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1.
PLoS Pathog ; 20(6): e1012333, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38935804

ABSTRACT

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.


Subject(s)
Endocytosis , Glycosyltransferases , Transferrin , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolism , Trypanosoma brucei brucei/genetics , Animals , Endocytosis/physiology , Mice , Transferrin/metabolism , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Trypanosomiasis, African/parasitology , Trypanosomiasis, African/metabolism , Mutation , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Receptors, Transferrin/metabolism , Receptors, Transferrin/genetics , Polysaccharides
2.
Mol Biochem Parasitol ; 256: 111598, 2023 12.
Article in English | MEDLINE | ID: mdl-37923299

ABSTRACT

Visualisation of genomic loci by microscopy is essential for understanding nuclear organisation, particularly at the single cell level. One powerful technique for studying the positioning of genomic loci is through the Lac Operator-Lac Repressor (LacO-LacI) system, in which LacO repeats introduced into a specific genomic locus can be visualised through expression of a LacI-protein fused to a fluorescent tag. First utilised in Trypanosoma brucei over 20 years ago, we have now optimised this system with short, stabilised LacO repeats of less than 2 kb paired with a constitutively expressed mNeongreen::LacI fusion protein to facilitate visualisation of genomic loci. We demonstrate the compatibility of this system with super-resolution microscopy and propose its suitability for multiplexing with inducible RNAi or protein over expression which will allow analysis of nuclear organisation after perturbation of gene expression.


Subject(s)
Trypanosoma brucei brucei , Trypanosoma brucei brucei/genetics , Lac Repressors/genetics , Promoter Regions, Genetic , Genomics
3.
Cell Rep Methods ; 2(8): 100274, 2022 08 22.
Article in English | MEDLINE | ID: mdl-36046624

ABSTRACT

Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. Using libraries of barcoded T. gondii, we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii.


Subject(s)
Toxoplasma , Mice , Animals , Toxoplasma/genetics , Protozoan Proteins/genetics , Mice, Inbred CBA , Virulence , Brain/metabolism
4.
PLoS Pathog ; 17(6): e1009696, 2021 06.
Article in English | MEDLINE | ID: mdl-34161395

ABSTRACT

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation.


Subject(s)
Adaptation, Physiological/physiology , Iron/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Cells, Cultured , Homeostasis/physiology , RNA-Binding Proteins/metabolism , Receptors, Transferrin/metabolism , Transcriptome , Trypanosomiasis, African/metabolism
5.
J Biol Chem ; 295(8): 2227-2238, 2020 02 21.
Article in English | MEDLINE | ID: mdl-31932305

ABSTRACT

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4-7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.


Subject(s)
Glycosylphosphatidylinositols/chemistry , Glycosylphosphatidylinositols/metabolism , Trypanosoma brucei brucei/metabolism , Glycosides/metabolism , Glycosylation , Lectins/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Protozoan Proteins/metabolism , Receptors, Transferrin/metabolism , Substrate Specificity
6.
mSphere ; 4(5)2019 09 04.
Article in English | MEDLINE | ID: mdl-31484744

ABSTRACT

Calvin Tiengwe works on posttranscriptional gene regulation and iron homeostasis in the parasitic protozoan Trypanosoma brucei In this mSphere of Influence article, he reflects on how the paper "Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS)" by Queiroz et al. (Nat Biotechnol 37:169-178, 2019, https://doi.org/10.1038/s41587-018-0001-2) influenced his research by providing a tool to capture RNA-protein complexes on a global scale using acid guanidinium thiocyanate-phenol-chloroform (AGPC), an old method hitherto applied for RNA, DNA, or protein purification.


