Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Evolution of increased complexity in a molecular machine

Abstract

Many cellular processes are carried out by molecular ‘machines’—assemblies of multiple differentiated proteins that physically interact to execute biological functions1,2,3,4,5,6,7,8. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection9,10,11 and manipulative genetic experiments to determine how the complexity of an essential molecular machine—the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump—increased hundreds of millions of years ago. We show that the ring of Fungi, which is composed of three paralogous proteins, evolved from a more ancient two-paralogue complex because of a gene duplication that was followed by loss in each daughter copy of specific interfaces by which it interacts with other ring proteins. These losses were complementary, so both copies became obligate components with restricted spatial roles in the complex. Reintroducing a single historical mutation from each paralogue lineage into the resurrected ancestral proteins is sufficient to recapitulate their asymmetric degeneration and trigger the requirement for the more elaborate three-component ring. Our experiments show that increased complexity in an essential molecular machine evolved because of simple, high-probability evolutionary processes, without the apparent evolution of novel functions. They point to a plausible mechanism for the evolution of complexity in other multi-paralogue protein complexes.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure and evolution of the V-ATPase complex.
Figure 2: Two reconstructed ancestral V 0 subunits functionally replace the three-paralogue ring in extant yeast.
Figure 3: Increasing complexity by complementary loss of interactions in the fungal V 0 ring.
Figure 4: Genetic basis for functional differentiation of Anc.3 and Anc.11.

Similar content being viewed by others

References

  1. Forgac, M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature Rev. Mol. Cell Biol. 8, 917–929 (2007)

    Article  CAS  Google Scholar 

  2. Pallen, M. J. & Matzke, N. J. From the origin of species to the origin of bacterial flagella. Nature Rev. Microbiol. 4, 784–790 (2006)

    Article  CAS  Google Scholar 

  3. Liu, R. & Ochman, H. Stepwise formation of the bacterial flagellar system. Proc. Natl Acad. Sci. USA 104, 7116–7121 (2007)

    Article  ADS  CAS  Google Scholar 

  4. Mulkidjanian, A. Y., Makarova, K. S., Galperin, M. Y. & Koonin, E. V. Inventing the dynamo machine: the evolution of the F-type and V-type ATPases. Nature Rev. Microbiol. 5, 892–899 (2007)

    Article  CAS  Google Scholar 

  5. Dolezal, P., Likic, V., Tachezy, J. & Lithgow, T. Evolution of the molecular machines for protein import into mitochondria. Science 313, 314–318 (2006)

    Article  ADS  CAS  Google Scholar 

  6. Clements, A. et al. The reducible complexity of a mitochondrial molecular machine. Proc. Natl Acad. Sci. USA 106, 15791–15795 (2009)

    Article  ADS  CAS  Google Scholar 

  7. Archibald, J. M., Logsdon, J. M., Jr & Doolittle, W. F. Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes. Mol. Biol. Evol. 17, 1456–1466 (2000)

    Article  CAS  Google Scholar 

  8. Gabaldón, T., Rainey, D. & Huynen, M. A. Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (complex I). J. Mol. Biol. 348, 857–870 (2005)

    Article  Google Scholar 

  9. Thornton, J. W. Resurrecting ancient genes: experimental analysis of extinct molecules. Nature Rev. Genet. 5, 366–375 (2004)

    Article  CAS  Google Scholar 

  10. Liberles, D., ed. Ancestral Sequence Reconstruction (Oxford Univ. Press, 2007)

  11. Harms, M. J. & Thornton, J. W. Analyzing protein structure and function using ancestral gene reconstruction. Curr. Opin. Struct. Biol. 20, 360–366 (2010)

    Article  CAS  Google Scholar 

  12. Frattini, A. et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nature Genet. 25, 343–346 (2000)

    Article  CAS  Google Scholar 

  13. Pérez-Sayáns, M., Somoza-Martìn, J. M., Barros-Angueira, F., Rey, J. M. & Garcìa-Garcìa, A. V-ATPase inhibitors and implication in cancer treatment. Cancer Treat. Rev. 35, 707–713 (2009)

    Article  Google Scholar 

  14. Xu, L. et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010)

    Article  Google Scholar 

  15. Hirata, T. et al. Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the g and c subunits. J. Biol. Chem. 278, 23714–23719 (2003)

    Article  CAS  Google Scholar 

  16. Imamura, H. et al. Rotation scheme of V1-motor is different from that of F1-motor. Proc. Natl Acad. Sci. USA 102, 17929–17933 (2005)

    Article  ADS  CAS  Google Scholar 

  17. Powell, B., Graham, L. A. & Stevens, T. H. Molecular characterization of the yeast vacuolar H+-ATPase proton pore. J. Biol. Chem. 275, 23654–23660 (2000)

    Article  CAS  Google Scholar 

  18. Umemoto, N., Yoshihisa, T., Hirata, R. & Anraku, Y. Roles of the VMA3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein transport. A study with VMA3-disrupted mutants of Saccharomyces cerevisiae . J. Biol. Chem. 265, 18447–18453 (1990)

    CAS  PubMed  Google Scholar 

  19. Umemoto, N., Ohya, Y. & Anraku, Y. VMA11, a novel gene that encodes a putative proteolipid, is indispensable for expression of yeast vacuolar membrane H+-ATPase activity. J. Biol. Chem. 266, 24526–24532 (1991)

    CAS  PubMed  Google Scholar 

  20. Taylor, J. W. & Berbee, M. L. Dating divergences in the fungal tree of life: review and new analyses. Mycologia 98, 838–849 (2006)

