A functional bacteria-derived restriction modification system in the mitochondrion of a heterotrophic protist

The overarching trend in mitochondrial genome evolution is functional streamlining coupled with gene loss; therefore, gene acquisition by mitochondria is considered to be exceedingly rare. Selfish elements in the form of self-splicing introns occur in many organellar genomes, but the wider diversity of selfish elements, and how they persist in the DNA of organelles, has not been explored. In the mitochondrial genome of a marine heterotrophic katablepharid protist, we identify a functional type II restriction modification (RM) system originating from a horizontal gene transfer (HGT) event involving bacteria related to flavobacteria. This RM system consists of an HpaII-like endonuclease and a cognate cytosine methyltransferase (CM). We demonstrate that these proteins are functional by heterologous expression in both bacterial and eukaryotic cells. These results suggest that a mitochondrial-encoded RM system can function as a toxin-antitoxin selfish element and that such elements could be co-opted by eukaryotic genomes to drive biased organellar inheritance.Peer reviewe

The Neurospora crassa TOB Complex: Analysis of the Topology and Function of Tob38 and Tob37

The TOB or SAM complex is responsible for assembling several proteins into the mitochondrial outer membrane, including all β-barrel proteins. We have identified several forms of the complex in Neurospora crassa. One form contains Tob55, Tob38, and Tob37; another contains these three subunits plus the Mdm10 protein; while additional complexes contain only Tob55. As previously shown for Tob55, both Tob37 and Tob38 are essential for viability of the organism. Mitochondria deficient in Tob37 or Tob38 have reduced ability to assemble β-barrel proteins. The function of two hydrophobic domains in the C-terminal region of the Tob37 protein was investigated. Mutant Tob37 proteins lacking either or both of these regions are able to restore viability to cells lacking the protein. One of the domains was found to anchor the protein to the outer mitochondrial membrane but was not necessary for targeting or association of the protein with mitochondria. Examination of the import properties of mitochondria containing Tob37 with deletions of the hydrophobic domains reveals that the topology of Tob37 may be important for interactions between specific classes of β-barrel precursors and the TOB complex

Comparative genomic analysis of the ‘pseudofungus’ Hyphochytrium catenoides

Eukaryotic microbes have three primary mechanisms for obtaining nutrients and energy: phagotrophy, photosynthesis and osmotrophy. Traits associated with the latter two functions arose independently multiple times in the eukaryotes. The Fungi successfully coupled osmotrophy with filamentous growth, and similar traits are also manifested in the Pseudofungi (oomycetes and hyphochytriomycetes). Both the Fungi and the Pseudofungi encompass a diversity of plant and animal parasites. Genome-sequencing efforts have focused on host-associated microbes (mutualistic symbionts or parasites), providing limited comparisons with free-living relatives. Here we report the first draft genome sequence of a hyphochytriomycete ‘pseudofungus’; Hyphochytrium catenoides. Using phylogenomic approaches, we identify genes of recent viral ancestry, with related viral derived genes also present on the genomes of oomycetes, suggesting a complex history of viral coevolution and integration across the Pseudofungi. H. catenoides has a complex life cycle involving diverse filamentous structures and a flagellated zoospore with a single anterior tinselate flagellum. We use genome comparisons, drug sensitivity analysis and high-throughput culture arrays to investigate the ancestry of oomycete/pseudofungal characteristics, demonstrating that many of the genetic features associated with parasitic traits evolved specifically within the oomycete radiation. Comparative genomics also identified differences in the repertoire of genes associated with filamentous growth between the Fungi and the Pseudofungi, including differences in vesicle trafficking systems, cell-wall synthesis pathways and motor protein repertoire, demonstrating that unique cellular systems underpinned the convergent evolution of filamentous osmotrophic growth in these two eukaryotic groups

Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the ER

The Voltage Dependent Anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes of the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii

The ubiquitous and ancient ER membrane protein complex (EMC): tether or not? [version 2; referees: 2 approved, 1 approved with reservations]

