Mitochondrial ATP production and energy-buffering in the human pathogen Trypanosoma brucei

Gao, Fei

Abstract

The mitochondrion plays a vital role in the cellular energy (ATP) provision of the human pathogen Trypanosoma brucei. ATP is produced in the mitochondrial matrix from ADP and inorganic phosphate by substrate-level and oxidative phosphorylation. To maintain the cellular energy provision, ATP has to be exported from the mitochondrial matrix, and ADP and Pi imported from the cytosol. This exchange is in eukaryotes facilitated by specific mitochondrial carrier family (MCF) proteins, i.e. the ADP/ATP carrier and the phosphate carrier located in the mitochondrial inner membrane.

The MCF protein inventory of T. brucei from our lab’s previous study showed two putative ADP/ATP carriers, i.e. TbMCP5 and TbMCP15, and two putative phosphate carriers, i.e. TbMCP8 and TbMCP11. In order to confirm the mitochondrial carrier family proteins and further study mitochondrial energy metabolism, proteomic analysis of mitochondria in both bloodstream form and procyclic form T. brucei were performed. The preliminary results showed that many enzymes required for TCA cycle, lipid metabolism, amino acid metabolism, nucleotide metabolism exist in both bloodstream form and procyclic form T.brucei. (Data were not shown in this thesis). Functional characterisation of TbMCP5 and TbMCP15 (Chapter II) revealed that only TbMCP5 functions as a mitochondrial ADP/ATP carrier, while the function of TbMCP15 remains unknown. TbMCP5 is able to functionally complement growth of ADP/ATP-carrier deficient Saccharomyces cerevisiae on a non-fermentable carbon source, and mitochondrial transport experiments (done by Ludovic Pelosi) revealed that TbMCP5 has similar biochemical ADP/ATP transport kinetics to the prototypical ADP/ATP carrier ScAnc2p from yeast. Silencing of TbMCP5 expression confirmed that this MCF protein is essential for the survival of the procyclic form T. brucei and represents the only ADP/ATP exchanger present in the procyclic form mitochondrion. Functional characterisation of the putative mitochondrial phosphate carriers TbMCP8 and TbMCP11 (Chapter III) revealed that only TbMCP11 is expressed in the bloodstream form and procyclic form of T. brucei. Silencing of TbMCP11 expression in the bloodstream form had no effect on growth, whereas in the procyclic form it resulted in a lethal growth phenotype. Heterologous expression of TbMCP11 in mitochondrial phosphate carrier-deficient S. cerevisiae restored its growth on a non-fermentable carbon source as well as the phosphate-dependent swelling of its mitochondria.

ATP exported from the mitochondrion is either directly used by the rest of the cell or stored as an energy buffer, which can be used during periods of high energy demand. The phosphoarginine/arginine kinase energy (ATP) buffering system of T. brucei consists of three different arginine kinase isoforms, i.e. TbAK1-3 (Chapter IV). The TbAK1-3 isoforms are localised in different subcellular compartments, here respectively the flagellum, glycosome and cytosol, and are dependent on the presence of specific organellar targeting signals. Silencing of total TbAK expression in the procyclic form of T. brucei resulted in a significant growth defect, and was even lethal in the presence of the oxidative challenging agent hydrogen peroxide. These results suggest an important role of the T. brucei phosphoarginine/arginine kinase energy-buffering system in oxidative stress defence. Additional roles of the TbAK isoforms in the different subcellular compartments are proposed.

In conclusion, the mitochondrial ADP/ATP carrier TbMCP5 and phosphate carrier TbMCP11, and the TbAK1-3-dependant energy buffering system play an essential role in the maintenance of the T.brucei energy metabolism.

Citation

Gao, F. (2013). Mitochondrial ATP production and energy-buffering in the human pathogen Trypanosoma brucei. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4215678

• Thesis Type: Thesis
• Deposit Date: Jun 6, 2014
• Publicly Available Date: Feb 23, 2023
• Keywords: Biological sciences
• Public URL: https://hull-repository.worktribe.com/output/4215678
• Additional Information: Department of Biological Sciences, The University of Hull
• Award Date: Mar 1, 2013