weeks.
Then 80 kBq I-labeled POS were added to each well and incubated for 6, 12, 18, or 24 hours and phagocytosis and degradation rates were assayed according to a published method. The following calculations were made:. The amount of lactate detected in the conditioned medium after three days of culture was 2. Thus, the cells are able to partially compensate for the atractyloside-induced reduction of mitochondrial ATP supply by nonmitochondrial ATP generation. In addition to moderately reduced cellular ATP levels, we intended to induce conditions of increased oxidative stress.
To this end, the cells were treated with tert-butyl hydroperoxide tBH. Neither treatment with atractyloside or tBH alone nor the combination of both induced significant changes in the morphology of the cells data not shown.
No increase of LDH leakage to the culture medium of the treated cell was observed, excluding acute cell death during the experiments. However, when ATP levels were reduced according to the conditions described above, a striking decrease of reduced glutathione was observed upon tBH treatment. In order to test whether these conditions of elevated oxidative stress may cause harm to cellular proteins, the amount of malondialdehyde MDA modifications on cellular protein was tested by ELISA.
MDA is a widely accepted marker for protein damage caused by oxidative stress. During oxidative stress, MDA is generated as a lipid peroxidation byproduct that covalently attaches to cellular proteins. However, when tBH, an inducer of oxidative stress, was included in the culture medium to induce conditions of elevated oxidative stress, an increase of protein damage was observed in cells with reduced ATP levels, whereas the amount of MDA modification was only moderately elevated in cultures with regular ATP levels Fig.
Again, 8OHdG was not elevated above the detection limit of 2. A major mechanism in post-mitotic cells to prevent aging and oxidative damage of cellular biomolecules and structures is autophagy. Since autophagy is a highly energy-dependent process, we assessed the effects of atractyloside-induced reduction of intracellular ATP levels on intracellular autophagic degradation rates by measuring 3-methyladenine-sensitive protein degradation after labeling intracellular proteins with 3H-leucine.
In the atractyloside-treated cells autophagic activity was approximately 3-fold lower as compared with the untreated controls Fig. For comparison, also ammonia-sensitive degradation total lysosomal degradation is shown. The differences between ammonia-sensitive and 3MA-sensitive degradation indicate the contribution of nonautophagic lysosomal degradation, which appears relatively small and not affected by the decreased ATP level.
However, phagocytic capacity for uptake and degradation of photoreceptor outer segments was reduced in cells with lowered ATP-levels Fig. Figure 1. View Original Download Slide. RPE cells were grown to confluence in well plates. Figure 2. Cells were treated with atractyloside as described in Figure 1 and total and reduced glutathione was assayed after homogenization of the cells at the indicated time points by ELISA. The percentage of reduced glutathione as compared to total glutathione is shown.
Figure 3. Cells were treated with atractyloside as described in Figure 2. Oxidative stress was induced by adding tBH to the culture medium. Figure 4. Cells were treated with tBH as described in Figure 3. Figure 5. Figure 6. This will be an overestimate if only a fraction of proteins are properly assembled and embedded in the cell membrane. 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. 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, 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.
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, ; Lynch and Marinov, Most cellular membranes are predominantly comprised of glycerophospholipids, which despite containing a variety of head groups e.
Although variants on glycerophospholipids are utilized in a variety of species Guschina and Harwood, ; Geiger et al.
The reduced direct cost, which ignores the loss of ATP-generating potential from the diversion of metabolic precursors, is. Application of the preceding expressions to the known membrane compositions of cells indicates that the biosynthetic costs of eukaryotic lipids are higher than those in bacteria Supplementary table.
The latter estimate is identical to the mean obtained for whole eukaryotic cells, but the cost of mitochondrial lipids is especially high, 5. These elevated expenses in eukaryotes are joint effects of the cost of mitochondrial export of oxaloacetate to generate acetyl-CoA and the tendency for eukaryotic lipids to have longer chains containing more desaturated carbons. Enough information is available on the total investment in mitochondrial membranes that a general statement can be made.
