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Endocytosis is a vital cellular process maintaining the cell surface, modulating signal transduction and facilitating nutrient acquisition. In metazoa, multiple endocytic modes are recognized, but for many unicellular organisms the process is likely dominated by the ancient clathrin-mediated pathway. The endocytic system of the highly divergent trypanosomatid Trypanosoma brucei exhibits many unusual features, including a restricted site of internalization, dominance of the plasma membrane by GPI-anchored proteins, absence of the AP2 complex and an exceptionally high rate. Here we asked if the proteins subtending clathrin trafficking in trypanosomes are exclusively related to those of higher eukaryotes or if novel, potentially taxon-specific proteins operate. Co-immunoprecipitation identified twelve T. brucei clathrin-associating proteins (TbCAPs), which partially colocalized with clathrin. Critically, eight TbCAPs are restricted to trypanosomatid genomes and all of these are required for robust cell proliferation. A subset, TbCAP100, TbCAP116, TbCAP161 and TbCAP334, were implicated in distinct endocytic steps by detailed analysis of knockdown cells. Coupled with the absence of orthologs for many metazoan and fungal endocytic factors, these data suggest that clathrin interactions in trypanosomes are highly lineage-specific, and indicate substantial evolutionary diversity within clathrin-mediated endocytosis mechanisms across the eukaryotes.
Membrane trafficking is essential for nutrient uptake, regulation of signal transduction, receptor expression and control, maintenance of cell polarity, antigen presentation and parasite virulence mechanisms among other functions [1-4]. The best-characterized endocytic pathway involves the coat protein clathrin, but additional mechanisms are known for metazoan cells. Endocytosis can thus be defined as either clathrin-mediated endocytosis (CME) [5-7] or clathrin-independent [2, 8, 9]; examples of the latter include RhoA-dependent, cdc42-dependent, Arf6-dependent and caveolin-dependent pathways . Comparative genomics suggests that clathrin is an ancient feature of eukaryotes , as the protein is present in all modern supergroups and hence likely also present in the last eukaryotic common ancestor (LECA), while the key proteins defining clathrin-independent pathways, where known, appear to be lineage-specific, and therefore are inferred as innovations that came post-LECA . In unicellular pathogens, the plasma membrane and the endo/exocytic system also represent the principal interface with the host and contribute to immune evasion, host cell invasion and defense, all vital processes for the effective infection and persistence of many pathogens, including Plasmodium, Toxoplasma, pathogenic fungi and trypanosomes [3, 4].
Trypanosoma brucei is a flagellate protozoan and the causative agent of sleeping sickness in humans and n'agana in cattle. The parasite is responsible for severe morbidity and mortality, significantly limiting human economic activities in sub-Saharan Africa. Related organisms cause a spectrum of morbidities including Chagas' disease in South America and Kala Azar in Asia, and combined have an impact that encompasses much of the globe. Trypanosoma brucei infections are characterized by the chronic presence of the parasite within the bloodstream and lymphatic systems. To evade immune clearance, bloodstream form parasites (BSFs) undergo antigenic variation by sequential expression of antigenically distinct glycosyl-phosphatidylinositol (GPI)-anchored variant surface glycoproteins (VSG; [11, 12]). By contrast, in the insect vector, the major proliferative procyclic form (PCF) expresses an invariant protease-resistant GPI-anchored protein, procyclin . The two life stages are marked by significant morphological, structural and biochemical changes, and of particular relevance is the developmental regulation of endocytosis, estimated at ∼20-fold higher in BSF than PCF .
The endocytic system of T. brucei has been comparatively well studied, and many protein participants are now known. The endomembrane system is extremely polarized, with most organelles of the exo- and endocytic pathways located at the posterior cell pole, while exchange with the plasma membrane is restricted to the flagellar pocket (FP), an invagination at the base of the flagellum. Rapid endocytosis in BSFs contributes to immune defense by efficient recycling of VSG and fast turnover of invariant surface proteins, both of which likely contribute to clearance of surface bound antibodies [14, 15]. All T. brucei endocytosis is clathrin mediated , but the AP-2 adaptin complex, a hallmark of plasma membrane clathrin recruitment and cargo selection in most organisms, is absent . Important proteins include several Rab GTPases [18-22], ADP-ribosylation factor 1 (ARF1) , clathrin [16, 24], actin  and myosin , and the importance of ubiquitylation in sorting and internalization has recently been demonstrated [15, 27], while the process appears dynamin-independent . However, orthologs of a number of critical endocytic proteins have not been identified; while this may reflect extreme sequence divergence, it is also possible that the failure to identify these genes reflects true absence .
In trypanosomes, surface proteins are packed into clathrin-coated pits (CCPs) that undergo scission into ∼135 nm diameter clathrin-coated vesicles (CCVs) . TbEpsinR, the only characterized trypanosome endocytic clathrin adaptor to date, has limited involvement in bulk membrane internalization, albeit with a general role in membrane protein endocytosis . The CCVs shed their clathrin coat and subsequently dock and fuse with Rab5 early endosomes. Cargo are sorted and packaged into 50–60 nm diameter CCVs [24, 31], but VSG is concentrated into disk-like Rab11-positive exocytic structures that subsequently fuse with the FP [24, 32-34, 19]. Clathrin is associated with the membranes of the FP/sorting endosomes and also the trans-Golgi cisterna [35, 24], an indication that the clathrin interaction network is likely complex and participates in multiple pathways.
