Evolution of increased complexity in a molecular machine

Journal name:
Nature
Volume:
481,
Pages:
360–364
Date published:
DOI:
doi:10.1038/nature10724
Received
Accepted
Published online

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.

At a glance

Figures

  1. Structure and evolution of the V-ATPase complex.
    Figure 1: Structure and evolution of the V-ATPase complex.

    a, In S. cerevisiae, the V-ATPase contains two subcomplexes: the octameric V1 domain is on the cytosolic side of the organelle membrane, and the hexameric V0 ring is membrane bound. Protein subunits Vma3, Vma11 and Vma16 are labelled and coloured. b, Maximum likelihood phylogeny of V-ATPase subunits Vma3, Vma11 and Vma16. All eukaryotes contain subunits 3 and 16, but Fungi also contain subunit 11. Circles show ancestral proteins reconstructed in this study. Colours correspond to those of subunits in panel a; unduplicated orthologues of Vma3 and Vma11 are green. Asterisks show approximate likelihood ratios for major nodes: ****, >103; ***, >102; **, >10; *, <10; ~, <2. The complete phylogeny is presented in Supplementary Information, section 2.

  2. Two reconstructed ancestral V0 subunits functionally replace the three-paralogue ring in extant yeast.
    Figure 2: Two reconstructed ancestral V0 subunits functionally replace the three-paralogue ring in extant yeast.

    S. cerevisiae were plated in decreasing concentrations on permissive medium (YEPD) buffered with elevated CaCl2. a, Expression of Anc.3-11 rescues growth in yeast that are deficient for endogenous subunit Vma3 (3Δ), subunit Vma11 (11Δ) or both (3Δ11Δ). Growth of wild-type (WT) yeast is shown for comparison. b, Anc.16 rescues growth in yeast that are deficient for subunit Vma16 (16Δ). c, Expression of Anc.3-11 and Anc.16 together rescues growth in yeast that are deficient for Vma3, Vma11 and Vma16. d, Anc.11 rescues growth in vma11Δ but not in vma3Δ yeast. e, Anc.3 rescues growth in vma3Δ but not vma11Δ yeast. f, Anc.3 and Anc.11 together rescue growth in vma3Δvma11Δ mutants. g,Yeast expressing reconstructed ancestral subunits properly acidified the vacuolar lumen. Red signal shows yeast cell walls; green signal (quinacrine) shows acidified compartments. Yeast were visualized by differential interference contrast microscopy.

  3. Increasing complexity by complementary loss of interactions in the fungal V0 ring.
    Figure 3: Increasing complexity by complementary loss of interactions in the fungal V0 ring.

    a, Model of the ancestral three-paralogue ring, arranged as in extant yeast24. Unique intersubunit interfaces are labelled P, Q and R. Subunits are colour-coded as in Fig. 1. b, Model of the ancestral two-paralogue ring, before duplication of Anc.3-11. c, To constrain the location of specific subunits, gene fusions were constructed by tethering an ancestral subunit to either the amino- or carboxy-terminal side of yeast Vma16. Roman numerals indicate the locations of transmembrane helices (I, II, III, IV and V)24. df, Growth assays of yeast with fused V0 subunits identify the interfaces that ancestral subunits can form. For each experiment, expressed V0 subunits are listed. Tethered subunits are in brackets and connected by a thick line. Cartoons show the constrained location of the tethered subunit relative to Vma16. Anc.3-11 can function on either side of Vma16 (d). Anc.3 can function only on the clockwise side of Vma16 (e). Anc.11 can function only on the anticlockwise side of Sc.16 (f). g, Interfaces that are formed by V0 subunits before and after duplication and complementary loss of interfaces, based on the data in panels df. Red crosses indicate lost interfaces. h, Schematic of interfaces formed by Anc.3-11 that were lost in Anc.3 and Anc.11, based on data in panels df.

  4. Genetic basis for functional differentiation of Anc.3 and Anc.11.
    Figure 4: Genetic basis for functional differentiation of Anc.3 and Anc.11.

    a, Experimental analysis of historical amino acid replacements. The table lists replacements that occurred on the branches leading from Anc.3-11 to Anc.11 (yellow) or to Anc.3 (blue) and that were subsequently conserved. Each derived residue was introduced singly into Anc.3-11; the variant genes were transformed into S. cerevisiae, and growth was assayed on elevated CaCl2. The table shows growth semiquantitatively from zero (none) to wild type (++++++). Bold mutations entirely or partly recapitulate the functional evolution of Anc.11 and Anc.3. b, Replacement V15F abolishes the capacity of Anc.3-11 to function as subunit 3 and enhances the capacity of Anc.3-11 to function as subunit 11. c, Replacement M22I impairs the capacity of Anc.3-11 to function as subunit 11 without affecting its capacity to function as subunit 3.

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Author information

  1. These authors contributed equally to this work.

    • Gregory C. Finnigan &
    • Victor Hanson-Smith

Affiliations

  1. Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA

    • Gregory C. Finnigan &
    • Tom H. Stevens
  2. Institute for Ecology and Evolution, University of Oregon, Eugene, Oregon 97403, USA

    • Victor Hanson-Smith &
    • Joseph W. Thornton
  3. Department of Computer and Information Science, University of Oregon, Eugene, Oregon 97403, USA

    • Victor Hanson-Smith
  4. Howard Hughes Medical Institute, Eugene, Oregon 97403, USA

    • Joseph W. Thornton
  5. Departments of Human Genetics and Ecology & Evolution, University of Chicago, Chicago, Illinois 60637, USA

    • Joseph W. Thornton

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Information (957K)

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

Additional data