For the tiny cells of Leptospira interrogans and Mycoplasma pneumoniae average volumes of 0. The picoplanktonic alga Ostreococcus , which has a cell volume of just 0. Ot denotes the green alga Ostreococcus tauri , Sc the yeast Saccharomyces cerevisiae , Ds the green alga Dunaliella salina , and Ss the pig Sus scrofa pancreas cell; references given in Supplementary material. The fraction of the total cell growth budget allocated to membranes is obtained by the ratio of Equations 1b and 4 , using the species-specific reduced costs in Table 1 where available, and otherwise applying the averages for eukaryotic species; this total cost is then apportioned into five different fractional contributions in the following lines.
Taken together, these observations imply that the use of internal membranes constitutes a major drain on the total energy budgets of eukaryotic cells, much more than would be expected in bacteria of comparable size. Finally, given that the observations summarized in Figure 1a,b are derived from a diversity of studies, likely with many unique inaccuracies, it is worth considering whether the overall conclusions are consistent with the known capacity of ATP synthase.
First, it bears noting that only a fraction of the energy invested in biosynthesis is derived directly from the chemiosmotic activity of ATP synthase. Thus, given that a substantial fraction of complexes are likely to be misassembled in artificial membranes, the energy-budget based estimates of the numbers of ATP turnovers generated per cell appear to be consistent with the known capacity of ATP synthase.
The ribosome content of a cell provides a strong indicator of its bioenergetic capacity. Owing to the large number of proteins required to build the complex, ribosomes are energetically costly, and the number per cell appears to be universally correlated with cellular growth rate Fraenkel and Neidhardt, ; Tempest et al.
We previously pointed out that the genome-wide total and mean number of transcripts per gene scale with cell volume as V 0. As with the transcripts they process and the proteins they produce, the numbers of ribosomes per cell also appear to scale sublinearly with cell volume, in a continuous fashion across bacteria, unicellular eukaryotes, and cells derived from multicellular species Figure 2. These observations are inconsistent with the idea that entry into the eukaryotic world resulted in an elevated rate of protein production.
Moreover, as noted previously Lynch and Marinov, , the absolute costs of producing individual proteins and maintaining the genes associated with them are substantially higher in eukaryotes than in bacteria, owing to the substantial increase in gene lengths, investment in nucleosomes, etc. Color coding as in previous figures. The data presented in this figure can be found in Figure 2—source data 1 ; see also Appendix 1—table 3. However, the scaling of the costs of building and maintaining cells is inconsistent with an abrupt shift in volumetric bioenergetic capacity of eukaryotic cells, and although the absolute costs of biosynthesis, maintenance, and operation of individual genes are much greater in eukaryotes, the proportional costs are less Lynch and Marinov, This means that evolutionary additions and modifications of genes are more easily accrued in eukaryotic genomes from a bioenergetics perspective, regardless of their downstream fitness effects.
The analyses presented here reveal a number of additional scaling features involving cellular bioenergetic capacity that appear to transcend the substantial morphological differences across the bacterial-eukaryotic divide. There is not a quantum leap in the surface area of bioenergetic membranes exploited in eukaryotes relative to what would be possible on the cell surface alone, nor is the idea that ATP synthesis is limited by total membrane surface area supported.
Although there is considerable room for further comparative analyses in this area, when one additionally considers the substantial cost of building mitochondria, it is difficult to accept the idea that the establishment of the mitochondrion led to a major advance in net bioenergetic capacity.
Most discussion of the origin of the mitochondrion by endosymbiosis starts and often ends with a consideration of the benefits gained by the host cell. This ignores the fact that the eukaryotic consortium consists of two participants. At least initially, the establishment of a stable symbiotic relationship requires that each member of the pair gain as much from the association as is lost by relinquishing independence.
For such a consortium to be evolutionarily stable as a true mutualism, both partners would have to acquire more resources than would be possible by living alone, in which case this novel relationship would be more than the sum of its parts.
This scenario certainly applies today, as all mitochondria have relinquished virtually all genes for biosynthesis, replication, and maintenance, and as a consequence depend entirely on their host cells for these essential metabolic functions. In contrast, all eukaryotes have relocated membrane bioenergetics from the cell surface to mitochondrial membranes.
Such an outcome represents a possible grand example of the preservation of two ancestral components by complementary degenerative mutations Force et al. Notably, this process of subfunctionalization is most likely to proceed in relatively small populations because the end state is slightly deleterious from the standpoint of mutational vulnerability, owing to the fact that the original set of tasks becomes reliant on a larger set of genes Lynch et al.
