FIGURE 4.

Tandem affinity-purified Coq complexes co-purify with Q₆ and late-stage Q₆ intermediates. For each strain, aliquots of the anti-PC eluates (29% of E1 and 29% of E2) were combined and subjected to lipid extraction with methanol and petroleum ether. Lipid extracts were analyzed by HPLC-MS/MS for Q₆ (A), DMQ₆ (C), and IDMQ₆ (E). Measured lipids were normalized by the extracted eluate volume. Bars represent the means of two measurements, and error bars represent the standard deviation. Statistical significance was determined with the two-tailed Student's t test, and the lowercase letters above the bars are indicative of statistical significance. In A, the content of Q₆ in CNAP3, CNAP6, and CNAP9 was 11.7-, 8.6-, and 22.6-fold higher, respectively, compared with wild type (a, p = 0.0001; b, p = 0.0040; c, p = 0.0006). In C, the content of DMQ₆ in CNAP3, CNAP6, and CNAP9 was 30-, 24-, and 97-fold higher, respectively, compared with wild type (a, p = 0.0110; b, p = 0.0113; c, p = 0.0010). In E, there was no detectable IDMQ₆ in the wild type. ND, not detected. Representative overlaid traces of all four strains (W303, purple; CNAP3, green; CNAP6, red; CNAP9, blue) are shown for Q₆ (B), DMQ₆ (D), and IDMQ₆ (F).