Insights into the molecular mechanisms of mitochondrial outer membrane tethering and fusion
Engelhart, Emily Anne
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Mitochondrial dynamics are crucial for cellular health as perturbations in these processes which include mitochondrial fusion, division, transport and mitophagy are associated with numerous diseases, cancer and neurodegeneration. The mechanisms of mitochondrial outer membrane tethering and fusion are poorly understood. It is known that Mitofusin proteins are required on both membranes of the fusion pair and that they are responsible for mitochondrial tethering, but the mechanism of tethering and the steps required to progress to lipid mixing are unknown. As members of the Dynamin related protein family, we predict that Mitofusin-mediated membrane fusion will be driven by the catalytic cycle, which will be coupled to self-assembly and conformational changes. To gain insight into the molecular steps of mitochondrial outer membrane tethering and fusion, I worked on three distinct but related projects: (1) purification and characterization of recombinant Mitofusin protein from Escherichia coli, (2) identification and characterization of a mutant variant of the Mitofusins that uncouples mitochondrial tethering and fusion, and (3) fractionation of mammalian cytosol to identify new factors that stimulate mitochondrial fusion. The purification of recombinant mouse Mitofusin protein from E. coli was extremely difficult and highly variable in both the quality and quantity of the final purified protein. The vast majority of protein purifications resulted in the formation of soluble aggregates as assessed by sucrose gradient ultracentrifugation and negative stain electron microscopy. This recombinant Mitofusin protein did potentially possess nucleotide binding activity and appeared to have some structural features by negative stain electron microscopy and limited trypsin digestion. Despite this encouraging data, purified recombinant Mitofusin protein lacked GTP hydrolysis activity and proteoliposomes with recombinant Mitofusin lacked tethering and fusion activity. Although functional recombinant Mitofusin protein was not obtained, this work lays the groundwork for future attempts at obtaining purified protein. To gain insights into functional features of the GTPase domain, we performed a screen of disease-associated mutant variants and identified a mutant variant of Mfn1 that caused mitochondrial hyperfusion and perinuclear collapse of the network. The substitution was Mfn1 Phe-202 to leucine, which is located in a conserved central beta-sheet. Despite the high degree of organelle connectivity observed in cells, our in vitro mitochondrial fusion assay revealed that Mfn1F202L lacked mitochondrial fusion activity. Further biochemical analysis indicated that this mutant variant uncouples mitochondrial tethering from fusion due to impaired higher-order assembly. This mutant variant of the Mitofusins has provided insight into the molecular steps of mitochondrial outer membrane fusion placing nucleotide dependent self-assembly after the initial tethering event. To find additional factors that stimulate mitochondrial fusion, we performed cytosol fractionation by ion exchange chromatography and tested for pro-fusion activity in our cell-free assay. We identified a discrete fraction of cytosolic factors that tightly bound to cation exchange resin that significantly stimulated mitochondrial fusion in vitro. Incubation of the stimulatory fraction with either RNase, protease or heat reduced the stimulatory activity suggesting that the stimulatory factor might have both RNA and polypeptide components. This work indicates that classical biochemical fractionation can identify novel pro-fusion proteins. Together, these three independent projects have given us insights into the molecular mechanisms of mitochondrial outer membrane tethering and fusion.
- Biological chemistry