Thus, a plausible scenario is that the full eukaryotic cell plan emerged at least in part by initially nonadaptive processes made possible by a very strong and prolonged population bottleneck Lynch, ; Koonin, The origin of the mitochondrion was a singular event, and we may never know with certainty the early mechanisms involved in its establishment, nor the order of prior or subsequent events in the establishment of other eukaryotic cellular features Koonin, However, the preceding observations suggest that if there was an energetic boost associated with the earliest stages of mitochondrial colonization, this has subsequently been offset by the loss of the use of the eukaryotic cell surface for bioenergetics and the resultant increase in costs associated with the construction of internal membranes.
Rather than a major bioenergetic revolution being provoked by the origin of the mitochondrion, at best a zero-sum game is implied. Thus, if the establishment of the mitochondrion was a key innovation in the adaptive radiation of eukaryotes, the causal connection does not appear to involve a boost in energy acquisition.
It is plausible, that phagocytosis was a late-comer in this series of events, made possible only after the movement of membrane bioenergetics to the mitochondrion, which would have eliminated the presumably disruptive effects of ingesting surface membranes containing the ETC and ATP synthase. The results in this paper are derived from an integration of bioenergetic analyses based on known biochemical pathways and existing morphological observations on a variety of cell-biological features.
The sources of this information, as well as the basic approaches employed can be found in the Appendix where not mentioned directly in the text. The central analyses involve: 1 estimation of the biosynthetic costs for lipid-molecule production in terms of ATP equivalents per molecule produced ; 2 mitochondrial surface areas and cell membrane areas; 3 investments in lipids at the cell-membrane and organelle levels; and 4 numbers of ATP synthase complexes, ETC complexes, and ribosomes per cell.
The vast majority of lipids in most membranes are phospholipids, with a polar hydrophilic head group attached to a negatively charged phosphate, which in turn is attached to a glycerolphosphate G3P , which links to two fatty-acid chains.
Common head groups are choline, ethanolamine, serine, glycerol, inositol, and phosphatidyl glycerol. In both bacteria and eukaryotes, fatty-acid chains usually contain 12 to 22 carbons, and only rarely harbor more than three unsaturated bonds. Evaluation of the total cost of synthesizing a lipid molecule requires a separate consideration of the investments in the three molecular subcomponents: the fatty-acid tails; head groups; and linkers.
As adhered to in Lynch and Marinov , such costs will be quantified in units of ATP usage, specifically relying on the number of phosphorus atoms released via hydrolyses of ATP molecules, the primary source of energy in most endergonic cellular reactions.
The following results are derived from observations cataloged in most biochemistry text books:. The starting point for the synthesis of most fatty acids is the production of one particular linear chain, palmitate, which contains 16 carbon atoms. Synthesis of this molecule takes place within a large complex, known as fatty-acid synthase. Each molecule of acetyl-CoA is generally derived from a pyruvate molecule, but each acetyl-CoA molecule diverted to lipid production deprives the cell of one rotation of the energy producing citric-acid cycle, which would otherwise yield 3 NADH, 1 FADH 2 , and 1 ATP per rotation; this leads to a net loss to the cell of the equivalent of 12 ATPs per acetyl-CoA molecule.
Fatty-acid production is slightly more expensive in nonphotosynthetic eukaryotes, where acetyl-CoA is produced in the mitochondrion and reacts with oxaloacetate to produce citrate, which must then be exported. Cleavage of oxaloacetate in the cytosol regenerates acetyl-CoA at the expense of 1 ATP, and a series of reactions serve to return oxaloacetate to the citric-acid cycle in an effectively ATP neutral way.
Each additional pair of carbons added to the palmitate chain requires one additional acetyl-CoA, one additional ATP, and two additional NADPHs, or an equivalent of 19 ATPs in bacteria, and accounting for mitochondrial export increases this to 20 in eukaryotes.
The G3P linker emerges from one of the last steps in glycolysis, and its diversion to lipid production deprives the cell of one further step of ATP production as well as a subsequent pyruvate molecule. All that remains now is to add in the cost of synthesis of the head group, which we do here still assuming 16 saturated bonds in each fatty acid.
