Characterization of land plant-specific proteins required for mitochondrial translation initiation

Abstract: Plant mitochondria produce the majority of adenosine triphosphate (ATP) – the cellular energy currency for metabolic reactions needed for plant growth, development, and maintenance. Despite a relatively thorough understanding of basic mitochondrial functions, many mitochondrial proteins and processes remain poorly understood. The aims of this thesis are to i) review and compare the mitochondrial unfolded protein response (UPRmt) and related signalling across eukaryotic kingdoms, to ii) describe an efficient isolation method of Arabidopsis mitochondria using continuous Percoll density gradients, and to iii) characterize two Arabidopsis thaliana genes, mTRAN1 and mTRAN2. Paper I summarizes the current knowledge of UPRmt across eukaryotic kingdoms, and describes a meta-analysis of UPRmt regulators and target genes. UPRmt is a mitochondria-to-nucleus “retrograde” response that regulates nuclear gene expression during mitochondrial dysfunction to maintain mitochondrial homeostasis. Although UPRmt has been extensively studied in animals, relatively little is known about the plant UPRmt and only few regulators have recently been identified. In yeast, very few unfolded protein responses that seem to be related to UPRmt have been described. Here, the UPRmt in animals, yeast and plants are compared. Our study indicates that each kingdom has evolved their own specific regulators, which however induce very similar groups of target genes. Our meta-analysis identifies homologs of known UPRmt regulators and responsive genes across eukaryotic kingdoms.Paper II describes a strategy for efficient purification of Arabidopsis mitochondria using continuous Percoll density gradients. By using this method, the purity of isolated mitochondria is greatly improved. Obtained mitochondria can be either used for assays requiring highly intact and functional mitochondria, e.g. import assay or respiration measurement, or be stored for later use, e.g. BN-PAGE or western blot.Paper III describes the characterization of two Arabidopsis thaliana genes that we named as named as mitochondrial TRANslation factor 1 (mTRAN1) and 2 (mTRAN2). Here, mTRAN1 and mTRAN2 are shown to be land plant-specific mitochondrial proteins that are critical for plant performance, but they are unlikely to be adenyl cyclases as previously annotated. Interestingly, knocking out both mTRAN1 and mTRAN2 results in low abundance and activity of mitochondrial oxidative phosphorylation complexes. Using co-immunoprecipitation, mTRAN1 and mTRAN2 are confirmed to be part of the mitoribosome small subunit (mtSSU). In organello protein synthesis and polysome fractionation assays suggest that mTRANs are required for mitochondrial translation initiation. Moreover, the RNA electrophoretic mobility shift assays suggest that mTRAN1 binds to the A/U-rich regions in the 5’-untranslated regions of mitochondrial mRNAs to mediate mtSSU-mRNA binding to initiate translation. Finally, our mitoribosome profiling suggests that mTRAN proteins are universal mtSSU-embedded translation initiation factors. Together, these findings suggest that mitochondrial translation initiation occurs via a unique mechanism in plants, as compared to in bacteria, yeast and mammals.