Mitochondrial translation in Toxoplasma gondii: Establishing tools, and characterizing essential mitoribosomal components

Lacombe, Alice (2020) Mitochondrial translation in Toxoplasma gondii: Establishing tools, and characterizing essential mitoribosomal components. PhD thesis, University of Glasgow.

Abstract

Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. These parasites are obligate intracellular pathogen and divergent organisms whose cellular machineries often composed of unique structures of functions compared to model organisms. Studying fundamental mitochondrial biology in these organisms means defining the ancestral core of eukaryotic pathways while simultaneously identifying organism specific traits, that may also inspire drug discovery. Organellar translation has been a focus for the latter in recent years. Due to extreme gene transfer to the nuclear genome, the apicomplexan mitochondrial genome encodes only three proteins: COXI, COXIII and COB, and a series of indirect evidence suggest active mitochondrial translation of these proteins. Evidence also point to several divergent features in this pathway compared to model organism, primarily the reliance on the import of the full set of tRNAs from the cytosol, and the unusual ribosome composition. Progress has been hampered by the lack of functional assays to detect apicomplexan mitochondrial translation, a shortage of knowledge of proteins involved in this process and the incapacity to detect mitoribosomes. We investigate the molecular detail of translation in apicomplexan organisms using Toxoplasma gondii as a model.
tRNA affinity pull down identified 7 candidate proteins with potential role in mitochondrial tRNA import. Using a bioinformatics screen based on mRNA expression patterns, 279 candidate mitochondrial housekeeping components were identified in Toxoplasma. 11 were validated, including the mitoribosomal small subunit protein 35 (TgmS35). The small subunit of the mitoribosome was detected for the first time in apicomplexans through TgmS35 triple HA tagging. A new analytical pipeline detected defects in mitochondrial translation upon TgmS35 depletion, while other mitochondrial functions remain unaffected. Our findings provide further support for the divergent nature of apicomplexan mitochondrial translation and lay a foundation for the study of apicomplexan mitochondrial translation.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Toxoplasma gondii, mitochondrial translation, mitoribosome, mitochondrial tRNA import, tRNA affinity assay, ribosomal protein, mitochondrial translation assay, cell lysis, crude mitochondria.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Parasitology
• Supervisor's Name: Sheiner, Dr Lilach and Meissner, Prof Markus
• Date of Award: 2020
• Embargo Date: 24 July 2023
• Depositing User: Miss Alice Lacombe
• Unique ID: glathesis:2020-81531
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Jul 2020 07:21
• Last Modified: 27 Jul 2020 07:21
• URI: https://theses.gla.ac.uk/id/eprint/81531

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