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.

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

“…Hence, the SIP approach is highly successful for analyzing discrete protein structures and regions of the cytoskeleton of cells. It adds to the existing repertoire of techniques, such as the proximity-dependent biotinylation approach (31).…”

SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament

“…Affinity Purification of Biotinylated Proteins and LC-MS/ MS-Affinity purification of biotinylated proteins was carried out essentially as described previously (35). Briefly, TbSAS-4-BirA*-HA was overexpressed by induction with 0.5 g/ml tetracycline for 24 h, and cells (ϳ5 ϫ 10 9 ) were incubated with 50 M biotin for an additional 24 h. Cells were collected by centrifugation, washed three times with PBS, and then treated with PEME buffer (100 mM PIPES, pH 6.9, 2 mM EGTA, 0.1 mM EDTA, 1 mM MgSO 4 ) containing 0.5% Nonidet P-40.…”

“…2C) were very likely to be derived from asymmetrical cytokinesis of the 2N2K cells. (35,38). We tagged TbSAS-4 with a C-terminal BirA* and an HA epitope and overexpressed the fusion protein in the procyclic form.…”

“…5C), and the purified biotinylated proteins from the cytosolic fraction and the cytoskeletal fraction were digested with trypsin and analyzed by LC-MS/MS. A total of 21 proteins, in addition to TbSAS-4 itself, were detected in the TbSAS-4-BirA*-HA overexpressed cells only, which contained four known FAZ proteins, FAZ6 (16), FAZ9 (16), FAZ10 (35), and FAZ11 (35); five known bilobe proteins (35); the tripartite attachment complex component p197 (39); KMP-11 (40); the flagellum targeting protein kharon1 (KH1) homolog (41); the putative centriole protein POC1 homolog (Tb927.10.2860); and eight hypothetical proteins ( Table 1). The identification of five bilobe proteins as TbSAS-4-binding partners and near neighbors likely was due to the fact that the new FAZ filament is assembled from the bilobe region (42) and, therefore, during the early S-phase when the FAZ filament just starts to elongate, TbSAS-4 at the tip of the newly assembled FAZ may make contact with these bilobe proteins.…”

Tubulin-binding cofactor C domain-containing protein TBCCD1 orchestrates cytoskeletal filament formation

“…Fluorescence and immunoelectron microscopy of known bi-lobe proteins, including centrin isoforms and various trypanosomatidspecific proteins, reveal the bi-lobe to possess a complex sub-structure (Esson et al, 2012;Gheiratmand et al, 2013;Morriswood et al, 2013). Immunofluorescence with either the monoclonal pan-centrin antibody 20H5 (Fig.…”