Figure 5.

CysSSH-mediated polysulfidation of CDKAL1. (A) Experimental design to detect ³⁴S-labeled polysulfide in the CDKAL1 protein. (B and C) HeLa cells overexpressing Myc-CDKAL1 were treated with vehicle (CysSSH donor) or the CysS³⁴SH donor for 1 h. Myc-CDKAL1 was immunoprecipitated and subjected to mass spectrometry to determine the isotopic distribution of polysulfidation in Cys residues. The isotopic distribution of CysSSH (abundance of CysS³⁴SH relative to that of CysSSH) is shown in (B). The isotopic distribution of the Cys residue (abundance of CysS³⁴H relative to that of CysSH) is shown in (C). ****P < 0.0001, n = 4. (D) HeLa cells expressing wild-type Myc-CDKAL1 or mutant CDKAL1 carrying Cys-to Ala mutations in the UPF0004 domain were treated with the CysS³⁴SH donor, followed by mass spectrometric analysis. The mutant CDKAL1 had less ³⁴S incorporation in Cys residues in CDKAL1, when compared with the wild-type CDKAL1. n = 3 each, **P < 0.01.