Investigating ATAD3 Function in Arabidopsis thaliana
Presenter: Nicholas James Cassidy
Faculty Sponsor: Elizabeth Vierling
School: UMass Amherst
Research Area: Biochemistry and Molecular Biology
Session: Poster Session 5, 3:15 PM - 4:00 PM, Auditorium, A86
ABSTRACT
The ATPase family AAA+ Domain-containing Protein 3 (ATAD3) genes encode mitochondrial membrane proteins crucial for mitochondrial function. These proteins span from the mitochondrial matrix to the cytosol, crossing both inner and outer mitochondrial membranes. The N-terminus faces the cytosol and contains a domain of unknown function, potentially interacting with the endoplasmic reticulum, while the C-terminus, facing the mitochondrial matrix, is a AAA+ ATPase that contains Walker A and B motifs, and forms a hexameric structure. Disruptions in ATAD3 in Arabidopsis thaliana impair mitochondrial DNA organization, delay growth, and reduce oxidative phosphorylation complex I activity. This research investigates the function of the four ATAD3 gene homologs in A. thaliana (A1, A2, B1, B2). My first objective is to generate null allele mutants for each homolog using CRISPR/Cas9-mediated mutagenesis and to then perform genetic crosses to examine protein interactions and identify essential gene combinations. To date, I have identified mutants with early frameshifts or large deletions in the A1, A2, B1, and B2 genes. The second objective is to generate transgenic plants with reduced A1 ATAD3 expression in an A2 knockout mutant. This aims to overcome the lack of phenotypic differences in single ATAD3 mutants and the lethality of double-knockout mutants. Reduced A1 expression will be assessed by immunoblot analysis, and plant phenotypes characterized. Additionally, I am expressing the C-terminal domain of ATAD3 A2 and B1 to assess coassembly, oligomeric states, and ATPase activity of the C-terminus.