Abstract
Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent.
Highlights
Transfer of proteins and lipids between intracellular compartments by vesicular transport is a fundamental process and central to many eukaryotic cellular functions[1]
To initiate a systematic and unbiased identification of proteins interacting with the clathrin in T. cruzi we created transgenic epimastigotes harbouring epitope-tagged forms of the clathrin light chain (CLC) and EpsinR, both of which interact with the clathrin heavy chain
Using TcCLC as affinity handle, coupled with cryomilling, we identified a large cohort of candidate interacting proteins using label-free proteomics
Summary
Transfer of proteins and lipids between intracellular compartments by vesicular transport is a fundamental process and central to many eukaryotic cellular functions[1]. Multiple compartments and pathways comprise the exoand endocytic arms of the endomembrane system Transport between these compartments involves budding of protein-coated vesicles from donor membranes, a process essential for cargo sorting[2]. Membrane transport is well characterised in African trypanosomatids and lacks multiple proteins that are otherwise widely conserved This includes the AP-2 complex, a major mediator of clathrin sorting in endocytic systems many organisms[14,15,16]. For example a feature differentiating T. brucei and T. cruzi is clathrin-independent endocytosis, that in the latter operates mainly through the cytostome/cytopharynx[20,21] This structure is an invagination of the plasma membrane close to the flagellar pocket and which penetrates deep into the cytoplasm, frequently terminating at the posterior end of the cell and distal to the nucleus[22,23,24]. The presence of orthologs of Rab proteins associated with early and intermediate endosomes of other organisms in T. cruzi argues for a complex endomembrane system, and this matter has yet to be resolved
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