Molecular interaction of Mitofusin 2 and its role in mitochondrial fusion

Abstract

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2008.Mitochondria change their shape dynamically, mainly through fission and fusion. Dynamin-related GTPases have been shown to mediate remodeling of mitochondrial membranes during these processes. Mitochondrial fission in mammals is mediated by the dynamin-like protein DLP1/Drp1 that is recruited to the outer mitochondrial surface through the membrane-anchored protein hFis1. Another GTPase, Mitofusin (Mfn), is anchored at the outer mitochondrial membrane and mediates fusion of the outer membrane. Mammalian cells have two Mfn isoforms, Mfn1 and Mfn2, that share a conserved molecular structure. Either Mfn1 or Mfn2 can functionally replace each other in Mfn-null cells, suggesting their conserved role as fusion proteins as well. This thesis research centers on the mitochondrial fusion protein Mfn2. Specifically, it focuses on studying the effect of the Mfn2-induced mitochondrial shape change on mitochondrial function and the molecular mechanisms of mitochondrial fusion mediated by the Mfn2 protein. We found that overexpression of Mfn2 drastically changes mitochondrial morphology, forming mitochondrial clusters. High-resolution microscopic examination indicated that the mitochondrial cluster consisted of small fragmented mitochondria. Inhibiting mitochondrial fission prevented the cluster formation, supporting the notion that mitochondrial clusters are formed by fission-mediated mitochondrial fragmentation and subsequent aggregation. Mitochondrial clusters displayed a decrease in inner membrane potential and proton pumping activity, suggesting functional compromise of small fragmented mitochondria by Mfn2 overexpression; however, mitochondrial clusters still retained mitochondrial DNA. We found that cells containing clustered mitochondria lost cytochrome c from mitochondria and underwent caspase-mediated apoptosis. These results demonstrate that mitochondrial deformation impairs mitochondrial function, leading to apoptotic cell death and suggest the presence of an intricate form-function relationship of mitochondria. Because intra- and inter-molecular interactions play an important role in the membrane remodeling action of dynamin family proteins, we analyzed domain interactions of the Mfn2 molecule using genetic and biochemical approaches. We found that two hydrophobic heptad-repeat (HR) domains, HR1 and HR2, interact with each other, in addition to the already reported HR2 and HR2 interaction. Interestingly, we discovered that the region of Mfn2-HR1 interacting with HR2 also interacts with the C-terminal coiled-coil domain of the fission protein DLP1 (DLP1-CC). We identified mutations in the Mfn2-HR1 region that selectively disrupt the HR1/HR2 interaction and the Mfn2/DLP1 interaction. Morphological analyses indicated that the Mfn2/DLP1 interaction participates in mitochondrial fusion whereas the association of HR1 and HR2 of Mfn2 is inhibitory in the fusion process. These data suggest that DLP1 functions as a regulatory factor interacting differentially with Mfn2 and hFis1, which provides a novel mechanism for efficient execution of mitochondrial fusion and fission. We also explored the possible mechanisms by which the differential interactions of Mfn2-HR1 with DLP1 and Mfn2-HR2 can be regulated. We found that Mfn2-HR1 and DLP1-CC can be phosphorylated in vitro. In addition, mutations mimicking phosphorylated and dephosphorylated HR1 showed opposite effects on interaction with HR2 and DLP1, suggesting that HR1 phosphorylation plays a role in regulating the differential interactions of Mfn2-HR1. In conclusion, our results indicate that regulated interactions of fission/fusion machineries constitute the controlling mechanisms of mitochondrial shape change, which has a critical role in maintaining the proper function of mitochondria