Sequence alignments of the catalytic motifs in strongly supported orthologs of biochemically characterized acyltransferases in each of the eGPATs, fungal GPATs (fGPATs-SACK), mitoGPATs and DHAPATs groups were compare to those of fungal GDPATs.

<p>The numbers in the boxes represent the distance between motifs in number of amino-acid residues. Proteins were aligned using Uniprot Align software and acyltransferase motifs recognized as proposed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684-Lewin1" target="_blank">[18]</a>. Positive, negative and aliphatic residues are highlighted in green, red and yellow respectively. Abbreviations: agos, <i>Ashbya gossypii</i>; amac, <i>Allomyces macrogynus</i>; anid, <i>Aspergillus nidulans</i>; bden, <i>Batrachochytrium dendrobatidis</i>; cgla, <i>Candida glabrata</i>; cimm, <i>Coccidioidies immitis</i>; gall, <i>Gallus gallus</i>; gzea, <i>Gibberella zeae</i>; hsap, <i>Homo sapiens</i>; klac, <i>Kluyveromyces lactis</i>; musm, <i>Mus musculus</i>; ncra, <i>Neurospora crassa</i>; scer, <i>Saccharomyces cerevisiae</i>; spun, <i>Spizellomyces punctatus</i>; xeno or xet, <i>Xenopus tropicalis</i>; ylip, <i>Yarrowia lipolytica</i>.</p

Phylogenetic Analysis of Glycerol 3-Phosphate Acyltransferases in Opisthokonts Reveals Unexpected Ancestral Complexity and Novel Modern Biosynthetic Components

<div><p>Glycerolipid synthesis represents a central metabolic process of all forms of life. In the last decade multiple genes coding for enzymes responsible for the first step of the pathway, catalyzed by glycerol 3-phosphate acyltransferase (GPAT), have been described, and characterized primarily in model organisms like <i>Saccharomyces cerevisiae</i> and mice. Notoriously, the fungal enzymes share low sequence identity with their known animal counterparts, and the nature of their homology is unclear. Furthermore, two mitochondrial GPAT isoforms have been described in animal cells, while no such enzymes have been identified in Fungi. In order to determine if the yeast and mammalian GPATs are representative of the set of enzymes present in their respective groups, and to test the hypothesis that metazoan orthologues are indeed absent from the fungal clade, a comparative genomic and phylogenetic analysis was performed including organisms spanning the breadth of the Opisthokonta supergroup. Surprisingly, our study unveiled the presence of ‘fungal’ orthologs in the basal taxa of the holozoa and ‘animal’ orthologues in the basal holomycetes. This includes a novel clade of fungal homologues, with putative peroxisomal targeting signals, of the mitochondrial/peroxisomal acyltransferases in Metazoa, thus potentially representing an undescribed metabolic capacity in the Fungi. The overall distribution of GPAT homologues is suggestive of high relative complexity in the ancestors of the opisthokont clade, followed by loss and sculpting of the complement in the descendent lineages. Divergence from a general versatile metabolic model, present in ancestrally deduced GPAT complements, points to distinctive contributions of each GPAT isoform to lipid metabolism and homeostasis in contemporary organisms like humans and their fungal pathogens.</p></div

Phylogeny of fungal GPAT homologues found in opisthokonts.

<p>Phylogenetic tree of the fungal GPATs. In this and all other phylogenies, node support values are shown in order of Bayesian posterior probabilities, PhyML bootstrap percentages and RaxML bootstrap percentages, or are symbolized as dots, colorized as per the inset to indicate statistical strength. Clades for orthologues of Sct1 and Gpt2 are shaded. For protein accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684.s006" target="_blank">Table S3</a>.</p

Acyltransferase motifs and distance between motifs (DBM) in microsomal yeast-like (fGPAT-A), metazoan ER-like (erGPAT), mitoGPATs, DHAPAT and fungal GDPAT (fGDPAT) sets.

<p>All proteins identified in this work for each category were aligned using Uniprot Align software and acyltransferase motifs recognized as proposed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684-Lewin1" target="_blank">[18]</a>. Number of proteins containing all four motifs used for each alignment is shown in brackets.</p>a<p>as proposed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684-Lewin1" target="_blank">[18]</a>.</p><p>Nomenclature used as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684-Aasland1" target="_blank">[54]</a>:</p><p>π  =  P, G, A and S (short chain).</p><p>φ  =  Hydrophobic amino acids (V, I, L, F, W, Y and M).</p><p>Residues highlighted in red are the hallmark of each motif.</p><p>* <i>N.vectensis</i> mGPAT [XP_001640722.1] and <i>C. owczarzaki</i> mGPAT gi|320164443| have 65 residues between Motifs 2&3.</p><p>Acyltransferase motifs and distance between motifs (DBM) in microsomal yeast-like (fGPAT-A), metazoan ER-like (erGPAT), mitoGPATs, DHAPAT and fungal GDPAT (fGDPAT) sets.</p

Phylogenetic tree of mammalian eGPATs in opisthokonts.

