LAUSR

Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial inner and outer membranes, respectively. Mutations in any of these proteins can lead to neuropathies including blindness and Charcot-Marie-Tooth, a disease characterized by progressive loss of distal muscle tissue. Currently, little is known structurally or biochemically about any of these proteins. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallographic studies. Both have comparable GTPase activity compared to full-length, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramer when assayed by size exclusion chromatography. In addition, the long, membrane-bound isoforms of Opa1 and Mfn1 have been expressed and purified in large quantities. To date, we have shown full length Mfn1 can be incorporated into proteoliposomes and in the presence of GTP forms dense tethers as seen by cryo-EM. This tethering is reversible as shown by confocal microscopy. Currently we are developing tethering assays for Opa1 and fusion assays for both Opa1 and Mfn1.