Fig. 1.

A disulfide cross-link is formed between subunits ND3 and PSST in mutant Q133C^PSST. Purified complex I from parental and mutant strains were incubated with either 5 mM dithiothreitol (DTT) or 0.1 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) for 5 min and then separated by non-reducing Tricine SDS-PAGE. a An additional band was observed in the sample from mutant Q133C^PSST treated with DTNB. b Mass spectrometric analysis of the corresponding gel slice confirmed the presence of cross-linked subunits PSST and ND3 and allowed label free quantification of their relative abundance as compared to the corresponding gel slice of the DTT treated sample (mean ± s.d.; n = 3 technical replicates; ***p < 0.001, ANOVA with Bonferroni correction). c Subunits PSST and ND3 were also identified by dSDS-PAGE, in which the cross-link was preserved in the first dimension (1D) and then reduced in the second dimension (2D) to separate both proteins at a shifted position in the mutant. Note that the spot containing subunit PSST does not disappear after cross-linking, because it also contains accessory subunits NUJM and NUPM³⁷