Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

Vidilaseris, Keni; Morriswood, Brooke; Kontaxis, Georg; Dong, Gang

doi:10.1074/jbc.m113.529032

Cited by 15 publications

(63 citation statements)

References 27 publications

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“…Cloning -Cloning of TbBILBO1-FL has been described previously (14). Briefly, this involved amplification of the ORF from T. brucei genomic DNA by PCR and ligation into the HM15b vector.…”

Section: Methodsmentioning

confidence: 99%

“…Transfection of cells was carried out as described previously (14). Transfected cells were cultured overnight in SDM79 medium supplemented with 7.5 g/ml hemin and 20% (w/v) heat-inactivated fetal calf serum (SigmaAldrich) at 27°C.…”

Section: Methodsmentioning

confidence: 99%

“…N-terminal YFP-tagged TbBILBO1 truncation constructs were subcloned from HM15b-TbBILBO1-FL into the pXS2-YFP vector as described previously (14). All TbBILBO1 truncation constructs carrying an N-terminal His 6 -MBP or MBPHis 10 tag were subcloned from HM15b-TbBILBO1-FL into either the HM15b or the custom expression vector MalpET.…”

Section: Methodsmentioning

confidence: 99%

“…Cloning of full-length TbBILBO1 (TbBILBO1-FL) and structure-based functional analysis of the N-terminal domain (NTD) have been described recently (14). Here, we report our follow-up structural dissection of the other domains of TbBILBO1 and their respective roles in protein oligomerization.…”

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confidence: 99%

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Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Vidilaseris

Shimanovskaya

Esson

et al. 2014

Journal of Biological Chemistry

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Background: TbBILBO1 is the only known protein component of the flagellar pocket collar, but its assembly remains unknown. Results: Structural dissections of the three different domains of TbBILBO1 revealed their roles in protein assembly. Conclusion: TbBILBO1 forms a linear filament that interacts laterally to form a fibrous bundle. Significance: The data show how two types of coiled coil act together to assemble TbBILBO1 into long filaments.

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“…Cloning -Cloning of TbBILBO1-FL has been described previously (14). Briefly, this involved amplification of the ORF from T. brucei genomic DNA by PCR and ligation into the HM15b vector.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Vidilaseris

Shimanovskaya

Esson

et al. 2014

Journal of Biological Chemistry

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“…Of these, the best characterized is an electrondense horseshoe-shaped structure named the flagellar pocket collar (FPC) (4). The only known component of the FPC is the protein TbBILBO1, which has been localized to the FPC by immunoelectron microscopy (immuno-EM) and shown to be essential for FP biogenesis (9)(10)(11)(12).…”

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confidence: 99%

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Morriswood

Schmidt

2015

Eukaryot Cell

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The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo-and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.T rypanosoma brucei is an important parasite of humans and domestic animals in sub-Saharan Africa, as the causative agent of sleeping sickness and nagana, respectively. Its complex life cycle involves transitions between tsetse fly vectors (its definitive hosts) and mammalian intermediate hosts. This life cycle involves a number of different cell stages, of which the procyclic form (found in the tsetse fly) and the slender bloodstream form (BSF) (found in the mammalian bloodstream) are the best studied in a laboratory setting. The procyclic form and the BSF of T. brucei share similar cytoskeletal architectures (1, 2).The principal feature of this cytoskeleton is a corset of microtubules that lie directly underneath the plasma membrane and impart to the cell its distinctive shape (3). A single invagination of the plasma membrane, termed the flagellar pocket (FP), constitutes a distinct subdomain and is found at the posterior end of the cell (4). The FP is the site of all endo-and exocytic traffic (5, 6). Abutting the FP membrane is a basal body that nucleates the single flagellum of the trypanosome cell. The flagellum exits the FP and is adhered longitudinally to the cell body along a left-handed helical path (7). Once outside the FP, the axoneme of the flagellum is paralleled by an associated intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of a paracrystalline lattice and is associated with cellular motility (8). Nucleated adjacent to the basal body is a specialized microtubule quartet that traces around the FP and then underlies the flagellum as far as the anterior end of the cell (4).The small cylinder of membrane that connects the FP to the rest of the plasma membrane constitutes a third subdomain and is called the flagellar pocket neck (FPN) (4). A number of discrete cytoskeletal structures cluster around the FPN membrane on its cytoplasmic face. Of these, the best characterized is an electrondense horseshoe-shaped structure named th...

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TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei

et al. 2022

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The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the flagellar pocket collar (FPC). TbBILBO1 is the first-described FPC protein of Trypanosoma brucei. It is essential for parasite survival, FP and FPC biogenesis. In this work, we characterize TbKINX1B, a novel TbBILBO1 partner. We demonstrate that TbKINX1B is located on the basal bodies, the microtubule quartet (a set of four microtubules) and the FPC in T. brucei. Down-regulation of TbKINX1B by RNA interference in bloodstream forms is lethal, inducing an overall disturbance in the endomembrane network. In procyclic forms, the RNAi knockdown of TbKINX1B leads to a minor phenotype with a small number of cells displaying epimastigote-like morphologies, with a misplaced kinetoplast. Our results characterize TbKINX1B as the first putative kinesin to be localized both at the basal bodies and the FPC with a potential role in transporting cargo along with the microtubule quartet.

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Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

Cited by 15 publications

References 27 publications

Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

TbKINX1B: a novel BILBO1 partner and an essential protein in bloodstream form Trypanosoma brucei

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