Subject(s)
Molecular Biology/methods , Proteins/metabolism , RNA/isolation & purification , RNA/metabolism , Trypanosoma brucei brucei/genetics , Chloroform , Guanidines , Humans , Phenol , Protein Interaction Domains and Motifs , Thiocyanates
7.
Mol Biol Cell ; 29(20): 2397-2409, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30091673

ABSTRACT

Misfolded secretory proteins are retained by endoplasmic reticulum quality control (ERQC) and degraded in the proteasome by ER-associated degradation (ERAD). However, in yeast and mammals, misfolded glycosylphosphatidylinositol (GPI)-anchored proteins are preferentially degraded in the vacuole/lysosome. We investigate this process in the divergent eukaryotic pathogen Trypanosoma brucei using a misfolded GPI-anchored subunit (HA:E6) of the trypanosome transferrin receptor. HA:E6 is N-glycosylated and GPI-anchored and accumulates in the ER as aggregates. Treatment with MG132, a proteasome inhibitor, generates a smaller protected polypeptide (HA:E6*), consistent with turnover in the proteasome. HA:E6* partitions between membrane and cytosol fractions, and both pools are proteinase K-sensitive, indicating cytosolic disposition of membrane-associated HA:E6*. HA:E6* is de-N-glycosylated and has a full GPI-glycan structure from which dimyristoylglycerol has been removed, indicating that complete GPI removal is not a prerequisite for proteasomal degradation. However, HA:E6* is apparently not ubiquitin-modified. The trypanosome GPI anchor is a forward trafficking signal; thus the dynamic tension between ERQC and ER exit favors degradation by ERAD. These results differ markedly from the standard eukaryotic model systems and may indicate an evolutionary advantage related to pathogenesis.


Subject(s)
Endoplasmic Reticulum-Associated Degradation , Glycosylphosphatidylinositols/metabolism , Protein Folding , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Gene Silencing , Genes, Reporter , Leupeptins/pharmacology , Models, Biological , Proteolysis/drug effects , RNA Interference , Receptors, Transferrin/metabolism
8.
PLoS Pathog ; 13(5): e1006366, 2017 May.
Article in English | MEDLINE | ID: mdl-28459879

ABSTRACT

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin-thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0-2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.


Subject(s)
Glycosylphosphatidylinositols/metabolism , Protein Transport , Receptors, Transferrin/metabolism , Trypanosoma brucei brucei/physiology , Trypanosomiasis, African/parasitology , Variant Surface Glycoproteins, Trypanosoma/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/metabolism , Dimerization , Glycosylphosphatidylinositols/genetics , Humans , Lysosomes/metabolism , RNA Interference , Receptors, Transferrin/genetics , Sequence Alignment , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/ultrastructure , Up-Regulation , Variant Surface Glycoproteins, Trypanosoma/genetics
9.
Cell Microbiol ; 18(11): 1673-1688, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27110662

ABSTRACT

Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG is efficiently synthesized and exported to the cell surface, it was recently claimed that 2-3 fold more is synthesized than required, the excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate VSG turnover and find no evidence for rapid degradation, consistent with a model whereby VSG synthesis is precisely regulated to match requirements for a functional surface coat on each daughter cell. However, using a mutated version of the ESAG7 subunit of the transferrin receptor (E7:Ty) we confirm functional ERAD in trypanosomes. E7:Ty fails to assemble into transferrin receptors and accumulates in the ER, consistent with retention of misfolded protein, and its turnover is selectively rescued by the proteasomal inhibitor MG132. We also show that ER accumulation of E7:Ty does not induce an unfolded protein response. These data, along with the presence of ERAD orthologues in the Trypanosoma brucei genome, confirm ERAD in trypanosomes. We discuss scenarios in which ERAD could be critical to bloodstream parasites, and how these may have contributed to the evolution of antigenic variation in trypanosomes.


Subject(s)
Antigens, Protozoan/metabolism , Endoplasmic Reticulum-Associated Degradation , Trypanosoma brucei brucei/metabolism , Variant Surface Glycoproteins, Trypanosoma/metabolism , Protein Folding , Protein Stability , Proteolysis
10.
Nucleic Acids Res ; 44(10): 4763-84, 2016 06 02.
Article in English | MEDLINE | ID: mdl-26951375

ABSTRACT

Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying.


Subject(s)
DNA Replication , Origin Recognition Complex/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/genetics , Cell Cycle , Cell Nucleus/genetics , Origin Recognition Complex/physiology , Protozoan Proteins/physiology , Trypanosoma brucei brucei/metabolism
11.
Eukaryot Cell ; 14(11): 1094-101, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26318397

ABSTRACT

The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.