From Akashi and Gojobori , the cost of a serine is 10 ATP, so the total cost of a phosphatidylserine is ATP, and because ethanolamine and choline are simple derivatives of serine, this closely approximates the costs of both phosphatidylethanolamine and phosphatidylcholine. The headgroup of phosphatidylinositol is inosital, which is derived from glucosephosphate, diverting the latter from glycolysis and depriving the cell of the equivalent of 9 ATPs, so the total cost of production of this molecule is ATP.
As a first-order approximation, we will assume all of the above molecules to have a cost of ATP when containing fully saturated fatty acids with chain length Finally, cardiolipin is synthesized by the fusion of two phosphatidylglycerols and the release of one glycerol, so taking the return from the latter to be 15 ATP, the total cost per molecule produced is ATP.
Information on absolute protein copy numbers per cell was collected from publicly available proteomics measurements Lu et al. The number of protein complexes N P C was calculated as follows:. Clear outliers i. As proteomics measurements may not be absolutely reliable, the raw estimates N P C , r a w were then further corrected where possible by taking advantage of the availability of direct counts of the number of ribosomes per cell:.
The composition of the E. The F O -particle has 10 copies of subunit 9 equivalent to c , and one copy each of subunits 6 equivalent to a , 8, 4 equivalent to b , d , h , f , e , g , i and k , where the individual subunits are encoded by the following genes:.
The F O -particle has 8 copies of subunit c , and one copy each of subunits a , 8, b , d , F 6 , f , e , and g , where the individual subunits are encoded by the following genes:. A Nonreduced costs including opportunity cost of precursors; B Reduced costs without precursors. Amino acid values are obtained from Akashi and Gojobori , assuming growth on glucose.
Cell volumes are from Lynch and Marinov , in some cases supplemented with additional references from the literature. ATP synthase surface area assumed to be maximum associated with the inner ring, 6. Direct estimates taken from microscopic examinations; proteomic estimates are from averaging of cell-specific estimates for each ribosomal protein subunit. See Figure 2—source data 1 for further details. The average cost per molecule is calculated for a variety of species using estimates of lipid compositions from the literature and the formulas described in the text.
The fraction of fatty acids of given length and saturation level is not shown. Cardiolipin costs are assumed to be evolutionary and reduced ATP. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Competing interests No competing interests declared. Author contributions Conceptualization, Data curation, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing.
Data curation, Formal analysis, Investigation, Methodology, Writing—original draft. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Paul Falkowski as the Reviewing Editor and Patricia Wittkopp as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Ron Milo Reviewer 2.
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Summary: Both reviewers identified many strengths of this work, but also have identified additional elements to consider. I hope you find their detailed and constructive reviews helpful. We anticipate that this work will be an important contribution to the field that will spark additional discussion and debate.
Essential revisions: Both reviewers have provided detailed reviews of this manuscript, and we believe that considering all of their comments will be beneficial in this case. These comments are provided in their entirety below. The most essential comment from the reviewers that must be addressed is: The "possibility that protein packing density in the membranes under consideration is a fundamental limitation needs to be taken into account.
This is an interesting analysis of the relative bioenergetic characteristics in the growth of prokaryotic and eukaryotic cells. The article addresses the basic conjecture, that the evolution of the eukaryotic type, specifically the development of mitochondrial systems, endowed the eukaryotes with energetic advantages over the prokaryotic cellular organization.
The authors are challenging this often-made, yet largely unsubstantiated, assumption that the presence of mitochondria in eukaryotes confers a large bioenergetic advantage owing to a corresponding increase in internal membrane surface area due to the presence of the mitochondrial inner membrane. To address this question, the authors perform an analysis based upon previous scaling relationships they have developed between quantities such as the volume of a cell and the rates of ATP consumption and combined these with a new analysis that includes protein and lipid abundances combined with estimations, from the literature, of their costs as expressed in terms of ATP equivalents.
The authors note that the energetics of the cell can be divided into maintenance costs and the costs of duplicating the parental cell and their analysis goes on from there. Basically, they are concluding that if there ever was an energetic advantage e. Overall, I think the article is sound, albeit, it is difficult for this reviewer to critically assess the validity of their calculations, which on the surface seems sound. On the other hand, the article is written in a with the tenor of a polemic and is a bit rambling.
