Gene duplication and gene conversion shape the evolution of archaeal chaperonins¹

Abstract

Chaperonins are multi-subunit double-ring complexes that mediate the folding of nascent or denatured proteins. Gene duplication has been a potent force in the evolution of chaperonins in Archaea. Here we show that gene conversion has also been an important factor. We utilized a novel maximum likehood-based phylogenetic method for scanning DNA sequence alignments for regions of anomalous phylogenetic signal, such as those affected by gene conversion. Our results suggest that in crenarchaeotes, where an ancient gene duplication producing α and β subunits took place in the common ancestor of the Pyrodictium, Aeropyrum, Pyrobaculum and Sulfolobus lineages, multiple independent gene conversions have occurred between the α and β genes independently in each of these groups. Significantly, the conversions have repeatedly homogenized the region of the gene encoding the substrate-binding domain. This suggests that while the α and β subunits in crenarchaeotes share only 50–60 % overall amino acid sequence identity, they do not possess distinct roles in the binding of substrate. Cryptic gene conversion between distantly related paralogs may be more common than is currently appreciated, and could be a significant factor in slowing the functional differentiation of proteins encoded by duplicate genes long after their duplication.

Introduction

Multigene family members often exhibit considerable sequence homogeneity within genomes compared to the similarity shared between orthologous genes in different genomes. This observation is usually attributed to the phenomenon of “concerted evolution”, the non-independent evolution of duplicate genes. One of the most important mechanisms underlying the concerted evolution of multigene families is gene conversion, a non-reciprocal recombination process that occurs between relatively small tracts of DNA, typically a few to a few thousand nucleotides in length.1

Gene conversion is an extremely well documented phenomenon. The best known example of a multigene family which evolves in a concerted fashion is ribosomal RNA (rRNA), and gene conversion plays a major role in the evolution of paralogous rRNA genes in eukaryotes,2 Bacteria and Archaea.3 Other multigene families whose evolution has been influenced by gene conversion include the visual pigment genes of primates,4 the silk moth chorion genes5 and the human globin gene families.6 Although generally viewed as a process of sequence homogenization, gene conversion can also generate molecular diversity. In the immunoglobulin genes of chicken, gene conversion plays a crucial role in the production of the huge diversity of peptides needed by the immune system.7

Here we show that gene conversion has been a major factor in the evolution of archaeal chaperonins. The chaperonins are a ubiquitous class of molecular chaperone involved in the folding of nascent or denatured proteins.8, 9 On the basis of sequence similarity, two distantly related (but clearly homologous) types of chaperonins are apparent. Group I chaperonins are found in Bacteria and eukaryotic organelles, while group II chaperonins are present in Archaea and the cytosol of eukaryotes. Individual 60 kDa chaperonin monomers assemble to form multi-subunit double-ring complexes that sequester substrates within a central cavity and mediate protein folding through ATP hydrolysis. The monomers themselves are composed of an apical and equatorial domain, linked by a small, flexible intermediate domain. The equatorial and intermediate domains provide most of the intra- and inter-ring subunit-subunit contacts and form the ATP-binding and hydrolysis site, and the apical domain is involved primarily in the binding of substrate.10

While bacterial chaperonins (e.g. GroEL in Escherichia coli) are generally homo-oligomeric, the cytosolic chaperonin complex in eukaryotes, called CCT (or TriC), is a completely hetero-oligomeric complex. Eight distinct CCT subunit genes evolved by duplication early in eukaryotic evolution,11, 12, 13 and each subunit occupies a unique position in the eight-membered CCT rings.14 Biochemical and electron microscopic studies have shown that the binding of actin and tubulin, two well-defined CCT substrates, within the central cavity of the chaperonin particle occurs in a subunit-specific manner.15, 16 In Archaea, chaperonins exhibit an intermediate degree of complexity. The chaperonin complex in the euryarchaeote Thermoplasma acidophilum is composed of two homologous subunits, α and β, that alternate in each of its eight-membered rings,17 a situation that also seems to exist in the crenarchaeote Pyrodictium.18, 19 Phylogenetic analyses of archaeal chaperonins reveal a complex pattern of lineage-specific gene duplications and gene losses, suggesting that hetero-oligomeric chaperonin complexes have arisen, and been lost, multiple times in the evolutionary history of this group.20, 21

The frequency with which gene conversion occurs is known to be negatively correlated with the divergence of the template sequences.22, 23 Gene conversions are thus typically identified between relatively closely related sequences. Here we show that in crenarchaeotes, discrete regions of the substrate-binding domain of the α and β chaperonin subunits, which overall share only 50–60 % amino acid sequence identity, are evolving in a highly concerted fashion, as a result of gene conversion. The data suggest that while the α and β subunits are the product of an ancient gene duplication, distinct roles for the duplicate subunits in substrate binding likely do not exist. More generally, cryptic gene conversions between divergent paralogs may be more common than previously assumed, and could play an important role in mediating the functional differentiation of duplicate proteins.