Subject(s)
Trypanosoma brucei brucei/metabolism , Unfolded Protein Response , Life Cycle Stages , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , SEC Translocation Channels , Trypanosoma brucei brucei/growth & development
12.
EuPA Open Proteom ; 7: 11-19, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25821719

ABSTRACT

We review and demonstrate how an empirical Bayes method, shrinking a protein's sample variance towards a pooled estimate, leads to far more powerful and stable inference to detect significant changes in protein abundance compared to ordinary t-tests. Using examples from isobaric mass labeled proteomic experiments we show how to analyze data from multiple experiments simultaneously, and discuss the effects of missing data on the inference. We also present easy to use open source software for normalization of mass spectrometry data and inference based on moderated test statistics.

13.
Trends Parasitol ; 30(1): 27-36, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24287149

ABSTRACT

Nuclear DNA replication is, arguably, the central cellular process in eukaryotes, because it drives propagation of life and intersects with many other genome reactions. Perhaps surprisingly, our understanding of nuclear DNA replication in kinetoplastids was limited until a clutch of studies emerged recently, revealing new insight into both the machinery and genome-wide coordination of the reaction. Here, we discuss how these studies suggest that the earliest acting components of the kinetoplastid nuclear DNA replication machinery - the factors that demarcate sites of the replication initiation, termed origins - are diverged from model eukaryotes. In addition, we discuss how origin usage and replication dynamics relate to the highly unusual organisation of transcription in the genome of Trypanosoma brucei.


Subject(s)
DNA Replication , DNA, Kinetoplast/genetics , Kinetoplastida/genetics , Biological Evolution , Cell Nucleus/genetics , Genome, Protozoan , Trypanosoma brucei brucei/genetics
14.
Mol Microbiol ; 87(4): 713-29, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23336702

ABSTRACT

In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock-down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.


Subject(s)
Membrane Proteins/metabolism , Mitochondria/metabolism , Protozoan Proteins/metabolism , Trypanosoma brucei brucei/metabolism , Trypanosomiasis, African/parasitology , DNA, Kinetoplast/genetics , DNA, Kinetoplast/metabolism , Humans , Membrane Proteins/genetics , Mitochondria/genetics , Protozoan Proteins/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/growth & development
15.
Cell Rep ; 2(1): 185-97, 2012 Jul 26.
Article in English | MEDLINE | ID: mdl-22840408

ABSTRACT

Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.


Subject(s)
DNA Replication/genetics , Genome, Protozoan , Replication Origin/physiology , Sequence Analysis, DNA , Transcription, Genetic/genetics , Trypanosoma brucei brucei/genetics , Binding Sites/genetics , Epistasis, Genetic , Gene Expression Regulation , Genome, Protozoan/genetics , Models, Biological , Origin Recognition Complex/analysis , Origin Recognition Complex/metabolism , Replication Origin/genetics
16.
PLoS One ; 7(3): e32674, 2012.
Article in English | MEDLINE | ID: mdl-22412905

ABSTRACT

DNA replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the pre-replication complex is composed of the Origin Recognition Complex (ORC), Cdc6 and the MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC is considered to be composed of six subunits, named Orc1-6, and monomeric Cdc6 is closely related in sequence to Orc1. However, ORC has been little explored in protists, and only a single ORC protein, related to both Orc1 and Cdc6, has been shown to act in DNA replication in Trypanosoma brucei. Here we identify three highly diverged putative T. brucei ORC components that interact with ORC1/CDC6 and contribute to cell division. Two of these factors are so diverged that we cannot determine if they are eukaryotic ORC subunit orthologues, or are parasite-specific replication factors. The other we show to be a highly diverged Orc4 orthologue, demonstrating that this is one of the most widely conserved ORC subunits in protists and revealing it to be a key element of eukaryotic ORC architecture. Additionally, we have examined interactions amongst the T. brucei MCM subunits and show that this has the conventional eukaryotic heterohexameric structure, suggesting that divergence in the T. brucei replication machinery is limited to the earliest steps in origin licensing.


Subject(s)
Cell Cycle Proteins/metabolism , Origin Recognition Complex/metabolism , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Cell Cycle Proteins/chemistry , DNA Helicases/chemistry , DNA Helicases/classification , DNA Helicases/genetics , Molecular Sequence Data , Origin Recognition Complex/chemistry , Phylogeny , Protein Binding , Protein Multimerization , Protein Subunits/genetics , RNA Interference , Sequence Alignment
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