Despite this level of understanding, it is unclear if the failure to detect endocytic protein genes in the trypanosome genome represents a secondary reduction accompanying a parasitic lifestyle, or reflects deep evolutionary divergence between trafficking mechanisms of the different eukaryotic supergroups, and the major proteins required to recruit clathrin to the FP membrane and other organelles remain unknown. Given the prominent role that the FP plays both in trafficking and as part of the spatial organization of organelles in many trypanosomatids, this is a crucial gap in understanding. Demonstration by RNAi of endocytic essentiality in vivo suggests these pathways offer therapeutic possibilities, while a recent phenocopy using chemical biology suggests that this may well be exploitable [16, 36].
Here we used co-immunoprecipitation (co-IP), mass spectrometry (MS) proteomics, genomic in situ-tagging, immunofluorescence and electron microscopy to identify and validate a cohort of trypanosome clathrin-associating proteins (TbCAPs). Functional analysis of a subset of TbCAPs confirmed roles in clathrin-mediated trafficking. Remarkably, many TbCAPs appear to be restricted to the trypanosomatids, suggesting considerable evolutionary divergence in clathrin-mediated transport mechanisms across taxa.
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- Materials and Methods
- Supporting Information
Understanding the evolutionary origins, diversity and functions of intracellular trafficking pathways across eukaryotes has benefited from extensive genome sequencing coupled to structural studies; these revealed unexpected connections between pathways, together with great complexity in the earliest eukaryotes, including those predating the LECA [84-87]. This, together with recognition that many critical players mediating trafficking are members of paralogous families, i.e. coatomer, Rabs, SNAREs, etc., provides a simple but powerful paradigm for the evolution of new pathways. Secondary loss is also common, with the result that a better understanding of the drivers sculpting the endomembrane system is emerging, despite the absence of insights into how selective pressure has been applied . These in silico analyses, however, cannot capture much mechanistic detail, and more critically, due to the superior datasets available for animals and fungi, our view is subject to sampling bias.
One very ancient pathway is CME, but multiple modes of endocytosis are now recognized, defined by differential morphology and specific protein requirements . We asked if CME is mechanistically equivalent or distinct in diverse evolutionary lineages, especially as morphological data are unavailable or non-discriminatory for many organisms. We selected proteomics for unbiased sampling of clathrin-interacting proteins in T. brucei, an organism with considerable prior characterization of trafficking . CME is the sole endocytic mechanism in trypanosomes, but clathrin is also present on the Golgi complex and additional endosomal structures, suggesting involvement in multiple pathways, but with many conspicuous absences by in silico analysis; this includes for example orthologs of well characterized clathrin interactors like auxilin [35, 16, 10, 29]; Grunfelder et al., 2003). Subcellular fractionation in mammalian cells indicates substoichiometric relationships between clathrin and other CCV polypeptides , suggesting we would encounter low levels of clathrin associating proteins. Consequently we designed a hierarchical validation strategy (Figure S1), incorporating localization, reverse immunoisolation and gene silencing for direct functional inference of bona fide clathrin association.
We identified a large cohort of proteins, many of which are likely clathrin-associated, and selected the most significant for initial validation by localization, and validated twelve proteins with high confidence as TbCAPs, including several expected proteins, i.e. TbHsc70, TbMyo1 and TbTOR2, based on known functions in other organisms (TbHsc70) or trypanosomes (TbMyoI and TbTOR2; [42, 26]). The location reported here for TbTOR1 and TbTORL1 is at variance with that reported recently using polyclonal antibodies [42, 43], and therefore the direct association of these molecules with clathin-containing endomembrane compartments is uncertain. Of the remaining proteins, only TbCAP100, had broad taxonomic representation, and the remainder appeared restricted to trypanosomatids. Surprisingly, this distribution excluded metamonads, a sub-branch of the Excavata, suggesting restricted distribution, which may reflect trypanosomatid-specific mechanisms subtending clathrin trafficking pathways. Interestingly, TbCAP100 bears a Vps51/67 domain, and is likely the ortholog of H. sapiens Vps51.
