Figure 1. The cytoplasmic domains of non-functional Fis1 variants self-associate.

(a) Cartoon representation of the cytoplasmic domain of yeast Fis1 (Fis1ΔTM, 3o48.pdb). Three non-functional variants of Fis1 (L80P (green), ΔN16 (red), and Fis1ΔTM-3 (cyan, E78D/I85T/Y88H)) are highlighted by stick representation of the wild type residues. These variants have previously been reported to impair mitochondrial fission. The two TPR-like domains are colored yellow and blue. This figure was made with PyMOL⁸⁶. (b) The cytoplasmic domains of non-functional Fis1 variants, but not wild type, interact by yeast two-hybrid. Yeast two-hybrid assays using the HIS3 reporter (sequential ten-fold yeast dilutions at 1, 0.1, and 0.01 OD₆₀₀ are shown) with the indicated bait and prey constructs. Cells were plated onto media containing histidine (growth control) and media lacking histidine to select for yeast two-hybrid interactions and photographed after incubation for 3 days at 30 °C. (c) Fis1ΔTM variants elute by gel filtration chromatography as dimers to differing degrees. 2.5 μM of each of the indicated recombinantly expressed proteins were separated by a Superdex 200 10/300 GL at 25 °C (50 mM sodium phosphate, 184 mM NaCl, 5 mM EDTA and 2 mM DTT, pH 7.4). Each mutant is well-folded by circular dichroism spectrapolarimetry (Supplemental Figure 1).