It may be true that the bioenergetic machinery responsible for ATP production only occupies several percent of the total area, but this may be the upper limit for the bioenergetic system reflecting and optimal allocation of different protein functions, such as transporters, also necessary for metabolism. Presumably, the other mitochondrial components especially are present in an optimal stoichiometric ratio with respect to the ATP synthase and may indeed occupy much more of the membrane area.
For example, if the ATP synthase has an intrinsically higher enzymatic turnover frequency than the enzymes powering the generation of proton motive force, then it's amount can be comparatively small on a stoichiometric basis and the other membrane complexes may occupy a large fraction of the membrane surface. The authors revisit the hypothesis that the mitochondria were essential for the development of eukaryotic complexity for energetic reasons.
The authors thoroughly analyze the ATP and other investments as performed by current eukaryotic cells and compare them to prokaryotes. They use empirical scaling laws to see if the observed changes are more than one would expect from simple scaling with cell volume. They find no strong evidence for a significant energetic benefit from mitochondria which leads them to cast doubt on high profile earlier reports. I find the study scientifically sound and interesting.
I have suggestions for improvement in terms of clarity and accuracy as given below. Main text, third paragraph: "This implies that the mitochondrion-host cell consortium that became the primordial eukaryote did not precipitate a bioenergetics revolution.
In order to say it did not cause a bioenergetics revolution I need to have a definition of what is the definition such a revolution in as rigorous terms as possible. Either by the authors or by them repeating in detail a definition from previous authors. Throughout the paper the scaling laws have no uncertainty ranges on their parameter values. This makes it hard to understand how predictive they are and should be corrected.
The authors do not seem to reflect more on this value they derive but it seems like a very high value to me. The volume of an E. Discussion, fifth paragraph: "because the end state is slightly deleterious owing to the additional investment required to carry out individual tasks Lynch et al. I found it hard to follow the logic here and I think other readers might have this problem.
It is worth explaining in a bit more detail what is meant. Discussion, last paragraph: "It is plausible, that phagocytosis was a late-comer in this series of events, made possible only after the movement of membrane bioenergetics to the mitochondrion, which would have eliminated the disruptive effects of surface membrane ingestion on the ETC and ATP synthase. Thank you for pointing this out; done.
Fully admit to not having read this before, and it is remarkable how similar his results are to those of Akashi and Gojobori. Although he did not deal with lipids to any great extent, the little he did seems to be compatible with our calculations, so that is gratifying as well. Our point is already that there is a slowdown in the growth rate of bacterial cells at the low end of the size range. We are less clear as to what species the reviewer is referring to at the large end, as we attempted to perform as thorough and as unbiased a survey as possible; we have emphasized that there if a broad range around the general pattern.
As noted below, in response to the second review, we have acknowledged the uncertainties in this area, but also note that protein packing issues will also apply to internal mitochondrial membranes and perhaps even more so, owing to the need for proteins involved in the maintenance membrane folding. Thus, because there is not a dramatic increase in mitochondrial membrane area relative to that of the cell surface, the packing uncertainty does not seem to weaken our general conclusion that eukaryotes have not experienced a major increase in bioenergetics capacity relative to prokaryotes.
Moreover, our goal throughout the paper has been to bring as many additional and independent lines of evidence to bear on this conclusion as possible — the smooth scaling of bioenergetics growth and maintenance requirements across the prokaryotic-eukaryotic divide, as well as the scaling of numbers of ATP synthase complexes and ribosomes, all support our general conclusion; and the substantial additional costs of building internal membranes in eukaryotic cells does as well.
The statements we have made are based on many made the Lane books, and also paraphrase the claims in the Lane and Martin paper. Given the quotes we provide from the Lane and Martin paper below, it seems unlikely that any reader would find that we are overstating the claims being made.
The source of their repeated statements about a ,fold expansion in genes and genome size eludes us, and makes no sense :. This vast leap in genomic capacity was strictly dependent on mitochondrial power, and prerequisite to eukaryote complexity: the key innovation en route to multicellular life. Mitochondria increased the number of proteins that a cell can evolve, inherit and express by four to six orders of magnitude, but this requires mitochondrial DNA.
