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

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 FIS1 turn suggesting a 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.

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission

“…Fis1 undergoes numerous posttranslational modifications ( 60 ), of which phosphorylation by DNA-PKcs and Met enhance mitochondrial fission ( 61 , 62 ), whereas ubiquitination ( 63 , 64 ), SUMOylation ( 65 ), and acetylation ( 66 , 67 ) decrease mitochondrial fission. While our data do not identify a regulatory mechanism, significant chemical shift perturbations from the E7A substitution are localized to a conserved, noncanonical insert in the first TPR of Fis1 that comprises residues S45-K46-Y47 (the SKY insert) that have been recently identified as being important in Fis1 activity ( 68 ). Collectively, our data would support a model in which posttranslational modifications to either arm or SKY residues could regulate this interaction.…”

The peroxisome: an update on mysteries 3.0

“…The division-promoting activity of PEX11β was dependent on amino acids at its C-terminus, which may be essential for the formation of an active division complex with FIS1 and DRP1. A conserved, non-canonical insert in FIS1 appears to be required for DRP1 recruitment and organelle fission (Ihenacho et al 2023 ). Interestingly, alterations of the N-terminus did not prominently impact on the ability of PEX11β to induce peroxisome division.…”