Many but not all Eukaryotes have protein-enclosed compartments called vaults. Vaults are composed of multiple copies of the major vault protein, symmetrically assembled into a basket-like shell. A human cell contains approximately 100,000 vault particles, the vast majority localized to the cytosol but also observed in the nucleus and at the nuclear pore complex. Whilst there is intriguing structural information of the vault shell, the function of vaults remains largely elusive, apart from a potential contribution to mRNA maturation. We set out to explore the vault interactome in the early branching eukaryote Trypanosoma brucei employing a combination of affinity capture and TurboID proximity labelling. T. brucei encodes three major vault protein (MVP) paralogs, which exhibit a considerable degree of divergence. Unexpectedly, affinity capture proteomics with one MVP as a bait precipitated the other two paralogs, detected with similar intensities, indicating the possibility that all three are incorporated into the same particle. Dual color fluorescence microscopy of MVP pairs fused with different GFP-variants confirmed that all three paralogs are incorporated into a single vault shell. Our combined interactome data, including immune-isolations with varying stringencies, suggest a vault particle core composition of three MVPs homologs and the telomerase-associated protein 1 (TEP1), which has been described as a vault component in various organisms. Further, we demonstrate the association of vtRNA with the particle and suggest a cohort of potential transient vault interactors, dominated by RNA-binding proteins and splicing factors, which were found enriched in both orthogonal interactome approaches.

Biocenter University of Würzburg Würzburg Germany

Department of Parasitology Faculty of Science Charles University Prague Biocev Vestec Czech Republic

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