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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 mitochondrion-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.

Many mitochondrial proteins contain N-terminal presequences that direct them to the organelle. The main driving force for their translocation across the inner membrane is provided by the presequence translocase-associated motor (PAM) which contains the J-protein Pam18. Here, we show that in the PAM of Trypanosoma brucei the function of Pam18 has been replaced by the non-orthologous euglenozoan-specific J-protein TbPam27. TbPam27 is specifically required for the import of mitochondrial presequence-containing but not for carrier proteins. Similar to yeast Pam18, TbPam27 requires an intact J-domain to function. Surprisingly, T. brucei still contains a bona fide Pam18 orthologue that, while essential for normal growth, is not involved in protein import. Thus, during evolution of kinetoplastids, Pam18 has been replaced by TbPam27. We propose that this replacement is linked to the transition from two ancestral and functionally distinct TIM complexes, found in most eukaryotes, to the single bifunctional TIM complex present in trypanosomes.

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 Bigelowiella natans and Ectocarpus siliculosis. 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 Capsaspora owczarzaki and Chlamydomonas reinhardtii that will help to answer the question of MAPL's ancestral function in ways that cannot be answered by studying animal cells alone.

The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the er to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. Saccharomyces cerevisiae and Neurospora crassa strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of β-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in N. crassa and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of β-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of N. crassa Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.

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.