Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition

Bringaud, Frédéric; Robinson, Derrick R.; Barradeau, Sébastien; Biteau, Nicolas; Baltz, D; Baltz, Théo

doi:10.1016/s0166-6851(00)00319-4

Cited by 56 publications

(77 citation statements)

References 39 publications

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“…Inhibition of FRDg and/or FRDm1 Genes Expression by RNAi-The inhibition by RNAi of FRDm1 and/or FRDg expression in the procyclic forms (9) was performed by expression of stem-loop "sense/antisense" molecules of the targeted sequences (5,10,22) introduced in the pLew79 expression vector (kindly provided by E. Wirtz and G. Cross) (35). Construction of the pLew-FRDg-SAS plasmid, which target FRDg expression, was described before (5).…”

Section: Methodsmentioning

confidence: 99%

“…The EATRO1125 procyclic form cell line (EATRO1125.T7T), constitutively expressing the T7 RNA polymerase gene and the tetracycline repressor under the dependence of a T7 RNA polymerase promoter for inducible control by tetracycline (10), was transformed with the plasmids. Trypanosome transfection and selection of phleomycin-resistant clones were performed as previously reported (10,36).…”

Section: Methodsmentioning

confidence: 99%

“…The pLew79 vector (35) was used to produce the hairpin double-stranded RNA molecules, as described before (5, 10, 22). The recombinant pLew79-derived plasmids were introduced into the EATRO1125.T7T cell line expressing the tetracycline repressor (10,35). We have selected five clonal cell lines, designated ⌬FRDm1-B4, ⌬FRDm1-G7, ⌬FRDg/m1-B5, ⌬FRDg/m1-C6, and ⌬FRDg-C2, the latter one being a ⌬FRDg clone different from the one previously analyzed (5).…”

Section: Three Frd Genes In T Brucei-we Previously Reportedmentioning

confidence: 99%

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A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

Coustou

Besteiro

Rivière

et al. 2005

Journal of Biological Chemistry

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115

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Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13 C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.Fumarate reductases (FRDs) 1 catalyze the reduction of fumarate to succinate and can be divided into two classes of enzymes: those belonging to a multimeric complex associated with the respiratory chain and transferring electrons from a quinol to fumarate and the soluble enzymes, which transfer electrons from a noncovalently bound cofactor (NADH or FADH 2 /FMNH 2 ) to fumarate. Most of the FRDs characterized so far belong to the first class (1). These FRDs are structurally similar to succinate dehydrogenases (SDH), complex II of the respiratory chain. The Shewanella putrefaciens FRD is functionally equivalent to the membrane-bound enzymes by accepting/transferring electrons from/to the respiratory chain but is a soluble enzyme lacking a membrane anchor (2). To date, only two examples of soluble FRDs not linked to the respiratory chain (second class) have been described. The yeast Saccharomyces cerevisiae expresses two soluble FRDs (cytosolic and promitochondrial) that use FADH 2 /FMNH 2 as an electron donor (3, 4), and the African trypanosome Trypanosoma brucei expresses a soluble NADH-dependent FRD located in the peroxisome-like organelle, called glycosome (5, 6). A phylogenetic analysis of FRDs and SDHs showed that the membrane-bound enzymes form a monophyletic group distantly related to the soluble enzymes, including the S. putrefaciens FRD (5). In 1980, Gest (7) proposed that the membrane-bou...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Three Frd Genes In T Brucei-we Previously Reportedmentioning

confidence: 99%

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A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

Coustou

Besteiro

Rivière

et al. 2005

Journal of Biological Chemistry

Self Cite

115

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“…Procyclic cells settled on microscope slides were fixed in −20°C methanol or, to extract cytoskeletons, incubated in 1% Igepal CA-630 and fixed. The mouse monoclonal AB1 antibody and the rabbit polyclonal anti-FTZC and anti-ClpGM6 (for the flagellum attachment zone length measurements) antibodies were used following published protocols (13,15,27). For more details, see SI Appendix.…”

Section: Two-dimensional Difference Gel Electrophoresis Comparison Ofmentioning

confidence: 99%

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

Varga

Moreira-Leite

Portman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.

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“…Initially we tagged one of the earliest described TZ proteins (TZPs), flagellar transition zone component (FTZC) (29), with enhanced YFP (eYFP) and confirmed the localization of the fusion protein at the TZ by microscopy (Fig. 1A).…”

Section: Ip-based Enrichment Of Tz Fraction Containing Ciliopathy Promentioning

confidence: 90%

Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

Dean

Moreira-Leite

Varga

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.transition zone | cilium/flagellum | BBSome | MKS/B9 complex | trypanosome C ilia, or flagella (the two terms are used here interchangeably), are multifunctional organelles that were present in the last common eukaryotic ancestor (1). Defects in cilia are responsible for pleiotropic human diseases (called ciliopathies) and their function is essential for pathogenesis in protozoan parasites (2, 3).During ciliogenesis, a microtubule organizing center called the "basal body" docks at the membrane. Basal bodies are built from nine microtubule triplets and two microtubules from each triplet extend to form a transition zone (TZ) and, ultimately, the axoneme. Thus, the TZ represents a structural junction between the basal body and the axoneme.As expected from its strategic position between the basal body and the axoneme, the TZ acts as a "ciliary gate" that controls ciliary composition and function (4). Many ciliopathies are caused by defects in complexes that associate with the TZ, including Meckel syndrome (MKS), Joubert syndrome and Bardet-Biedl syndrome (BBS). Studies using murine kidney epithelial cells and embryos suggest that the MKS complex demarcates the ciliary membrane by forming a diffusion barrier at the base of the cilium (5-7). On the other hand, the BBS complex (BBSome) is an adapter for intraflagellar transport (IFT) complexes that cross the TZ barrier to transport sensory proteins between the ciliary membrane and the cell body (8, 9).The TZ has distinctive structural features. At the proximal boundary of the TZ, where the basal body C tubule terminates, a "terminal plate" crosses the TZ. Studies in Tetrahymena suggest that the terminal plate contains pores for the passage of IFT "trains" that deliver axonemal components to the distal tip of flagella (10). Striated transitional fibers radiate from the distal end of the basal body triplets to join the plasma membrane (11-14), forming blades thought to create a physical barrier preventing vesicles from entering the ciliary lumen. Electron microscopy (EM) studies in Chlamydomonas suggest that...

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Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition

Cited by 56 publications

References 39 publications

A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

A Mitochondrial NADH-dependent Fumarate Reductase Involved in the Production of Succinate Excreted by Procyclic Trypanosoma brucei

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum

Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

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