Functional characterization of novel mitochondrial outer membrane proteins involved in mitochondrial biogenesis in Arabidopsis thaliana

Submission note: This thesis is submitted by published work in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora, Victoria, Australia.

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The mitochondrial outer membrane (MOM) is the interface with the cytosol and other organelles. It allows the passage of metabolites, proteins, lipids and nucleic acids into mitochondria and signals from mitochondria must pass through the MOM to alter gene expression in mitochondrial retrograde signaling. Yet, the MOM is the least well characterised sub-compartment in plant mitochondria. The studies outlined in this thesis characterize the function of two novel proteins on the MOM. Functional characterization of a plant-specific β-barrel protein, OUTER MEMBRANE PROTEIN 47 (OM47) found it has no effect on protein import but likely functions in transport of molecules and metabolites. T-DNA insertion om47 mutants accumulate more starch and show delayed senescence compared to wild-type. These results suggest that it functions in metabolite transport to support catabolic breakdown of chloroplasts during senescence. DGS1 is a MOM protein that can affect chloroplast galactolipids biosynthesis under phosphate starvation and Alternative Oxidase (AOX) expression. Here we show that DGS1 is in a multi-subunit protein complex with Mic60 and is involved in lipid import into mitochondria, most probably galactolipids. A point mutation of DGS1 resulting in a single amino acids change (dgs1 EMS) affects the stability, composition and protease accessibility of subunits of this complex. The DGS1 EMS protein also targets to the ER. This results in altered mitochondrial and chloropalst lipid composition, changes in organelle size and morphology, with an apparent decrease in mitochondria fission. Furthermore, dgs1 EMS allele affects plant growth and development and confers plants greater tolerance to drought stress, probably through distinct mitochondrial retrograde signaling by mediating lipid metabolism and homostasis to cope with water depletion. A dgs1 knock-out mutant is indistinguishable from wild type under non-limiting growth conditions revealing the dgs1 EMS represents a gain of-function mutant, providing functional insights into the role of DGS1 in plant mitochondria.