Lineage-specific proteins essential for endocytosis in trypanosomes

Clathrin-mediated endocytosis (CME) is the most ancient endocytic mechanism known, and in many lineages the sole mechanism for internalization. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes we previously demonstrated that CME is AP-2 independent, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here we used a novel cryo-milling affinity isolation strategy to preserve transient low affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, TbEpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites.