Hoppins, SuzanneSamanas, Nyssa Becker2020-02-042020-02-042019Samanas_washington_0250E_20870.pdfhttp://hdl.handle.net/1773/45238Thesis (Ph.D.)--University of Washington, 2019Mitofusin 2 (Mfn2) is an outer mitochondrial membrane protein responsible for mitochondrial fusion. Mitochondrial fusion is essential for cellular health and survival. Mutations in Mfn2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A, however, the etiology of the disease as well as the molecular mechanism of Mfn2-mediated mitochondrial fusion are not fully understood. Comparison to related proteins indicates a potential model of mitofusin activity, but details have yet to be filled in. Due to the lack of structural and biochemical information available about Mfn2, I used disease-associated alleles of Mfn2 to perform a structure-function analysis to learn about the molecular mechanism of Mfn2. I first sought to identify domains of Mfn2 essential to its fusion activity. I found that the C-terminus of Mfn2 is essential to mitochondrial fusion, while the N-terminus and the middle stalk domain can be modified without gross morphological changes of mitochondria. I next examined the role of Mfn2 in response to oxidative stress and found that this response, at least in our system, is different dependent on the exact stressor and is dependent on Mfn2. I also report that oxidative environments cause a change in Mfn2 assembly in isolated mitochondria. Finally, I report that the integrity of a major predicted hinge region of Mfn2 is essential for correct mitochondrial fusion using a series of microscopy based and biochemical assays. This is likely due to its contribution to the ability of Mfn2 to assemble within mitochondrial membranes to form oligomers capable of mitochondrial fusion.application/pdfen-USCC BY-NCBiochemistryCellular biologyMolecular biologyMolecular and cellular biologyThesis