Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa

<div><p>Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for <i>Guillardia</i><i>theta</i> and <i>Bigelowiella</i><i>natans</i>. The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate <i>Perkinsus marinus</i>. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.</p> </div

Phylogenetic analysis separating Vps3 family proteins from Vps39.

<p>This figure demonstrates that the duplication into Vps3 and 39 occurred early in eukaryotic evolution and that both are widely retained proteins. The best Bayesian topology is shown and values for critical nodes are shown in the order of Bayesian posterior probabilities, PhyML bootstrap and RAxML bootstrap values. All other nodes are symbolized as inset.</p

Comparative genomic survey of selected MTC proteins across the diversity of eukaryotes.

<p>The analysis demonstrates the conserved nature of the newly described TRAPPII subunit Tca17/TRAPPC2L, the CORVET complex, and the Dsl1 complex. In this and subsequent Coulson plots, filled pie sectors indicate an identified homologue, while unfilled indicate that no homologue was identified. Asterisks indicate that Vps39 is also present in these taxa (See Table S2). Infinity symbols represent organisms with divergent peroxisomes, or in which peroxisomes have not been identified. Colour-coding is arbitrary and for visual purposes only.</p

Phylogenetic analysis of Vps3, Vps39, and Vps41 proteins from select taxa.

<p>This figure demonstrates the separation of select Vps proteins from taxa spanning the breadth of eukaryotic diversity into distinct clades. Colour-coding is arbitrary and for visual purposes only.</p

A Characterization of the <i>Manduca sexta</i> Serotonin Receptors in the Context of Olfactory Neuromodulation

<div><p>Neuromodulation, the alteration of individual neuron response properties, has dramatic consequences for neural network function and is a phenomenon observed across all brain regions and taxa. However, the mechanisms underlying neuromodulation are made complex by the diversity of neuromodulatory receptors expressed within a neural network. In this study we begin to examine the receptor basis for serotonergic neuromodulation in the antennal lobe of <i>Manduca sexta</i>. To this end we cloned all four known insect serotonin receptor types from <i>Manduca</i> (the Ms5HTRs). We used phylogenetic analyses to classify the Ms5HTRs and to establish their relationships to other insect serotonin receptors, other insect amine receptors and the vertebrate serotonin receptors. Pharmacological assays demonstrated that each Ms5HTR was selective for serotonin over other endogenous amines and that serotonin had a similar potency at all four Ms5HTRs. The pharmacological assays also identified several agonists and antagonists of the different Ms5HTRs. Finally, we found that the Ms5HT1A receptor was expressed in a subpopulation of GABAergic local interneurons suggesting that the Ms5HTRs are likely expressed heterogeneously within the antennal lobe based on functional neuronal subtype.</p> </div

Phylogenetic relationships between the Ms5HTRs and other amine receptors.

<p>Ms5HTRs are indicated with bold text (A) Phylogenetic tree of all available serotonin, tyramine, octopamine and dopamine receptors from 7 species of insects. This figure is arbitrarily rooted on the 5-HT2 clade. Strongly supported clades for 5-HT1, 2, 7 Dopamine 1, 2, Octopamine A, B, and Tyramine are generated as shown. The newly characterized Ms5HT2R sequence is present in the 5-HT2 clade and Ms5HT7R sequence in the 5-HT7 clade. In this and all subsequent phylogenetic trees, the best Bayesian topology is shown with node support values listed in the order of Posterior probability values, Maximum Likelihood bootstrap support values for PhyML and Maximum Likelihood bootstrap support values for RAxML. Other values are replaced with symbols as shown in the figure. Vertical bars with labels denote the reconstructed clades. The scale bar represents the number of changes per site. (B) Phylogenetic analysis of serotonin receptors from insects (I) and vertebrates (V), with the 5-HT2 receptors excluded. The figure is arbitrarily rooted on the vertebrate 5-HT3 clade, but should be treated as unrooted. Note the reconstruction of the various named insect and vertebrate receptor sub-types.</p

The Ms5HT1A receptor is expressed in a subset of GABA-like immunoreactive local interneurons.

<p>(A) Western blots of the AM3-A antibody against <i>Manduca</i> protein and in which the AM3-A antibody was omitted (no 1^o) produced no labeling. (B) Frontal section through an AL labeled using an antibody against a 5-HT_1A receptor from prawn (AM3-A) generously provided by Dr. Maria Sosa. Note cell bodies (arrowheads; 24±2) and fine processes (arrows). (C) <i>Manduca</i> ALs labeled with AM3-A antibody pre-adsorbed with the Ms5HT1A receptor sequence (KDPDYLARITQQQKCLVSQD) homologous to the antigenic sequence used to generate the AM3-A antibody (KDPDFLVRVNEHKKCLVSQD) resulted in no labeling. (D) Double labeling against the Ms5HT1A receptor (green) and 5-HT (magenta) show no overlap. (E) Backfills of projection neurons (PNs) (magenta) reveal no co-localization of the Ms5HT1A receptor (green) in the lateral cell cluster of the AL. Magenta arrows indicate PNs and green arrows indicate Ms5HT1A-ir neurons. (F) Double labeling against the Ms5HT1A receptor (green) and GABA (magenta) revealed a subset of Ms5HT1A-ir cell bodies co-localizing GABA (arrows). All scale bars=100µm.</p

Responses of the Ms5HTRs for 5-HT and the dose response curves for selective agonists.

<p>(A) Currents (normalized to maximal responses) evoked by increasing concentrations of 5-HT (from 0.1nM to 10 µM) in <i>Xenopus laevis</i> oocytes expressing the Ms5HT1A (black circle), Ms5HT1B (grey triangle), Ms5HT2 (open triangle), Ms5HT7 (black diamond). (B) <i>Xenopus</i> oocytes expressing the Ms5HT2 receptor (open triangle) respond to 5-nonyloxytryptamine at 0.1 µM and above, while oocytes expressing the Ms5HT1A (black circle), Ms5HT1B (grey triangle) or Ms5HT7 (black diamond) receptors do not respond at any of the concentrations tested. The symbols for the Ms5HT1A, 1B and 7 receptors have been staggered for clarity. In (A) and (B) the 5HT_1A responses were measured in oocytes co-injected with receptor cRNA and cRNA for the CFTR channel whilst the 5HT_1B, 5HT2 and 5HT7 responses were measured in oocytes injected with the receptor cRNA alone. Oocytes were exposed to 2 min pulses of the agonists at various concentrations. (C) HEK293 cells expressing the Ms5HT2 receptor respond (measured as nM concentration of IP-1/well of a 96 well plate) to 5-HT and DOI (4-Iodo-2,5-dimethoxy-α-methylbenzene ethanamine). (D) HEK293 cells expressing the Ms5HT7 receptor respond (measured as nM concentration of cAMP/well of a 96 well plate) to 5-HT, but not DOI. Shades of grey in (C) and D) indicate concentration of pharmacological agent applied. All error bars represent SEM.</p

All four Ms5HTRs are expressed in both the antennal lobes and the antennae.

<p>RT-PCR for the four Ms5HTRs using cDNA from (A) antennal lobes and (B) the antennae. For each receptor, the left-hand lane depicts the RT-PCR with the reverse transcriptase included during cDNA production and right-hand lane depicts the RT-control to ensure no genomic DNA contamination.</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