In eukaryotic cells, peroxisomes and mitochondria are ubiquitous organelles playing essential roles in development. Peroxisomes and mitochondria not only house a number of organelle-specific biochemical reactions, but also function together in accomplishing several intracellular metabolic pathways. As multifunctional organelles, they are highly dynamic and plastic, in which the conserved fission proteins, Dynamin-related protein (DRP) and Fission1 (FIS1), govern the division of both organelles. In addition, several lineage-specific factors also exist to mediate the division of peroxisomes and mitochondria. To further dissect the division machinery of peroxisomes and mitochondria in Arabidopsis, I explored novel division factors and examined the regulation of known division factors. First, I identified and characterized two novel plant-specific proteins, Peroxisomal And Mitochondrial Division Factor1 (PMD1) and its homologue PMD2. We demonstrated that PMD1 is a C-terminal tail-anchored (C-TA) protein of peroxisomes and mitochondria and involved in the division and morphogenesis of these organelles. PMD2, on the other hand, is targeted only to mitochondria and involved specifically in mitochondrial division/morphogenesis. As there is no detectable physical interaction between PMD1 and the known division proteins DRP3 and FIS1, PMD1 is suggested to be involved in peroxisomal and mitochondrial division in a DRP3/FIS1-independent manner. DRPs are large GTPases, functioning as molecular scissors during vesicle fission. In Arabidopsis, DRP3A and DRP3B play critical roles in peroxisomal and mitochondrial division, whereas DRP5B mediates the division of chloroplasts and peroxisomes. In this study, I further characterized these three DRP proteins for their roles in organelle division. Our results showed that DRP5B is not physically associated with mitochondria, but its mutants exhibit an elongated mitochondrial phenotype similar to that of the drp3 mutants, suggesting that DRP5B is indirectly involved in the division or morphogenesis of mitochondria. Genetic and biochemical analyses showed that DRP5B may function in a manner that is independent from DRP3A and DRP3B, as DRP5B is not crucial for forming the DRP3 protein complexes. Furthermore, I studied post-translational regulation of DRP3, the major division factor of peroxisomes and mitochondria. I showed that the function of DRP3 is regulated by protein phosphorylation at DRP3ASer575 and DRP3BSer560. Overexpression of phospho-mimic DRP3s fails to rescue the drp3 mutants and causes peroxisomal and mitochondrial division deficiencies in wild-type Col-0, suggesting that DRP3 phosphorylated at these serine residues is inactive. Lastly, I discovered that the absence of the mitochondrial and peroxisomal adenine nucleotide transporter, AAC1, or the peroxisomal NAD+ carrier PXN, alters the morphologies and abundance of the organelles. These findings suggest that metabolic homeostasis within peroxisomes and mitochondria is also involved in the morphogenesis and proliferation of these organelles. In summary, my work has provided significant insights into the molecular control of the morphogenesis and proliferation of peroxisomes and mitochondria in Arabidopsis.
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