A Designed Point Mutant in Fis1 Disrupts Dimerization and Mitochondrial Fission

Organelle fission and fusion control the size and distribution of mitochondria and peroxisomes, which are fine-tuned in many adult cell types to meet their local demand for ATP or Ca2+. These membrane remodeling events may also be coordinated to ensure the fidelity of essential components that incur damage in their oxidative environments found in these organelles1-8. Supporting the importance of these processes, mutations in fission and fusion genes cause or contribute to mammalian disease9-14. For organelle fission only Fis1 and dynamin-related protein 1 (called Drp1 in mammals and Dnm1 in budding yeast) are conserved across species. Fis1 is a tail-anchored transmembrane protein that is thought to be involved in recruiting soluble fission factors, including Drp1, to sites of membrane scission from the cytoplasm2, 15-25. In budding yeast, Fis1 cooperates with Mdv1 and Caf4, proteins found only in fungi, to recruit the yeast dynamin-related protein Dnm117-19, 24-27. Both blue-native PAGE and chemical cross-linking studies show that Fis1 can form homooligomeric complexes in mammalian cells2, 28, 29. However, seven of the eight available structures of the isolated cytoplasmic domains of Fis1 from human, mouse, and yeast are monomeric27, 30-35. In the eighth structure, human Fis1 crystallizes as a multimer, although it is a monomer in solution32. Thus, there were no previous reports that Fis1 multimerizes in solution and the exact basis of the high molecular weight species of Fis1 by blue-native PAGE analysis is unclear and may involve other proteins. The structures of the cytoplasmic domain of Fis1 are similar; each with a core six-helix bundle, where the central four helices consist of two tandem tetratricopeptide repeat (TPR)-like motifs. The individual TPR-motifs adopt a helix-turn-helix motif and together form a right-handed superhelical structure creating a concave surface. The structures differ in the relative orientation of the Fis1 arm (residues 1-16 in budding yeast and 1-8 in human)27, 30-35. Cell biological experiments, primarily in budding yeast, have determined that both the concave surface and the Fis1 arm are functionally important. NMR and chemical modification experiments suggest that the Fis1 arm is innately dynamic and, in some structures, physically occludes the concave surface suggesting a possible role in modulating function by mediating access to the concave surface36 (Figure 1a). Consistent with this idea, removal of the Fis1 arm (ΔN) impairs yeast mitochondrial morphology producing net-like organelles from a defect in fission33, 37. An important role for the Fis1 arm is supported by two other non-functional Fis1 alleles identified in yeast genetic screens37, 38, L80P and fis1-3 (derived from E78D/I85T/Y88H) on helix 4 of the concave surface. These alleles contain substitutions to residues adjacent to the arm in the tertiary structure, and similar to ΔN-fis1, expression of either L80P or fis1-3 does not restore proper mitochondrial morphology in fis1Δ cells37, 38. Overexpression of Mdv1 can rescue the fission defect caused by L80P and ΔN37, but not fis1-3. The fis1-3 allele requires a mutant in the Mdv1 coiled coil domain (E250G) that may enhance the Fis1 interaction either directly or indirectly39. These results suggest Fis1 variants impair fission by different means. Here we isolated the cytoplasmic domains from these variants with the initial goal of determining whether they share a common mechanism for disrupting fission. Surprisingly, we found that expression of these domains revealed a previously undetected state of yeast Fis1 that is dimeric. Further, we discovered that the wild type domain also forms a dimer, but is kinetically trapped as both a monomer and a dimer. This is the first example of the cytoplasmic domain mediated dimerization in solution. We designed a point mutant (A72P) that strongly disrupts mitochondrial fission and dimerization of isolated protein. Since variants that act either as obligate monomers or dimers in vitro are impaired for fission in vivo, our results suggest a model in which the interconversion between monomeric and dimeric species may play a role in fission and reveals a new mechanism for how TPR motifs can mediate self-association. Figure 1 The cytoplasmic domains of non-functional Fis1 variants self-associate