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 analysis of SAR BAM proteins.

<p>In this analysis the BAM proteins from <i>E</i>. <i>siliculosis</i> and <i>B</i>. <i>natans</i> group with prokaryotic sequences suggesting that these eukaryotic proteins have a different origin than other MAPL and might be derived from recent HGT events. The RING domain was excluded from this analysis. Analysis and node support as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128795#pone.0128795.g002" target="_blank">Fig 2</a>.</p

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

Phylogenetic analysis of Archaeplastid MAPL proteins.

<p>This analysis demonstrates that <i>A</i>. <i>thaliana</i> and <i>C</i>. <i>rubella</i> BAM proteins that lack the RING domain group within a weakly supported clade comprising sequences from multicellular plants that retain a RING domain. The RING domain was excluded from this analysis. Node support as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128795#pone.0128795.g002" target="_blank">Fig 2</a>.</p

Distribution of BAM domain proteins across the tree of life.

<p>A. BAM domain distribution across the three domains of life. BAM domain proteins are present in all three domains of life, but only plants, animals, and a single fungus contain BAM proteins that are followed by a RING domain. Filled circles indicate many taxa contain at least one BAM protein. Open circles indicate only one or two species were identified with BAM proteins. B. Distribution of MAPL (BAM-RING) in holozoa (clade comprising animals and their closest single-celled relatives), with particular focus on non-vertebrates. Most species contain MAPL, but several instances of loss are recorded. C. Expansion of MAPL in the vertebrate lineage followed by loss in mammals. Multiple MAPL paralogues are present in non-mammalian vertebrates (MAPL2 and MAPL2-like). D. Expansion of MAPL in multicellular plants. Green algae contain a single MAPL whereas multicellular plants have gained a paralogue. The <i>Capsella</i>-<i>Arabidopsis</i> clade has further gained a paralogue that has lost the RING domain (MAPL-R).</p

Phylogenetic analysis of Opisthokont MAPL proteins.

<p>This analysis demonstrates that all vertebrate MAPL proteins group together to the exclusion of all other opisthokont MAPL proteins. MAPL has been retained in all major vertebrate clades. MAPL-like is an ancient vertebrate protein lost in the mammalian lineage. MAPL-like2 is specific to fishes. The RING domain was included in the alignment in this analysis as the vast majority of predicted proteins contained this domain. Node support as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128795#pone.0128795.g002" target="_blank">Fig 2</a>.</p

Analysis of Mutations in <i>Neurospora crassa</i> ERMES Components Reveals Specific Functions Related to β-Barrel Protein Assembly and Maintenance of Mitochondrial Morphology

<div><p>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. <i>Saccharomyces cerevisiae</i> and <i>Neurospora crassa</i> 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 <i>N. crassa</i> 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 <i>N. crassa</i> Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.</p></div

Characterization of <i>N. crassa</i> Cys to Ser Mmm1 mutants.

<p>A. The Mmm1-HA protein engages in disulphide bonding. Mitochondria (30 µg) were treated with cracking buffer that either did (+BME) or did not (-BME) contain β-mercaptoethanol. Samples were subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting for the indicated proteins. B. Coimmunoprecipitation of two tagged forms of Mmm1 from a heterokaryon. An unforced heterokaryon consisting of strains Mmm1-HA3 and Mmm1-Myc10 (HA/Myc) was constructed as described in the Methods. Mitochondria isolated from the heterokaryon were dissolved and treated with anti-Myc agarose beads. Elutions from the beads, or total mitochondrial proteins (mito load, to monitor the input level of proteins), were electrophoresed, blotted, and immunodecorated with the antibodies indicated on the right. Controls were an untagged wild type NCN251 strain (control), the homokaryotic Mmm1-HA3 strain (HA), and the homokaryotic Mmm1-Myc10 strain (Myc). Arrowheads on the left indicate the relevant bands in panels containing non-specific background bands. C. Tenfold dilutions of conidiaspores from strains expressing control (Mmm1-HA) and mutant HA-tagged versions of Mmm1 were spotted on plates containing Vogel’s sorbose medium. The plates were incubated at 30°C for 48 h and then photographed. D. Strains expressing control (Mmm1-HA) and mutant HA-tagged versions of Mmm1 were grown on solid Vogel’s media, stained with MitoTracker Green FM and examined by confocal fluorescence microscopy. Mitochondria in the <i>Δmmm1</i> strain are shown for comparison. Bar represents 10 µm. E. Western blot analysis of Mmm1 Cys mutant crude mitochondria. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3A</a>, but mitochondria were only analyzed by non-reducing SDS-PAGE. F. Cell fractionation of the indicated strains as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g001" target="_blank">Figure 1A</a> except that Tom22 was the mitochondrial marker.</p

Characterization of the <i>Δgem1</i> strain.

<p>A. Examination of mitochondrial morphology as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3D</a>. B. Growth rates as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3C</a>. C. Steady state levels of mitochondrial proteins as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g005" target="_blank">Figure 5A</a>. D. β-barrel protein (Tom40 and porin) assembly as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g004" target="_blank">Figure 4A and B</a>, respectively. E. Tom22 assembly as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g004" target="_blank">Figure 4C</a>. F. Mitochondrial phospholipid content as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g006" target="_blank">Figure 6G</a>.</p

Alignments of fungal Mmm1 proteins.

<p>A. Alignment of Mmm1 N-terminal regions from several Ascomycetes. Known <i>S. cerevisiae</i> (N50, N55 and N59) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone.0071837-Stroud1" target="_blank">[12]</a> and potential N-glycosylation sites in other species are highlighted in black. Cys residue conserved in Sordariomycetes is shaded in grey. B. Predicted transmembrane domain of Mmm1is present in most fungi, but absent in Mucormycotina and Chytridiomycota. The predicted <i>N. crassa</i> transmembrane domain is highlighted in grey. Identity/similarity symbols are for the alignment of the Ascomycota and Basidiomycota only. * indicates conserved residues, : indicates conservation of groups with strongly similar properties (score of >0.5 in the Gonnet PAM 250 matrix),. indicates conservation of groups with weakly similar properties (score of <0.5 in the Gonnet PAM 250 matrix). C. Alignment of the highly conserved region of Mmm1 chosen for mutation analysis. The nine amino acid region that was chosen for mutation is highlighted in the <i>N. crassa</i> protein (residues 116-124). Symbols (as in panel B) are for the alignment of all proteins. Abbreviations: N.c., <i>Neurospora crassa</i>; G.z., <i>Gibberella zeae</i>; C.g., <i>Chaetomium globosum</i>; S.m., <i>Sordaria macrospora</i>; M.o., <i>Magnaporthe oryzae</i>; P.a., <i>Podospora anserina</i>; F.o. <i>Fusarium oxysporum</i>; V.d., <i>Verticillium dahliae</i>; A.n., <i>Aspergillus nidulans</i>; T.t.,<i>Trichophyton tonsurans</i>; C.i., <i>Coccidioides immitis</i>; P.b., <i>Paracoccidioides brasiliensis</i>; S.s., <i>Sclerotinia sclerotiorum</i>; B.f., <i>Botryotinia fuckeliana</i>; P.t., <i>Pyrenophora teres</i>; S.c., <i>Saccharomyces cerevisiae</i>; K.l., <i>Kluyveromyces lactis</i>; C.a., <i>Candida albicans</i>; S.p., <i>Schizosaccharomyces pombe</i>; U.m., <i>Ustilago maydis</i>; C.n., <i>Cryptocuccus neoformans</i>; R.o., <i>Rhizopus oryzae</i>; B.d., <i>Batrachochytrium dendrobatidis</i>.</p