title: Lineage-specific proteins essential for endocytosis in trypanosomes.
abstract: Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. 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 independent of the AP-2 adaptor protein complex, 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 cryomilling 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,Trypanosoma brucei(Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. 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.
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Manna
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: Samson O
authors.initials: SO
authors.lastname: Obado
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK.
authors.forename: Cordula
authors.identifier: 0000-0002-1746-2450
authors.initials: C
authors.lastname: Boehm
authors.affiliation: School of Life Sciences, University of Nottingham, Nottingham NG2 7UH, UK.
authors.forename: Catarina
authors.initials: C
authors.lastname: Gadelha
authors.affiliation: California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.affiliation: School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK mfield@mac.com.
authors.forename: Mark C
authors.identifier: 0000-0002-4817-6035
authors.initials: MC
authors.lastname: Field
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D005075:Biological Evolution; D002966:Clathrin / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004705:Endocytosis; D006801:Humans; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014346:Trypanosoma brucei brucei; D014352:Trypanosomiasis / Q000378:metabolism*
chemical_list: D033962:Adaptor Protein Complex 2; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C475910:CLINT1 protein, human; D002966:Clathrin; D003598:Cytoskeletal Proteins; C484407:KIFAP3 protein, human; D015800:Protozoan Proteins; D050600:SNARE Proteins; D018808:Transcription Factor AP-1; C451304:AAK1 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Clathrin; Cytoskeleton; Evolution; Flagellum; Morphology; Trafficking; Trypanosoma