BILBO1 is a scaffold protein of the flagellar pocket collar in the pathogen Trypanosoma brucei

PLoS Pathog. 2015 Mar 30;11(3):e1004654. doi: 10.1371/journal.ppat.1004654. eCollection 2015 Mar.

Abstract

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 annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Cell Line, Tumor
  • Flagella / genetics
  • Flagella / metabolism*
  • Humans
  • Mutation
  • Protein Multimerization / physiology*
  • Protein Structure, Tertiary
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / metabolism*

Substances

  • Calcium-Binding Proteins
  • Protozoan Proteins

Grants and funding

This work was funded by the Centre National de la Recherche Scientifique (CNRS), Aquitaine Regional Council Grant - 20111301014, (ANR-09-BLAN-0074), Preciput-ANR grant from the University of Bordeaux and a Labex - “ParaFrap” - Alliance Française Contre les Maladies Parasitaires. CF was a recipient of the Région Martinique Ph.D. fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.