Identification of key components involved in mtDNA inheritance during cell division (965.1)

Abstract

Majority of cellular DNA is located in the cell nucleus, however the mitochondrion also possesses DNA. Mitochondrial DNA (mtDNA) is packaged into protein DNA complexes called nucleoids required for production of ATP. Mutations in mtDNA inheritance contribute to human diseases. Despite its importance, little is known about the mechanisms. Using live cell microscopy in yeast, it was established that mitochondrial nucleoids localize to the tips of mitochondrial tubules, which migrate into the daughter cell during cell division, ensuring mtDNA inheritance. Tip localization of mtDNA lead us to hypothesize that there is protein or complexes of proteins that anchor mtDNA to mitochondrial tips. To identify and characterize these molecular factors, a novel genetic approach was designed using Saccharomyces cerevisiae that relies on synthetic biology. mtDNA was artificially tethered to the inner mitochondrial membrane by expression of a synthetic protein. This synthetic protein consists of Yme2, a transmembrane protein of the inner mitochondrial membrane, and the LacI protein that binds LacO repeats, which were inserted into the mtDNA. The presence of this synthetic tether renders the endogenous protein complex that anchors mtDNA to the membrane dispensable. Using a genetic tool known as white and red markers, a genetic sectoring screen was performed to identify tethering components. White, sector and red colonies were obtained, but only red colonies indicated dependence on the artificial tether for mtDNA. Red colonies were isolated for further identification of the molecular factors that tether mtDNA to mitochondrial tips. This novel genetic approach will impact our understanding of mtDNA metabolism.