    Article  Google Scholar 

  21. Yang, Z., Kumar, S. & Nei, M. A new method of inference of ancestral nucleotide and amino acid sequences. Genetics 141, 1641–1650 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kane, P. M. The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. Microbiol. Mol. Biol. Rev. 70, 177–191 (2006)

    Article  CAS  Google Scholar 

  23. Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H. & Anraku, Y. Vma11 and vma16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase. J. Biol. Chem. 272, 4795–4803 (1997)

    Article  CAS  Google Scholar 

  24. Wang, Y., Cipriano, D. J. & Forgac, M. Arrangement of subunits in the proteolipid ring of the V-ATPase. J. Biol. Chem. 282, 34058–34065 (2007)

    Article  CAS  Google Scholar 

  25. Ohno, S. Evolution by Gene Duplication (Springer, 1970)

    Book  Google Scholar 

  26. Jacob, F. Evolution and tinkering. Science 196, 1161–1166 (1977)

    Article  ADS  CAS  Google Scholar 

  27. Lynch, M. The frailty of adaptive hypotheses for the origins of organismal complexity. Proc. Natl Acad. Sci. USA 104, 8597–8604 (2007)

    Article  ADS  CAS  Google Scholar 

  28. Hietpas, R. T., Jensen, J. D. & Bolon, D. N. Experimental illumination of a fitness landscape. Proc. Natl Acad. Sci. USA 108, 7896–7901 (2011)

    Article  ADS  CAS  Google Scholar 

  29. Tong, A. H. Y. et al. Global mapping of the yeast genetic interaction network. Science 303, 808–813 (2004)

    Article  ADS  CAS  Google Scholar 

  30. Pereira-Leal, J. B., Levy, E. D., Kamp, C. & Teichmann, S. A. Evolution of protein complexes by duplication of homomeric interactions. Genome Biol. 8, R51 (2007)

    Article  Google Scholar 

  31. Ryan, M., Graham, L. A. & Stevens, T. H. Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum. Mol. Biol. Cell 19, 5131–5142 (2008)

    Article  CAS  Google Scholar 

  32. Löytynoja, A. & Goldman, N. An algorithm for progressive multiple alignment of sequences with insertions. Proc. Natl Acad. Sci. USA 102, 10557–10562 (2005)

    Article  ADS  Google Scholar 

  33. Löytynoja, A. & Goldman, N. Phylogeny-aware gap placement prevents errors in sequence alignment and evolutionary analysis. Science 320, 1632–1635 (2008)

    Article  ADS  Google Scholar 

  34. Whelan, S. & Goldman, N. A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 18, 691–699 (2001)

    Article  CAS  Google Scholar 

  35. Abascal, F., Zardoya, R. & Posada, D. Prottest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105 (2005)

    Article  CAS  Google Scholar 

  36. Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003)

    Article  Google Scholar 

  37. Anisimova, M. & Gascuel, O. Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552 (2006)

    Article  Google Scholar 

  38. Aguinaldo, A. M. A. et al. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387, 489–493 (1997)

    Article  CAS  Google Scholar 

  39. Yang, Z. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007)

    Article  CAS  Google Scholar 

  40. Fitch, W. M. Toward defining the course of evolution: minimum change for a specific tree topology. Syst. Zool. 20, 406–416 (1971)

    Article  Google Scholar 

  41. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994)

    Article  CAS  Google Scholar 

  42. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)

    Article  CAS  Google Scholar 

  43. Do, C. B., Mahabhashyam, M. S., Brudno, M. & Batzoglou, S. ProbCons: Probabilistic consistency-based multiple sequence alignment. Genome Res. 15, 330–340 (2005)

    Article  CAS  Google Scholar 

  44. Fletcher, W. & Yang, Z. Indelible: a flexible simulator of biological sequence evolution. Mol. Biol. Evol. 26, 1879–1888 (2009)

    Article  CAS  Google Scholar 

  45. Sambrook, J. & Russel, D. W. Molecular Cloning: A Laboratory Manual 3rd edn (Cold Spring Harbor Laboratory Press, 2001)

    Google Scholar 

  46. Goldstein, A. L. & McCuster, J. H. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae . Yeast 15, 1541–1553 (1999)

    Article  CAS  Google Scholar 

  47. Zheng, L., Baumann, U. & Reymond, J. L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004)

    Article  Google Scholar 

Download references

Acknowledgements

This study was supported by National Institutes of Health (NIH) grants R01-GM081592 (to J.W.T.) and R01-GM38006 (to T.H.S.), National Science Foundation (NSF) grants IOB-0546906 (to J.W.T.) and DEB-0516530 (to J.W.T.), NIH Genetics Training grant T32-GM007257 (to G.C.F.), NSF IGERT grant DGE-9972830 (to V.H.-S.) and the Howard Hughes Medical Institute (J.W.T.). We thank L. Graham, G. Butler and B. Houser for generating yeast strains and other assistance. We thank members of the Stevens and Thornton laboratories for helpful comments.

Author information

Authors and Affiliations

Authors

Contributions

V.H.-S. performed the phylogenetic analysis and statistical reconstructions. G.C.F. performed functional experiments. All authors conceived the experiments, interpreted the results and wrote the paper.

Corresponding author

Correspondence to Joseph W. Thornton.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains the Protein Sequences and Yeast Strains used in this study, together with 7 Supplementary Figures with legends. (PDF 957 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Finnigan, G., Hanson-Smith, V., Stevens, T. et al. Evolution of increased complexity in a molecular machine. Nature 481, 360–364 (2012). https://doi.org/10.1038/nature10724

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10724

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing