A conserved, non-canonical insert in mitochondrial fission protein 1 (FIS1) is required for DRP1 and TBC1D15 recruitment and fission

Abstract

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 ({Delta}SKY), did not substantially alter the fold or thermal stability of the protein. Replacing SKY with a canonical turn ({Delta}SKYD49G) 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 {Delta}SKY variants elongated mitochondrial networks and reduced mitochondrial DRP1. Co-expression of YFP-TBC1D15 partially rescued mitochondrial morphology and DRP1 recruitment for {Delta}SKY variants, although {Delta}SKY 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.