Dynamics of Mitochondrial Inheritance

Mitochondria are organelles involved in the cellular energetics in all eukaryotes. Defects in their dynamics, fission, fusion or localisation can lead to disease. S. cerevisiae is a model organism that has been used to identify factors regulating mitochondrial dynamics. In this study, strains overexpressing kinases, phosphatases and ubiquitin ligases were crossed with a strain expressing a mitochondrial marker. The resulting diploids were screened microscopically for strains showing changes in their mitochondrial distribution or appearance. One of the most striking mitochondrial phenotypes was observed in the overexpression of the gene encoding Cla4 (Cyclin activity dependent) kinase, which is one of the PAK (p21- activated kinases) family of Ser/Thr protein kinases. Cells overexpressing CLA4 showed a delay in mitochondrial transport to the bud. Previous work showed Cla4 phosphorylates Vac17 - the vacuole adaptor that binds to the Myo2 at the mother, and transports portions of the vacuole to the bud. Vac17 is phosphorylated by Cla4 which targets it for degradation. This mechanism ensures one-way traffic to the bud. We hypothesised that Cla4 could regulate mitochondrial inheritance in a similar way through phosphor regulation and through degradation of the mitochondrial adaptor Mmr1 (Mitochondrial Myo2p Receptor-related). Our research showed that the Mmr1 protein level is decreased upon CLA4 overexpression, and that small buds are devoid of Mmr1 accumulation. Using a range of genetic, biochemical approaches we investigated the interaction of Mmr1 with myosin, and generated mutants disrupted in this interaction. Mutant Mmr1 was not impacted by CLA4 overexpression. Intriguingly, mutant Mmr1 was still trafficked to the bud, suggesting that resistance to the effects of Cla4 overexpression is not solely due to Mmr1 being retained in the mother, and that additional mechanisms are responsible. Finally, the Mmr1 degradation mechanism was investigated, and a region of Mmr1 responsible for the Dma1 and Dma2 (E3 ubiquitin ligases)-dependent ubiquitination and degradation was identified.