Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
Endomembrane system compartments are significant elements in virtually all eukaryotic cells, supporting functions including protein synthesis, post-translational modifications and protein/lipid targeting. In terms of membrane area the endoplasmic reticulum (ER) is the largest intracellular organelle, but the origins of proteins defining the organelle and the nature of lineage-specific modifications remain poorly studied. To understand the evolution of factors mediating ER morphology and function we report a comparative genomics analysis of experimentally characterized ER-associated proteins involved in maintaining ER structure. We find that reticulons, REEPs, atlastins, Ufe1p, Use1p, Dsl1p, TBC1D20, Yip3p and VAPs are highly conserved, suggesting an origin at least as early as the last eukaryotic common ancestor (LECA), although many of these proteins possess additional non-ER functions in modern eukaryotes. Secondary losses are common in individual species and in certain lineages, for example lunapark is missing from the Stramenopiles and the Alveolata. Lineage-specific innovations include protrudin, Caspr1, Arl6IP1, p180, NogoR, kinectin and CLIMP-63, which are restricted to the Opisthokonta. Hence, much of the machinery required to build and maintain the ER predates the LECA, but alternative strategies for the maintenance and elaboration of ER shape and function are present in modern eukaryotes. Moreover, experimental investigations for ER maintenance factors in diverse eukaryotes are expected to uncover novel mechanisms.
- MeSH
- Endoplasmic Reticulum * metabolism MeSH
- Eukaryotic Cells * MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.
- MeSH
- Exocytosis genetics MeSH
- Lysosomes metabolism MeSH
- Organelles metabolism MeSH
- Secretory Vesicles genetics metabolism MeSH
- Tetrahymena thermophila * genetics MeSH
- Tetrahymena * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.
The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration.
Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.
Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.
- MeSH
- Aquaporin 2 * chemistry genetics metabolism MeSH
- Aquaporins chemistry genetics metabolism MeSH
- Drug Resistance drug effects genetics MeSH
- Melarsoprol pharmacology MeSH
- Mutation MeSH
- Pentamidine pharmacology MeSH
- Trypanocidal Agents pharmacology MeSH
- Trypanosoma brucei brucei * drug effects genetics metabolism MeSH
- Trypanosomiasis, African drug therapy MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.
Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.
- MeSH
- Aquaglyceroporins chemistry metabolism MeSH
- Drug Resistance * MeSH
- Amino Acid Sequence MeSH
- Protein Stability MeSH
- Trypanocidal Agents pharmacology MeSH
- Trypanosoma brucei brucei * MeSH
- Trypanosomiasis, African parasitology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Energy Metabolism drug effects MeSH
- Flagella drug effects metabolism ultrastructure MeSH
- Glycolysis drug effects MeSH
- Pyruvic Acid metabolism MeSH
- Membrane Potential, Mitochondrial drug effects MeSH
- Metabolome drug effects MeSH
- Microbodies drug effects metabolism ultrastructure MeSH
- Mitochondria drug effects metabolism ultrastructure MeSH
- Models, Molecular MeSH
- Proline metabolism MeSH
- Proteome metabolism MeSH
- Proton-Translocating ATPases metabolism MeSH
- Protozoan Proteins metabolism MeSH
- Suramin pharmacology MeSH
- Trypanosoma brucei brucei metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
