title: Novel bilobe components in Trypanosoma brucei identified using proximity-dependent biotinylation.
abstract: The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.
authors.affiliation: Max F. Perutz Laboratories, University of Vienna and Medical University of Vienna, Vienna, Austria. brooke.morriswood@mfpl.ac.at
authors.forename: Brooke
authors.initials: B
authors.lastname: Morriswood
authors.forename: Katharina
authors.initials: K
authors.lastname: Havlicek
authors.forename: Lars
authors.initials: L
authors.lastname: Demmel
authors.forename: Sevil
authors.initials: S
authors.lastname: Yavuz
authors.forename: Marco
authors.initials: M
authors.lastname: Sealey-Cardona
authors.forename: Keni
authors.initials: K
authors.lastname: Vidilaseris
authors.forename: Dorothea
authors.initials: D
authors.lastname: Anrather
authors.forename: Julius
authors.initials: J
authors.lastname: Kostan
authors.forename: Kristina
authors.initials: K
authors.lastname: Djinovic-Carugo
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Roux
authors.forename: Graham
authors.initials: G
authors.lastname: Warren
mesh_terms: D019921:Biotinylation; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
chemical_list: D003598:Cytoskeletal Proteins; D015800:Protozoan Proteins