Proteomic Analysis of Clathrin Interactions in Trypanosomes Reveals Dynamic Evolution of Endocytosis

Abstract

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.

Missing Pieces of an Ancient Puzzle: Evolution of the Eukaryotic Membrane-Trafficking System

“…They, therefore, depend greatly on endocytosis so much so that its inhibition is lethal. Multiple adaptations have now been reported for the trypanosome endocytic system, including loss of the AP-2 complex (Manna et al 2013) and presence of apparently trypanosome-specific proteins that associate with clathrin and regulate the budding of clathrin-coated pits from the plasma membrane (Adung'a et al 2013). Although the function of these novel factors is not yet well characterized, this finding raises the possibility of new aspects of endocytic regulation that are not found in other eukaryotes.…”

Exploiting the Achilles’ heel of membrane trafficking in trypanosomes

“…Furthermore, the widely conserved heterotetrameric (AP)-2 adaptin complex is absent and is inversely correlated with the presence of VSG and thus antigenic variation, the principal mechanism of immune evasion [16, 12•]. Additional clathrin adaptor proteins are present and include both ENTH and ANTH-domain phosphoinositide-binding proteins [17] and a cohort of trypanosomatid-specific proteins [18]. Sorting surface trans -membrane proteins requires ubiquitylation, likely performed by divergent ubiquitin ligases, although these remain unidentified [19 •• ].…”

The Ancient Small GTPase Rab21 Functions in Intermediate Endocytic Steps in Trypanosomes

“…Endocytosis and recycling are extremely rapid processes and heavily reliant upon clathrin (CLH)-mediated mechanisms (2). Evolutionary modifications to the molecular machinery that subtend endocytosis include loss of the AP-2 cargo adaptor complex (3) and the presence of a cohort of lineage-specific clathrin-associated proteins (CAPs) (4). These adaptations, together with a very high endocytic flux, serve to maintain VSG surface density and rapidly remove surface-bound immune effectors, including anti-VSG antibodies (5).…”