![Distinct Mid51 mutants differentially modulate Fis1 oligomerization. (A) IP of myc-tagged Mid51(WT) or Drp1-binding domain mutant Mid51(Y240N) and co-IP of Flag-tagged Fis1, with corresponding input. *, Nonspecific bands. (B and C) Protein quantification and stoichiometry of the immunoprecipitated mitochondrial Mid51/Fis1 complex with Mid51(WT) or Mid51(Y240N) revealing Mid51 species (monomer, dimer, tetramer, and HMW; B) and Fis1 species (monomer, tetramer; C), normalized to monomer levels per condition, quantified from IP immunoblot (n = 3 independent experiments). (D–F) Quantification showing Drp1-binding domain mutant Mid51(Y240N) does not disrupt Fis1 oligomerization in a Mid51/Fis1 complex. Mid51(Y240N) shows normal Mid51/Fis1 binding (Fis1 IP monomer/Mid51 IP monomer ratio; D), Mid51 oligomerization (Mid51 IP [HMW/monomer ratio]; E), and Fis1 oligomerization (Fis1 IP [tetramer/monomer ratio]; F); quantified from IP immunoblot; n = 3 independent experiments. (G) IP of myc-tagged Mid51(WT) or oligomerization domain mutant Mid51(R169W) and co-IP of Flag-tagged Fis1, with corresponding input. *, Nonspecific bands. (H and I) Protein quantification and stoichiometry of the immunoprecipitated mitochondrial Mid51/Fis1 complex with Mid51(WT) or Mid51(R169W), revealing Mid51 species (monomer, dimer, tetramer, and HMW; H) and Fis1 species (monomer, tetramer; I), normalized to monomer levels per condition, quantified from IP immunoblot (n = 3 independent experiments). (J–L) Quantification showing oligomerization domain mutant Mid51(R169W) disrupts Fis1 oligomerization in a Mid51/Fis1 complex. Mid51(R169W) shows normal Mid51/Fis1 binding (Fis1 IP monomer/Mid51 IP monomer ratio; J), increased Mid51 oligomerization (Mid51 IP [HMW/monomer ratio]; K), and decreased Fis1 oligomerization (Fis1 IP [tetramer/monomer ratio]; L); quantified from IP immunoblot; n = 3 independent experiments. Mean ± SEM; unpaired two-tailed t test (D–F and J–L); N.S., not significant (D–F and J); *, P = 0.017 (K); *, P = 0.021 (L). Source data are available for this figure: SourceData F7.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/10/10.1083_jcb.202206140/3/jcb_202206140_fig7.png?Expires=1771043123&Signature=QponchtAfvEwEOw3R9iUlMkZsrZGR5qOY5xAeaOMBqT4Ci8BeLq7BfYcFKFrCxQpc~UgU26RUA43xd-LEVAtgnCxcY-gvkFcB8f0fNJfEtg3572BZgjj~ol58oxoW9CsxSaocHddXtQxdG0vZoMEdTuX6wPD1LIcmdDOB9Hti4MLSVLzm3GriesU-UUmmOCtBd~5rq9v0SZJIArrfPjC2Dq3cjOL~TxNKgXqrzpuBMlXl7RIaiX3CuefKPf3Cqkfncl3wwRnqe-2ChHTJhFvrvFmBS50P-L33-qqFoFObYDFpr5HLjFH3wfrugxuNiS3ywsiht-oDuIsLt1tTOuAmw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Distinct Mid51 mutants differentially modulate Fis1 oligomerization. (A) IP of myc-tagged Mid51(WT) or Drp1-binding domain mutant Mid51(Y240N) and co-IP of Flag-tagged Fis1, with corresponding input. *, Nonspecific bands. (B and C) Protein quantification and stoichiometry of the immunoprecipitated mitochondrial Mid51/Fis1 complex with Mid51(WT) or Mid51(Y240N) revealing Mid51 species (monomer, dimer, tetramer, and HMW; B) and Fis1 species (monomer, tetramer; C), normalized to monomer levels per condition, quantified from IP immunoblot (n = 3 independent experiments). (D–F) Quantification showing Drp1-binding domain mutant Mid51(Y240N) does not disrupt Fis1 oligomerization in a Mid51/Fis1 complex. Mid51(Y240N) shows normal Mid51/Fis1 binding (Fis1 IP monomer/Mid51 IP monomer ratio; D), Mid51 oligomerization (Mid51 IP [HMW/monomer ratio]; E), and Fis1 oligomerization (Fis1 IP [tetramer/monomer ratio]; F); quantified from IP immunoblot; n = 3 independent experiments. (G) IP of myc-tagged Mid51(WT) or oligomerization domain mutant Mid51(R169W) and co-IP of Flag-tagged Fis1, with corresponding input. *, Nonspecific bands. (H and I) Protein quantification and stoichiometry of the immunoprecipitated mitochondrial Mid51/Fis1 complex with Mid51(WT) or Mid51(R169W), revealing Mid51 species (monomer, dimer, tetramer, and HMW; H) and Fis1 species (monomer, tetramer; I), normalized to monomer levels per condition, quantified from IP immunoblot (n = 3 independent experiments). (J–L) Quantification showing oligomerization domain mutant Mid51(R169W) disrupts Fis1 oligomerization in a Mid51/Fis1 complex. Mid51(R169W) shows normal Mid51/Fis1 binding (Fis1 IP monomer/Mid51 IP monomer ratio; J), increased Mid51 oligomerization (Mid51 IP [HMW/monomer ratio]; K), and decreased Fis1 oligomerization (Fis1 IP [tetramer/monomer ratio]; L); quantified from IP immunoblot; n = 3 independent experiments. Mean ± SEM; unpaired two-tailed t test (D–F and J–L); N.S., not significant (D–F and J); *, P = 0.017 (K); *, P = 0.021 (L). Source data are available for this figure: SourceData F7.
