Mining for protein S-sulfenylation in <i>Arabidopsis</i> uncovers redox-sensitive sites

Abstract

Hydrogen peroxide (H₂O₂) is an important messenger molecule for diverse cellular processes. H₂O₂ oxidizes proteinaceous cysteinyl thiols to sulfenic acid, also known as S-sulfenylation, thereby affecting the protein conformation and functionality. Although many proteins have been identified as S-sulfenylation targets in plants, site-specific mapping and quantification remain largely unexplored. By means of a peptide-centric chemoproteomics approach, we mapped 1,537 S-sulfenylated sites on more than 1,000 proteins in <i>Arabidopsis thaliana</i> cells. Proteins involved in RNA homeostasis and metabolism were identified as hotspots for S-sulfenylation. Moreover, S-sulfenylation frequently occurred on cysteines located at catalytic sites of enzymes or on cysteines involved in metal binding, hinting at a direct mode of action for redox regulation. Comparison of human and <i>Arabidopsis</i> S-sulfenylation datasets provided 155 conserved S-sulfenylated cysteines, including Cys181 of the <i>Arabidopsis</i> MITOGEN-ACTIVATED PROTEIN KINASE4 (AtMAPK4) that corresponds to Cys161 in the human MAPK1, which has been identified previously as being S-sulfenylated. We show that, by replacing Cys181 of recombinant AtMAPK4 by a redox-insensitive serine residue, the kinase activity decreased, indicating the importance of this noncatalytic cysteine for the kinase mechanism. Altogether, we quantitatively mapped the S-sulfenylated cysteines in <i>Arabidopsis</i> cells under H₂O₂ stress and thereby generated a comprehensive view on the S-sulfenylation landscape that will facilitate downstream plant redox studies.