Exploring Knockdown Phenotypes and Interactions between ATAD3 Proteins in Arabidopsis thaliana

Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. ATAD3A is a mitochondrial membrane protein with unique topology. It comprises an N-terminal DUF (Domain of unknown function) domain that contains two transmembrane sequences the are inserted or interact with both the inner and outer mitochondrial membranes, two coiled-coil domains thought to help in oligomerization, and a region that is exposed to the cytosol, proposed to interact with the ER. It has a C-terminal AAA domain exposed to the mitochondrial matrix. ATAD3 proteins in A. thaliana have undergone two gene-duplication events, resulting in two clades, both of which are required for plant viability. I created artificial microRNA to knockdown expression of ATAD3A1 in the atad3b1 mutant background to assess the growth and mitochondrial phenotypes and found these plants displayed delayed and deficient growth and deformed mitochondria. I utilized Bi-Molecular Complementation Fluorescence and Laser-Scanning Confocal Microscopy to assess oligomeric patterns of A. thaliana ATAD3 proteins in vivo and discovered that ATAD3 proteins hetero-oligomerize with each other. I also created multiple constructs encoding ATAD3A1 fusion proteins to elucidate the amino acid sequence required to target ATAD3A1 to the mitochondria, and ATAD3A1 fusions with TurboID to identify protein-protein interactions using proximity-based labeling