The possession of mitochondria enabled eukaryotes to tunnel through this mountainous energetic barrier. Mitochondria allowed their host to evolve, explore and express ,fold more genes with no energetic penalty. If evolution works like a tinkerer, evolution with mitochondria works like a corps of engineers. This is a good point that we had not made clear enough, so we now have added a sentence to this paragraph to make the SA:V expectation explicit.
We do not think that these uncertainties upset our general conclusions, as the more general and compelling evidence derives from the absolute surface areas of the cell vs. 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.
In general, we have reduced the numbers of digits used throughout, with no resultant changes in the conclusions. We have tried to word this in a clearer way — the basic issue is that a cell would have a difficult time maintaining cell-membrane bioenergetics if the membrane and its resident ATP synthases was constantly being ingested.
National Center for Biotechnology Information , U. Journal List eLife v. Published online Mar Michael Lynch and Georgi K Marinov. Author information Article notes Copyright and License information Disclaimer. Michael Lynch: ude. Received Aug 7; Accepted Jan This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.
This article has been corrected. See eLife. This article has been cited by other articles in PMC. Research organism: E. 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. 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.
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, Open in a separate window.
Figure 1. Scaling features of membrane properties with cell size. 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. Table 1. Contributions of membranes to total cellular growth costs. The cellular investment in ribosomes The ribosome content of a cell provides a strong indicator of its bioenergetic capacity. Figure 2.
Figure 2—source data 1. Source data for Figure 2. Click here to view. Materials and methods The results in this paper are derived from an integration of bioenergetic analyses based on known biochemical pathways and existing morphological observations on a variety of cell-biological features.
Appendix The biosynthetic costs of lipid molecules The vast majority of lipids in most membranes are phospholipids, with a polar hydrophilic head group attached to a negatively charged phosphate, which in turn is attached to a glycerolphosphate G3P , which links to two fatty-acid chains. The following results are derived from observations cataloged in most biochemistry text books: The starting point for the synthesis of most fatty acids is the production of one particular linear chain, palmitate, which contains 16 carbon atoms.
Estimation of absolute protein copy numbers per cell Information on absolute protein copy numbers per cell was collected from publicly available proteomics measurements Lu et al. Appendix 1—figure 1. Appendix 1—table 1. Features of mitochondrial membranes. Article Google Scholar. Biochem Biophys Res Commun. Protein Sci. Ann N Y Acad Sci.
Download references. We thank Dr. Charles M. Rice for providing the Con1 cells, Dr. King-Song Jeng for providing the 1. George G. Brownlee for providing 12 plasmids to generate influenza A virus.
Shih-Yen Lo. You can also search for this author in PubMed Google Scholar. Correspondence to Shih-Yen Lo. All authors read and approved the final manuscript. Additional file 1: Supplementary Fig. DNA samples in different lanes represent the outcomes of different sets of primers for PCR only four sets of primers out of 20 are shown. Additional file 2: Supplementary Fig. Additional file 3: Supplementary Fig. Western blotting was performed 24 hr after oligomycin treatment.
Erk-2 protein was used as a loading control. Additional file 4: Supplementary Fig. Experiments were performed in duplicate. The ATP level was reduced to Additional file 5: Supplementary Fig. Additional file 6: Supplementary Fig.
HeLa cells were transfected with expression vector only pcDNA3. Cell lysates were analyzed by Western blot 48 hr after transfection. The cell lysate from HeLa cells 28 hr after vaccinia virus infection was used as a positive control, and ERK2 protein was used as a loading control.
Reprints and Permissions. Chang, CW. Increased ATP generation in the host cell is required for efficient vaccinia virus production. J Biomed Sci 16, 80 Download citation. Received : 25 December Accepted : 02 September Published : 02 September Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative.
Skip to main content. Search all BMC articles Search. Download PDF. Background Vaccinia virus VV , a member of the Poxviridae family, is an enveloped, DNA virus with a genome of kb encoding about proteins [ 1 ]. Virus infection Vaccinia virus WR strain was used to infect HeLa cells in this study, following previously published procedures for virus amplification and plaque assay [ 23 , 24 ].
Western blotting analysis Our previous procedures were followed for Western blotting analysis [ 27 , 28 ]. Figure 1. Full size image. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. References 1.
0コメント