| Popis: |
The mitochondrion is an essential organelle in eukaryotic cells. The major functions of mitochondria are ATP production, calcium homeostasis, heme and lipid biosynthesis and apoptosis. Mitochondria form a dynamic tubular spaghetti-like network that fuses, divides and moves within the cell. The molecular events that regulate mitochondrial dynamics are important for various cellular and developmental processes and are linked to cellular dysfunction and disease. This dissertation focuses on the molecular machineries that carry out mitochondrial fission in yeast and human. Mitochondrial fission requires both dynamin-related GTPases (called Dnm1/Drp1 in yeast/human) and membrane adaptors. In yeast, a tail-anchored protein called Fis1 and an adaptor protein called Mdv1 recruit Dnm1 to the membrane. Mdv1 interacts with Dnm1 and stimulates Dnm1 self-assembly. Although Fis1 is conserved in humans, an Mdv1 ortholog is absent. Instead, humans have at least three other membrane anchored proteins (Mff, MiD49, MiD51/MIEF1) whose roles in fission are poorly defined. Studies presented in this thesis address two key issues related to mitochondrial fission. First, experiments in Chapter 2 address how the structure of the yeast mitochondrial adaptor (Mdv1) affects Dnm1 function. A structural study of the Mdv1 central domain shows that this region forms an unusually long antiparallel coiled coil which positions the P-propeller domains of Mdv1 to interact with Dnm1 as it transitions from the cytoplasm to mitochondria. In vivo studies with altered coiled coil domains demonstrates the importance of this domain in Mdv1-Fis1 binding, Dnml mitochondrial recruitment and Dnm1-mediated mitochondrial fission. Second, experiments in Chapter 3 determine whether multiple human adaptors are capable of working independently to elicit Drp1-mediated membrane fission. In vivo studies using the yeast and human adaptors in a minimal yeast system show that Fis1 is dispensable for mitochondrial membrane scission. These studies also demonstrate that Mdv1, Mff, or MiDs can work in parallel with their corresponding DRPs to catalyze membrane fission. Importantly, coassembly of MiD49 protein with Drp1 dramatically decreases the diameter of assembled Drp1 structures. Together, these studies advance our understanding of how adaptors and fission dynamins work together to achieve membrane constriction and fission, and raise important questions for future studies. |