Of the trypanosome-specific cohort, four have no assignable domains while the remainder lack an obvious functional signature. The presence of WD40 and ankyrin domains in TbCAP116 and TbCAP334 suggests roles in protein–protein interactions, and evidence from gene silencing does indicate specific functions in endocytosis. The frequent emergence of a cytokinesis failure phenotype is one that is associated with many trafficking blocks and likely reflects incorrectly targeted protein and/or lipids to the cytokinesis furrow or other replicating structures at cell division. A specific impact on ConA internalization was also found on silencing of TbHsc70, TbCAP100, TbCAP116, TbCAP161 and TbCAP334, suggestive of a failure to endocytose VSG. This conclusion is supported by a decreased intracellular pool of ISG75 in Tb Hsc70, TbCAP100, TbCAP116 and TbCAP161 KD cells, which indicates defects in early stages of endocytosis. Whereas increased internal levels of ISG75 for TbCAP334 suggest functions later in the endosomal system, allowing internal accumulation but decreased turnover of ISG75, by either defects in recycling or late endocytic steps [27, 41]. The presence of a BigEye phenotype is both consistent with a role in CME, but also suggests a less prominent role than clathrin itself, and is more reminiscent of the phenotype of TbEpsinR silenced cells .
Our electron microscopy analysis revealed finer details of the effect of protein ablation, and allowed us to start to assign TbCAPs into different molecular functions: TbCAP100, TbCAP161 and TbCAP334 seems to be required for the curving of flat areas of clathrin-coated FP membrane to give rise to CCPs. Indeed, in the absence of these proteins, the FP can still recruit and assemble clathrin, but CME is blocked by prevention of CCP formation. TbCAP116 seem to play a different role: upon ablation of the protein, cells are competent for CCP formation, although the process is somehow delayed. Importantly, all of these data support roles for TbCAP100, TbCAP116, TbCAP161 and TbCAP334 at post-recruitment steps, consistent with the isolation protocol used, which included detergent, and hence indicating that these TbCAPs likely function in the removal of clathrin from the membrane. Interestingly, many of the proteins known to be involved in CCP neck constriction and scission are unidentified in the trypanosome genome , suggesting that TbCAPs represent a mechanism that arose specifically in the trypanosome lineage. Ongoing investigations are directed at exploring clathrin membrane anchors and interactions with the actin system.
A unifying aspect of ablation of different TbCAPs is that the absolute amount of clathrin triskelion being recruited to the FP is increased, and much of this additional clathrin is assembled into large flat sheets that fail or delay to mature into pits. Clathrin, as well as F-Bar and ENTH-domain containing proteins have been implicated as initiators of membrane curvature. Our genetic analysis coupled with ultra-structural studies suggests that TbCAPs function in the same pathway, although their precise mechanistic action remains to be elucidated. The presence of large expansions of flat clathrin-coated membrane is distinct from a model of gradual addition of triskelions to a growing, highly curved invagination , and favors a mechanism of pre-assembly of flat lattices as clathrin reservoirs to sustain the formation of a large number of coated pits and vesicles [91, 92]. This may reflect a specific adaptation to support the extremely fast endocytic flux at the FP.
Why does ablation of TbCAPs cause a severe cytokinesis defect? It is relevant here to recognize that TbCAPs may participate in functions beyond CME. A cytokinesis failure is unlikely to be a result of starvation as RNAi-induced cells are growing in size, divide internal organelles and complete mitosis. The defect is specific to cell division, and raises the possibility that TbCAPs function in signaling, recruiting division machinery components, and/or assembly of the cleavage furrow. Alternatively, the cytokinesis failure could be a secondary effect resulting from morphological defects. Specifically, an enlarged FP may perturb the highly ordered spatial polarization of the cell and impair the precise positioning and migration necessary for cell division. Similar effects have been observed by RNAi-mediated ablation, in this particular life cycle stage, of proteins involved in flagellar motility , and as well as for silencing of other endocytic components.
Overall, while the precise functions of most TbCAPs remain to be fully elucidated, our data indicate that a highly divergent clathrin interactome in trypansomes and consistent with earlier in silico predictions. However, the system is not necessarily simplified, but rather may comprise a cohort of pan-eukaryotic proteins together with a lineage-specific group, precisely as the animal and fungal systems. This suggests that CME and other clathrin-mediated functions are mechanistically divergent, and the simple designation of pathways as clathrin-dependent or independent fails to capture the true functional diversity between lineages. Of interest here is that several of the conserved interaction sites in the N-terminal region of the clathrin heavy chain remain well conserved between trypanosomes and higher eukaryotes including clathrin box motif (CBM) (H. sapiens TLQIF/KMK, T. brucei NLQIF/RLK) and the recently described fourth site (H. sapiens EHLQLQN versus T. brucei EVFGLNS) . By contrast the AP-2 binding site does not appear retained (H. sapiens 188-RKVSQ versus T. brucei NNSGR), fully consistent with the loss of AP-2.
The uncovering of the TbCAPs also provides potential for detailed mechanistic analysis of CME in trypanosomes. This mechanistic diversity has several parallels: e.g. (i) the independent evolution of ARF proteins between Trypanosoma and Opistokhonta , where the functions of these small GTPases are likely highly novel; and (ii) perhaps more similarly, the unique compartmentalization of glycolysis by Trypanosoma into the glycosome, such that an otherwise conventional pathway is sequestered and controlled by highly distinct mechanisms compared with mammalian cells, and which offers therapeutic potential . We suggest that the mechanistic details of trypanosome CME may also offer such opportunity due to high levels of diversity between the parasite and host.