Fig. 1. S-glutathionylation of Drp1 Cys644 by electrophilic glutathione.

A Drp1 S-glutathionylation in the hearts from sedentary mice (Sed) or voluntary exercise mice (Ex). S-glutathionylated Drp1 was immunoprecipitated and quantified. (n = 4 mice per condition). NC indicates immunoprecipitation without antibody as a negative control. B, C Quantification of total GSSG content B and GSSG/GSH ratio C in the hearts. (n = 4 mice per condition). D In vitro S-glutathionylation of Drp1. Recombinant His-Drp1 was reacted with GSH, GSSG or GSNO. (n = 3 independent experiments). E S-glutathionylation of Drp1 WT and C644W mutant by GSSG. (n = 3 independent experiments). F MS/MS spectrum for identifying polysulfidation of Cys644 in Drp1. Drp1-FLAG was purified from HeLa cells. The identified fragment ions shown in the spectra and the peptide sequence (b and y ions) evidenced the presence of persulfidated Cys labeled with HPE-IAM (marked in red). G Basal modification status of Cys644 in Drp1. The proportion of CysSH, CysS-SH (persulfidation), CysS-SSH (trisulfidation), CysS-SG (S-glutathionylation) and CysS-NO (S-nitrosylation) at Cys644 of Drp1-FLAG isolated from HeLa cells was quantified by mass spectrometry analysis. (n = 3 independent experiments). H MS/MS spectra for identifying S-glutathionylation of Cys644 in Drp1. Drp1-FLAG was treated with GSSG. I Quantification of S-glutathionylation of each cysteine residue in Drp1. Drp1-FLAG was treated with or without GSSG (3 mM). Analyzed peptide fragments containing cysteine residue are shown. (n = 3 independent experiments). J Quantification of polysulfidation and S-glutathionylation of Cys644 in Drp1. Depolysulfidated His-Drp1 was reacted with Na₂S₄ to prepare highly polysulfidated Drp1, and then treated with or without GSSG (1 mM). The proportion of CysSH, polysulfidation (CysS-SH, CysS-SSH and CysS-SSSH) and S-glutathionylation (CysS-SG, CysS-SSG and CysS-SSSG) at Cys644 was quantified. (n = 3 independent experiments). K Quantification of GSSG/GSH ratio in NRCMs treated with GSSG or GSH. (n = 3 independent experiments). L PLA assay using anti-Drp1 and anti-S-glutathione antibodies. PLA signals (green), counterstained with actinin (red) and DAPI (blue) were quantified. (n = 3 independent experiments). Scale bar, 20 µm. Data are shown as the means ± SEM. Significance was determined using two-sided unpaired t-test A–C,L; one-way ANOVA followed by Tukey’s post-hoc test E, J, K. Source data are provided as a Source Data file.