A conserved, non-canonical insert in FIS1 is required for mitochondrial fission and recruitment of DRP1 and TBC1D15

Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes yet its activity in metazoans is thought divergent from lower eukaryotes like fungi. To address this discrepancy, structure-based sequence alignments revealed a conserved but non-canonical, three-residue insert in a turn of FIS1, suggesting conserved activity. In vertebrate FIS1 this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of this “SKY insert”, three variants were evaluated for their fold, and tested in HCT116 cells for altered mitochondrial morphology and recruitment of effectors, DRP1 and TBC1D15. Substitution of the SKY insert with three alanine residues (AAA), or deletion of the insert (ΔSKY), did not substantially alter the fold or thermal stability of the protein. Replacing SKY with a canonical turn (ΔSKYD49G) introduced significant conformational heterogeneity by NMR that was removed upon deletion of a known regulatory region, the FIS1 arm. Expression of AAA fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1 similar to the wild-type protein. In contrast, expression of ΔSKY variants elongated mitochondrial networks and reduced mitochondrial DRP1. Co-expression of YFP-TBC1D15 partially rescued mitochondrial morphology and DRP1 recruitment for ΔSKY variants, although ΔSKY variants were markedly unable to support TBC1D15 assembly into punctate structures found upon co-expression with wildtype FIS1 or the AAA variant. Collectively these results show that FIS1 activity can be modulated by conserved residues supporting a generalized model whereby FIS1 is governed by intramolecular interactions between the regulatory FIS1 arm and SKY insert that may be conserved across species. ### Competing Interest Statement R. B. H. and R.T. have financial interest in Cytegen, a company developing therapies to improve mitochondrial function. A portion of the salary for R.T. is paid for by the company. The authors declare that they have no conflicts of interest with the contents of this article.