The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis.

Cordula M Boehm,Mary Munson,Mark C Field,Paul T Manna,Michael P Rout,Samson Obado,Brian T Chait,Gwyn W Gould,Catarina Gadelha,Alexandra Kaupisch

doi:10.1371/journal.ppat.1006063

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 isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

Highlights

Nutrient uptake, immune evasion, developmental progression and sensitivity to several drugs all require the action of the membrane transport system in trypanosomes [1]
The membrane transport system of Trypanosoma brucei is comparatively well studied, and whilst generally conserved with other eukaryotes in overall architecture [3], the system has been adapted to the parasitic life style and the specific challenges associated with survival in the mammalian bloodstream [4,5,1]
The membrane trafficking system is mediated by a core of paralogous protein families, Rabs, SNAREs, vesicle coats and tethering complexes

Summary

Introduction

Immune evasion, developmental progression and sensitivity to several drugs all require the action of the membrane transport system in trypanosomes [1]. In African trypanosomes, maintaining the composition and homeostasis of the surface proteome is closely connected to many of these processes, including clearance of acquired immune effectors from the surface coat, resistance to innate immune factors, uptake of heme and iron together with sensitivity to suramin and possibly additional trypanocides [2,1]. The membrane trafficking system is mediated by a core of paralogous protein families, Rabs, SNAREs, vesicle coats and tethering complexes. The SNARE and Rab families are large, and individual members associated with distinct subcellular organelles controling transport between compartments [6,7,8,9,10]. None of the MTC complexes have been studied in trypanosomes and their functions and compositions remain uncharacterised

Methods

Results

Conclusion