22 23 24 2 Summary 25 26Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The 27 apicomplexan mitochondrion shows striking differences from common model organisms,
28including in fundamental processes such as mitochondrial translation. Despite evidence 29 that mitochondrial translation is essential for parasites survival, it is largely understudied.
30Progress has been restricted by the absence of functional assays to detect 31 apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the 32 process and the inability to identify and detect mitoribosomes. 33 Using mRNA expression patterns, 279 candidate mitochondrial housekeeping 34 components were identified in Toxoplasma. 11 were validated, including the 35 mitoribosomal small subunit protein 35 (TgmS35). TgmS35 tagging enabled the 36 detection of a macromolecular complex corresponding to the mitoribosomal small 37 subunit for the first time in apicomplexans. A new analytical pipeline detected defects in 38 mitochondrial translation upon TgmS35 depletion, while other mitochondrial functions 39 remain unaffected. Our work lays a foundation for the study of apicomplexan 40 mitochondrial translation. 41 42 43 Abbreviated summary 44 45 46The apicomplexan mitochondrion is divergent and essential yet poorly studied.
47Mitochondrial translation is predicted to utilize ribosomes assembled from fragmented 48 rRNA but this was never shown. Knowing the mitochondrial protein content is critical for 49 these studies. We identified 11 new mitochondrial proteins via in-silico searches. 50 Tagging and depletion of a mitoribosomal small subunit protein enabled the first 51 detection of a macromolecular ribosomal complex, and provided proof of principle for our 52 new mitochondrial translation analytic pipeline. 53 54 55 3 Introduction 56 57 Mitochondria are organelles of central importance to eukaryotic cells, providing key 58 nutrients and metabolites. Mitochondria are present in virtually all eukaryotic cells, 59 excluding a rare case of secondary loss (Karnkowska et al., 2016; Karnkowska and 60 Hampl, 2016). The recent interest in evolutionary cell biology has resulted in an 61 increasing appreciation of the diverse features of mitochondria found in divergent 62 organisms, which includes differences in fundamental pathways such as protein 63 translation (e.g. Ramrath et al., 2018). However, most of our knowledge of mitochondrial 64 biology is still based on studies in organisms that represent a small proportion of the 65 range of eukaryotic diversity, thus limiting our understanding. One of the challenges to 66 the study of mitochondrial biology in diverse organisms is the lack of tools to identify and 67 functionally characterise genes encoding species or phylum specific mitochondrial 68 proteins.
70Apicomplexa is a phylum of parasitic protists with high impact on human health globally, 71 which includes the malaria causing Plasmodium spp and the causative agent of 72 toxoplasmosis, Toxoplasma gondii. Apicomplexans represent o...