APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins inTrypanosoma brucei

Abstract

25Trypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne 26 disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and 27pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into 28 subdomains, each having specialized composition and function. Overall flagellum proteome has been 29 well-studied, but a critical gap in knowledge is the protein composition of individual flagellum 30 subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition 31 of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused 32 APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed 33 along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification 34 of biotinylated proteins yields a DRC1 "proximity proteome" showing good overlap with proteomes 35 obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that 36 is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is 37 excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of 38 biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. 39Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is 40 enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our 41 combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can 42 be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily 43 purified. 44 IMPORTANCE 45Sleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. 46The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different 48 specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is 49 need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that 50 APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and 51 has allowed identification of proximity proteomes for different subdomains. This capacity opens the 52 possibility to study the composition and function of other compartments. We further expect that this 53 approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a 54 model organism to study ciliopathies, heritable human diseases in which cilia function is impaired. 55 INTRODUCTION 56Trypanosoma brucei is a flagellated parasite that is transmitted between mammalian hosts by a 57 hematophagous vector, the tsetse fly, ...

The distinctive flagellar proteome of Euglena gracilis illuminates the complexities of protistan flagella adaptation

“…Flagellar proteomes as determined for various eukaryotes. Numbers of identified proteins for Euglena gracilis , Trypanosoma brucei (Broadhead et al ., 2006; Zhou et al ., 2010; Oberholzer et al ., 2011; Subota et al ., 2014; Dean et al ., 2017; Velez‐Ramirez et al ., 2021), Leishmania mexicana (Beneke et al ., 2019), Homo sapiens (Baker et al ., 2012), Mus musculus (Liu et al ., 2007), Rattus norvergicus (Mayer et al ., 2009), Xenopus laevis (Sim et al ., 2020), Chlamydomnas reinhardtii (Pazour et al ., 2005; Yang et al ., 2006; Lechtreck et al ., 2009; Zhao et al ., 2019), Dunaliealla salina (Jia et al ., 2010), Colpomenia bullosa (Fu et al ., 2014) and Tetrahymena thermophilia (Smith et al ., 2005) proteomes. Shaded regions indicate multiple studies performed in T .…”

“…gracilis orthologues of proteins with known locations in T . brucei (Varga et al ., 2017; Velez‐Ramirez et al ., 2021). The nonemergent flagellum of E .…”