Figure 8.

Yeast coq10 null mutants have decreased de novo synthesis of ¹³C₆-labeled Q₆ compared to wild type, most notably during early-log phase growth. Wild-type and coq10 null yeast strains were cultured in SD-complete medium overnight and diluted to an OD_600nm of 0.05 in DOD-complete medium. Ethanol (vehicle-control), ¹³C₆-4HB (white bars), or ¹³C₆-pABA (black bars) were added to yeast cultures at an OD_600nm of 0.5 (early-log phase), 1.5 (late-log phase), or 3.0 (late-log phase). Prior to lipid extraction a known amount of Q₄ was added as an internal standard to each sample and to the Q₆ calibration standards. ¹³C₆-labeled precursor-to-product ion transitions are as follows: (A) ¹³C₆-HAB, 552.4/156.0; (B) ¹³C₆-HHB, 553.4/157.0; (C) ¹³C₆-IDMQ₆, 566.6/172.0; (D) ¹³C₆-DMQ₆, 567.6/173.0; (E) ¹³C₆-Q₆, 597.4/203.1. Dashed arrows leading from HAB to IDMQ₆ and from HHB to DMQ₆ indicate multiple steps of Q biosynthesis (requiring Coq3, Coq4, Coq5, and Coq6). Solid arrows indicate the deimination of IDMQ₆ to DMQ₆ (requiring Coq9), and the final two steps converting DMQ₆ to Q₆ (requiring Coq7 and Coq3). Error bars depict the average ± standard deviation (n=4). (The total content of Q₆ and Q₆-intermediates (¹³C₆-labeled + unlabeled), is depicted in Fig. S2). Statistical significance between pairs of samples was determined with the Student's t-test and lower-case letters above bars are indicative of statistical significance. In (A) and (B) the relative content of ¹³C₆-labeled HAB or HHB in the coq10 null during early-, mid-, or late-log phase growth were compared to the corresponding growth phase of the wild type (a, p < 0.0001; b, p = 0.0001; c, p = 0.0112). The significance level α was adjusted to 0.0167 according to the Bonferroni correction for both (A) and (B). In (E) the content of ¹³C₆-labeled Q₆ in the coq10 null during early-, mid-, or late-log phase growth was compared to amounts present in the corresponding growth phase of the wild type (a, p ≤ 0.0004). Additionally ¹³C₆-labeled Q₆ in mid- and late-log phase was compared to early-log phase for both wild type and the coq10 null. Labeled ¹³C₆-Q₆ in wild type at late-log phase was significantly different compared to early-log phase (b, p ≤ 0.0004), and labeled Q₆ in the coq10 null in mid-log phase was significantly different compared to early-log phase (c, p = 0.0008 for ¹³C₆-pABA). For all analyses in (E) the significance level α was adjusted to 0.0033 according to the Bonferroni correction.