(A) The proposed path of mtClpX unfolding of ALAS to the active site is indicated in blue on the structure of yeast ALAS (PDB: 5TXR (Brown et al., 2018); image created with UCSF Chimera (Pettersen et al., 2004). The positions of cysteine pairs (indicated by amino acid number) introduced into ALAS are indicated by orange bonds. (B) Samples of ALAS cysteine variants (wt, 68–243, and 88–427) were separated by nonreducing SDS-PAGE and stained with Sypro Red, after oxidation (induced by addition of copper phenanthroline) and after reduction of oxidized samples with TCEP. Molecular weight marker (MW) sizes are indicated in kD. Cartoons indicate expected migration of species with intermolecular crosslinks (top right), uncrosslinked (middle right), and intramolecular crosslinks (bottom right). (C) mtClpX stimulation of PLP binding to indicated ALAS variants after oxidation and after re-reduction, monitored by fluorescence (ex. 434 nm, em. 515 nm). ALAS variants were present at 10 μM (monomer), PLP at 150 μM, ATP at 2 mM + regenerating system, and mtClpX, when included, at 0.5 μM (hexamer). Error bars represent SD from three independent experiments. p=0.002 for suppression of mtClpX action by oxidation of ALAS68x243 and 10−4 for ALAS88x427 (compared to reduced form of same variant). (D) Representative traces of PLP binding to ALAS variants. 100% PLP-bound value was set to observed plateau of PLP fluorescence.