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. 2017 Jul 24;292(36):14851–14866. doi: 10.1074/jbc.M117.798397

Figure 9.

Figure 9.

Ptc7 isoforms have differing and opposing effects on CoQ6 synthesis. A, levels of steady-state CoQ6 (12C-CoQ6, blue bars) and de novo synthesized CoQ6 (13C6-CoQ6, orange bars) in strains expressing distinct Ptc7 isoforms (PTC7gHA, genomic, both isoforms expressed; PTC7sHA, exclusively expresses the isoform from spliced mRNA; PTC7nsHA, exclusively expresses the isoform from nonspliced pre-mRNA) and ptc7Δ. Labeling with 13C6-4HB was allowed to proceed for 3 h. Error bars, S.D. of n = 3 biological replicates (unpaired Student's t test; *, p < 0.05). B, expression and splicing of PTC7 for samples corresponding to A. PCR products representing the spliced and nonspliced forms are indicated. C, steady-state levels of HA-tagged Ptc7 proteins were determined by immunoblotting for samples corresponding to A. Proteins derived from the nonspliced and spliced forms of the PTC7 RNA are denoted as Ptc7ns and Ptc7s, respectively. Pgk1 (phosphoglycerate kinase 1) served as a loading control. Immunoblots for Snf2 and Coq7 are also included. D, levels of steady-state (12C-CoQ6, blue bars) and de novo synthesized (13C6-CoQ6, orange bars) CoQ6 in PTC7sHA and PTC7nsHA yeast cells were determined at the designated hours after labeling with 13C6-4HB. Error bars, S.D. of n = 3 biological replicates (unpaired Student's t test between corresponding bars for PTC7sHA and PTC7nsHA; *, p < 0.05; **, p < 0.005). E, RT-qPCR measurement of COQ1–COQ11 and PTC7 transcript levels between strains expressing different Ptc7 isoforms (PTC7gHA, genomic, both isoforms expressed; PTC7sHA, exclusively expresses the isoform from spliced mRNA; PTC7nsHA, exclusively expresses the isoform from nonspliced pre-mRNA) and ptc7Δ. Shown is the mean of three biological replicates. Error bars, S.D. F, summary analysis of transcript levels of Q-biosynthetic factors from E. G, RT-qPCR measurement of COQ1-COQ11 and PTC7 transcript levels between WT and snf2Δ yeast. Shown is the mean of three biological replicates. Error bars, S.D. H, summary analysis of transcript levels of Q-biosynthetic factors from G.