Characterization of Ciliate Mitochondrial Function


Naqvi, Anjum Zehra, 2017 Characterization of Ciliate Mitochondrial Function, Flinders University, School of Biological Sciences

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The electron transport chain and oxidative phosphorylation are two major phenomena which occur in mitochondria simultaneously. These processes are facilitated by the protein complexes of the electron transport chain, located in the inner membrane of mitochondria in eukaryotes. Among these complexes, ATP synthase is an enzyme of a crucial importance. A typical eukaryote ATP synthase consist of an F1 domain and an Fₒ domain. The F1 domain consists of five subunits including α, β, γ, δ and ε. The Fₒ domain consists of a, b, c, d, OSCP and F6. This enzyme couples oxidation of respiratory substrates to ATP synthesis in the mitochondria. The core subunits of the ATP synthase were thought to be well conserved across all forms of life. However, the availability of genome data has revealed that organisms belonging to the alveolate super phylum which includes Plasmodium, dinoflagellate algae and ciliates (including Tetrahymena thermophila) do not have genes homologous to those in other organisms that code for subunits that are critical for the function of the Fo domain. One of the apparently missing subunit is ‘a’ which plays a crucial role in the proton translocation during the process of ATP synthesis in mitochondria. It was hypothesized that as alveolates appear to have a functional ATP synthase, they must have some alternative subunits, with low homology to other species that fulfill the same function. This study involves the purification and identification of mitochondria in T. thermophila, investigation of the respiratory pathways and ATP synthesis, purification of ATP synthase and identification of its component subunits. In this study electron micrograph images showed the presence of mitochondria in T. thermophila and purified fractions. Biochemical and oxygen consumption assays confirmed that these purified mitochondria were intact, coupled, and able to synthesize ATP. The ATP synthase was affinity purified using a recombinant expressed ATP synthase inhibitor protein and an anti-beta subunit antibody. The purifications were carried out with mitochondria which were bot treated with and without the chemical crosslinker DTBP. ATP synthase subunits were resolved by SDS-PAGE and the bands were excised and analyzed by MALDI-ToF mass spectrometry. Altogether 56 different proteins, including all the annotated ATP synthase subunits, were isolated and identified through MS. After an extensive range of bioinformatics analysis, one protein (A2-32, XP_001010660.1) was identified as a putative a subunit of ATP synthase of T. thermophila mitochondria. A2-32 is a membrane-bound protein with a molecular weight of 37 kDa, which is targeted to the mitochondria. Another protein (G25-1, XP_001015517.2) was identified as a putative b subunit, this protein had a molecular weight of 39 kDa, with a hydrophobic N-terminus and hydrophilic C-Terminus In this study, none of the putative ATP synthase proteins suggested by Nina et al (2010) were identified. Also the putative Plasmodium subunit a (XP_001347344) identified by the bioinformatics study of Mogi and Kita (2009) did not show any homology to the proteins identified here. Hence this study has successfully identified a mitochondrially-targeted a subunit protein, which is of expected size and contains key conserved residues necessary for the passage of protons during ATP synthase.

Keywords: ATP synthase, mitochondria, ciliate, alveolate
Subject: Biology thesis

Thesis type: Doctor of Philosophy
Completed: 2017
School: School of Biological Sciences
Supervisor: Ian Menz