<p>Phylogenetic tree of the emergence of mammalian GPAT3 and GPAT4 homologues (called eGPATs, then enumerated). For protein accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684.s008" target="_blank">Table S5</a>.</p

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) Persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3) P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes. </p

Glycerolipid biosynthesis in eukaryotes.

<p>Cartoons highlighting differences and similarities between the glycerolipid biosynthetic pathways in humans and yeast <i>Saccharomyces cerevisiae</i>.</p

Opisthokont relationships and GPAT evolution.

<p><b>A</b>) Tree illustrating the relative evolutionary history for the eukaryotic emergence, loss and diversification/duplication of GPATs in opisthokonts, in lineages with genome sequences available in the fungi and metazoans with additional taxa from the base of the Opisthokonta supergroup. The apusozoan, <i>T. trahens</i>, was used as the outgroup. The group labeled “SACK” belongs to Saccharomycotina and involves <i>S. cerevisiae, A. gossypii, C.glabrata</i>, and <i>K. lactis</i> To note the Pezizomycotina and Taphrinomycotina form a paraphyletic group of ‘non- Saccharomycotina ascomycota’ which we found to be a useful grouping when considering the evolution of GPATs and are thus colour coded identically. Similarly, all non-tetrapoda metazoans are coded in the same colour. In order to avoid false negative or false positives in our evolutionary deductions, we do not infer any events on lineages where only a single genome was examined. <b>B</b>) Results from the comparative genomic survey and phylogenetic analysis. Individual species from the survey are color coded as in A) and grouped according to established taxonomic classification. The dendrogram is schematic of relationships only and the branch length is not representative of evolutionary distance. Symbols indicate the presence of <i>at least</i> one isoform of a given protein as verified by BLAST, Reciprocal BLAST, hidden Markov model searches, and phylogenetic data as follow: orthologs of Sct1/Gat2(), Gpt2/Gat1(), as well as new fungal fGPAT-A () or fGPAT-B (<b>□</b>) and orthologs of human mitochondrial GPATs (mitoGPATs) GPAT1(▴), GPAT2(▾), putative mitoGPATs (▵), GDPAT (), DHAPATs (⧫) and putative DHAPATs (pDHAPAT ◊), and orthologs of human microsomal GPATs (eGPATs) GPAT3(▸) and GPAT4(◂) and putative eGPATs (→). Putative orthologs of Sct1 and Gpt2 are shown in grey while strongly supported orthologs are shown in solid black. Blank cells indicate no hits. (^p) denotes GDPATs with PTS1 prediction. Designation in a particular orthologous group denotes a prediction of substrate specificity, based on assumed retention between orthologues, and provides hypotheses for future functional testing. (*) The Rhizopus acyltransferase gene groups with moderate support with the DHAPAT genes, but this placement may well be due to its highly divergent sequence. Protein sequences used to initiate the searches: yeast Sct1 (NP_009542.1), yeast Gpt2 (NP_012993.1), human GPAT1 (NP_065969.3), human GPAT2 (NP_997211.2), human DHAPAT (NP_055051.1), human GPAT3 (NP_116106.2), human GPAT4 (NP_848934.1). <i>Note</i>: Putative fungal GDPATs display a peroxisomal target sequence (PTS1) and were hits in a search initiated with mitochondrial human GPAT1.</p

Intracellular distribution of phosphatidic acid biosynthetic and fatty acid oxidation pathways in vertebrate and fungi.

<p>Intracellular distribution of phosphatidic acid biosynthetic and fatty acid oxidation pathways in vertebrate and fungi.</p

Phylogenetic tree of GDPAT-related genes in opisthokonts.

<p>The clades of mitochondrial GPATs (called mitoGPAT, then enumerated), putative DHAPATs (called pDHAPAT, then enumerated) and fungal GDPATs are shaded. Darker shading in each cluster highlights biochemically characterized enzymes from vertebrates, which includes mammalian GPAT1 and DHAPAT homologues. For protein accession numbers see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110684#pone.0110684.s007" target="_blank">Table S4</a>.</p