The recently discovered endoplasmic reticulum (ER) membrane protein complex (EMC) has been implicated in ER-associated degradation (ERAD), lipid transport and tethering between the ER and mitochondrial outer membranes, and assembly of multipass ER-membrane proteins. The EMC has been studied in both animals and fungi but its presence outside the Opisthokont clade (animals + fungi + related protists) has not been demonstrated. Here, using homology-searching algorithms, I show that the EMC is truly an ancient and conserved protein complex, present in every major eukaryotic lineage. Very few organisms have completely lost the EMC, and most, even over 2 billion years of eukaryote evolution, have retained a majority of the complex members. I identify Sop4 and YDR056C in Saccharomyces cerevisiae as Emc7 and Emc10, respectively, subunits previously thought to be specific to animals. This study demonstrates that the EMC was present in the last eukaryote common ancestor (LECA) and is an extremely important component of eukaryotic cells even though its primary function remains elusive

The persistent homology of mitochondrial ATP synthases

Summary: Relatively little is known about ATP synthase structure in protists, and the investigated ones exhibit divergent structures distinct from yeast or animals. To clarify the subunit composition of ATP synthases across all eukaryotic lineages, we used homology detection techniques and molecular modeling tools to identify an ancestral set of 17 ATP synthase subunits. Most eukaryotes possess an ATP synthase comparable to those of animals and fungi, while some have undergone drastic divergence (e.g., ciliates, myzozoans, euglenozoans). Additionally, a ∼1 billion-year-old gene fusion between ATP synthase stator subunits was identified as a synapomorphy of the SAR (Stramenopila, Alveolata, Rhizaria) supergroup (stramenopile, alveolate, rhizaria). Our comparative approach highlights the persistence of ancestral subunits even amidst major structural changes. We conclude by urging that more ATP synthase structures (e.g., from jakobids, heteroloboseans, stramenopiles, rhizarians) are needed to provide a complete picture of the evolution of the structural diversity of this ancient and essential complex

The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins

<div><p>MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists <i>Bigelowiella natans</i> and <i>Ectocarpus siliculosis</i>. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves the way for future studies into MAPL function in alternative model organisms like <i>Capsaspora owczarzaki</i> and <i>Chlamydomonas reinhardtii</i> that will help to answer the question of MAPL’s ancestral function in ways that cannot be answered by studying animal cells alone.</p></div

Phylogenetic reconstruction of BAM domain-containing proteins from opisthokonts, archaeplastida, and prokaryotes.

<p>BAM domain-containing protein sequences were aligned using MUSCLE, Sites that could not be aligned with confidence (including the eukaryote-specific RING domains) were removed manually. The resulting alignment was subjected to phylogenetic analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128795#sec002" target="_blank">methods</a> section for details). In this analysis, prokaryotic BAM proteins group together to the exclusion of all eukaryote proteins. Thus, the BAM domain-containing proteins present in various eukaryotes cannot be traced to independent HGT events. In this and all following phylogenetic analyses, numerical values represent Bayesian posterior probabilities and maximum-likelihood bootstrap values (Bayesian/PhyML/RAxML). Node values are given to highlight the clades of interest, denoted by coloured boxes and annotated by protein name. All other node support is iconized as inset.</p

Losing Complexity: The Role of Simplification in Macroevolution

Macroevolutionary patterns can be produced by combinations of diverse and even oppositional dynamics. A growing body of data indicates that secondary simplifications of molecular and cellular structures are common. Some major diversifications in eukaryotes have occurred because of loss and minimalisation; numerous episodes in prokaryote evolution have likewise been driven by the reduction of structure. After examining a range of examples of secondary simplification and its consequences across the tree of life, we address how macroevolutionary explanations might incorporate simplification as well as complexification, and adaptive as well as nonadaptive dynamics.M.A.O.’s research and the ISHPSSB symposium on which this paper is based were funded by a University of Sydney Bridging Support Grant and a University of Bordeaux IdEx Chair of Excellence. J.G.W. is supported by European Molecular Biology Organization ALTF 761-2014, and EMBOCOFUND2012, GA-2012-600394. I.R.T.’s research is supported by ERC-2012-Co-616960, BFU2014-57779-P, and Project 2014 SGR 619.Peer reviewe