Abstract:Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity is… Show more
“…2B; ). This is in contrast to a known exocytic protein TbSec15, which has been shown by SIM to form punctae interspersed with clathrin-positive structures at the flagellar pocket, but with essentially no colocalisation (Boehm et al, 2017). Fig.…”
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.
“…2B; ). This is in contrast to a known exocytic protein TbSec15, which has been shown by SIM to form punctae interspersed with clathrin-positive structures at the flagellar pocket, but with essentially no colocalisation (Boehm et al, 2017). Fig.…”
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.
“…The violin plot in Figure 3B shows specific enrichment of IFT and dynein knockdowns in the >4C pool, relative to other cohorts of knockdowns. Exocyst components, primarily involved in exocytosis (35), were included as a negative control cohort; indeed, none of the exocyst components register enrichment in the >4C pool, nor in any other pool (see below). Enrichment of individual chaperonin T-complex components, dyneins, and IFT factors in the >4C pool is illustrated in Figure 3C.…”
Section: Cytokinesis Defects Associated With Endoreduplicationmentioning
Trypanosomatids, which include major pathogens of humans and livestock, are divergent eukaryotes for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle regulators in bloodstream form Trypanosoma brucei. We induced massive parallel knockdown and sorted the perturbed population into cell cycle stages using flow cytometry. RNAi-targets were deep-sequenced from each stage and cell cycle profiles were digitally reconstructed at a genomic scale. We identify hundreds of proteins that impact cell cycle progression; glycolytic enzymes required for G1S progression, DNA replication factors, mitosis regulators, proteasome and kinetochore complex components required for G2M progression, flagellar and cytoskeletal components required for cytokinesis, mRNA-binding factors, protein kinases and many previously uncharacterised proteins. The outputs facilitate functional annotation and drug-target prioritisation and provide comprehensive functional genomic evidence for the machineries, pathways and regulators that coordinate progression through the trypanosome cell cycle.The data can be searched and browsed using an interactive, open access, online data visualization tool (https://tryp-cycle.onrender.com).
“…In yeast, late steps in exocytosis require the exocyst and octameric complex. Significantly, a novel subunit of exocyst is present in trypanosomes and is conserved across the entire lineage …”
Section: Exocytosis: Simplicity With Variationmentioning
The kinetoplastida and their close relatives are unicellular organisms prevalent within the biosphere and important for significant impacts on global health, economy and ecosystems. They are, under most models, an early branching lineage. Individual species adapted to highly diverse environments by adopting complex life styles; parasitic species can infect a wide range of eukaryotic hosts, while many relatives are free-living and some autotrophic from acquiring a plastid for photosynthesis. Adaptation is especially evident in the evolution of kinetoplastid cell surface architecture and is supported by endomembrane trafficking and serves as a platform for interaction with its environment. Here we summarize and discuss recent genomic and experimental studies of the protein trafficking system in kinetoplastids, with focus on the composition and function of the surface as well as mechanisms for constructing, maintaining and regulating the cell surface proteome. We hope this provides a broad view of how protein trafficking contributes to the intricate and dynamic host-parasite interfaces that are critical for successful environmental adaptation of this highly important lineage.
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