The cytosolic iron-sulphur cluster assembly machinery in Trypanosoma brucei

: characterisation and function of the targeting complex

Abstract

Cytosolic and nuclear proteins containing iron-sulphur clusters (Fe-S) are implicated in essential cellular processes. The biogenesis of Fe-S and their insertion into these proteins is accomplished through the Cytosolic Iron-Sulphur Cluster Assembly Machinery (CIA). Most of our knowledge on the modus operandi of the CIA derives from studies in yeast and human cells, but recently, the proteins comprising the early part of this pathway have been characterised in Trypanosoma brucei, a protozoan parasite of animals and humans. However, an outstanding question in this organism is the way by which the CIA machinery directs and inserts Fe-S clusters into client apo-proteins. The main goal of this study was to answer this question, bridging this gap in the knowledge.

Homologues of human and yeast CIA proteins are present in T. brucei. The proteins TbMIP18, TbMMS19, and TbCIA1 were found to associate and form a complex, called the CIA targeting complex, or CIATC. Proteomic analysis revealed that Fe-S proteins interact with the CIATC. Affinity pull-downs confirmed some of these interactions, and also revealed that the CIATC associates with TbNAR1, a previously described CIA member, suggesting that this complex has a role in transferring Fe-S from the upstream CIA members to client apo-proteins.

Functional analyses demonstrated that silencing TbMIP18 impairs the activity of cytosolic Fe-S proteins, interferes with intracellular iron levels, and impairs the cell growth of procyclic stage parasites. Furthermore, overexpressing the C-terminal domain of TbMMS19 led to a severe defect in cell growth. TbMIP18 and TbCIA1 were shown to interact with TbMMS19 through its C-terminal domain, and in vitro evidence suggested that a region of ca. 200 amino acids is the binding site for a tight interaction between TbMIP18 and TbMMS19, a feature that could be exploited for disruption of the protein- protein interactions of this complex.

Date of Award	7 Dec 2017
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Terry K Smith (Supervisor) & Stuart MacNeill (Supervisor)