The dynamic N-terminal arm of Fis1 regulates mitochondrial fission

Mitochondrial dynamics regulate the size, number and placement of these energy-producing organelles. Mitochondrial division is important in the response to changing energy needs, or apoptotic stimuli, and is important in human health. The proteins involved in mitochondrial fission are conserved from yeast to humans. Fis1 is a C-terminally membraneanchored protein. The conserved protein-protein interaction motif of Fis1 is responsible for the recruitment of the other conserved protein in mitochondrial fission, Dnm1. Dnm1, a dynamin family protein, a large mechanoenzyme that is known to deform and divide membrane bilayers. In fungi, a third protein is involved in mitochondrial fission. Mdv1/Caf4 is a protein that may mediate interactions between Fis1 and Dnm1. However, there are indicators that Fis1 may bind Dnm1 directly, and Mdv1 is a late-acting factor in fission after the Fis1-Dnm1 complex is formed. The N-terminus, or "arm" of Fis1, may act as an inhibitor of interaction with Dnm1. The N-terminal arm binds to a surface of Fis1 that is lined with residues important for the interaction with Dnm1. It is possible that the N-terminal arm is displaced at sites of fission, allowing Dnm1 to be recruited.;The N-terminal arm of Fis1 is in different conformations in structures previously solved of different homologues. We hypothesize that Fis1 protein from S. cerevisiae exists in more than one conformation. We tested this by looking at the backbone dynamics of Fis1 by NMR spectroscopy. Those experiments, along with chemical modification experiments, indicate that the N-terminal arm and the first and sixth helices of Fis1 undergo long timescale motions. These data can be accounted for by a low-populated conformation of Fis1 where the N-terminal arm is displaced from the surface with which it normally interacts. This rare conformation may be important for Fis1 to regulate the recruitment of the mitochondrial fission complex.;We also hypothesized that the small population of "open" Fis1 may be the active Fis1 conformation. We tested this by designing point mutants in Fis1 that would be able to increase the population of Fis1 that has the N-terminal arm displaced from the surface of Fis1. Some of these point mutants were unable to rescue mitochondrial fission in two in vivo assays. The point mutants in Fis1 also changed the pattern of protein-protein interactions when compared to wild-type Fis1, and also increased the population of Fis1 that exists as a homodimer.;The experiments outlined above led us to propose a revised scheme of how the mitochondrial fission complex comes together to divide mitochondria. Fis1 is normally in an inhibited state for fission, and displacement of the auto-inhibitory N-terminal arm allows for dimer formation. Dimeric Fis1 recruits Dnm1, which in turn recruits Mdv1. Mdv1 may stimulate oligomerization of Dnm1 and dissociation from Fis1. The machinery then divides the membrane and is disassembled. Specific experiments can be designed to test this model, and contribute to a more complete and detailed mechanism of mitochondrial fission.