Intragenic suppressor mutations of the COQ8 protein kinase homolog restore coenzyme Q biosynthesis and function in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.

Fig 1. Coenzyme Q biosynthetic pathway in S. cerevisiae and the formation of the high-molecular mass CoQ synthome.

A, The pathway for the enzymatic formation of Q₆ in yeast, starting with 4-hydroxybenzoic acid as the ring precursor. Questions marks indicate unknown steps involved in the decarboxylation and hydroxylation of the intermediate(s) leading to reduced coenzyme Q₆H₂. B, The schematic of a high-molecular mass complex in yeast, termed the CoQ synthome. Coq1, Coq2, and Coq10 polypeptides are not observed as members of the high-molecular mass complex. Coq1 and Coq2 produce HHB (designated by the dark gray hexagon). This early CoQ-intermediate is converted by subsequent action of Coq6 and other Coq polypeptides to an essential lipid component of the CoQ synthome (shown as a light gray hexagon in association with Coq4). Coq8 physically associates with Coq6 and is an ancient atypical kinase, thought to be responsible for the regulatory phosphorylation of Coq3, Coq5 and Coq7 and/or ATPase activity. Q₆, the product of the CoQ synthome, is designated by the orange hexagon.

Fig 2. Spontaneous revertants of NP-183AL expressing Coq8-A197V show respiratory growth on YPG plate medium.

The designated yeast strains (Table 1) were each grown overnight in 5 mL of YPD, diluted to an A_600nm of 0.2 with sterile PBS, and 2 μL of 5-fold serial dilutions were spotted onto the designated plate medium, corresponding to a final A_600nm of 0.2, 0.04, 0.008, 0.0016, and 0.00032. Plates were incubated at 30 °C, and growth is depicted after four days. Results shown are representative of two independent biological replicate experiments (see S8 Fig).

Fig 3. The Coq8-S232N substitution present in Rev-CL, Rev-DL, and Rev-EL is dominant, and its presence is sufficient to restore growth of the Coq8-A197V mutant on YPG.

A, Each isolated revertant (Rev-AL, Rev-BL, Rev-CL, Rev-DL, and Rev-EL) was mated with parental mutant, NP-183BH and the respective diploid strains (NPD-A, NPD-B, NPD-C, NPD-D, and NPD-E) were isolated as described in Materials and Methods. As a control, NP-183AL was mated with NP-183BH to form a diploid strain NPD-NP containing two copies of the coq8-3 mutation. The derived diploid strains were then plated on YPD, a fermentable carbon source, and YPG, a non-fermentable carbon source. The haploid wild-type strain, W303-1A, was also included. Plates were incubated at 30 °C for four days Panel A is representative of two independent biological replicate experiments (see S9 Fig). B, Expression of Coq8 harboring the dominant SupRC mutation present in Rev-CL rescues the growth of the NP183AL coq8-3 mutant on non-fermentable carbon source medium. Yeast plate dilution assay was conducted on parental mutant, NP-183AL, transformed with the designated plasmids: plc-Coq8 (p3HN4), yeast low-copy COQ8; plc-A197V, yeast low-copy Coq8-A197V; plc-S232N, yeast low copy Coq8-S232N; or plc-A197V/S232N, yeast low copy Coq8-A197V/S232N. Each strain was cultured overnight in SD-Ura selective plate media, and the optical density (A_600nm) adjusted to 0.2 with sterile PBS, and 2 μL of 5-fold serial dilutions were spotted onto each type of plate medium, as described in Fig 2. Cells were incubated at 30°C for three days. Panel B is representative of two independent biological replicate experiments (see S7 Fig).

Fig 4. Structural prediction of S. cerevisiae Coq8 and sequence alignment with Coq8 homologs depict the sites of the A197V and suppressor mutations.

A, PHYRE2 homology prediction of yeast Coq8, modeled to a 44% identity to PDB 4PED, corresponds to the partial structure of crystallized human COQ8A [13,41]. The structural features of Coq8 and COQ8A are color-coded as described previously [13]: The N-lobe folds into β-sheets, gray and a single helix αC, red; inserted between these features are the GQα5 and GQα6 helices, green; the C-lobe is shown in yellow; an N-terminal extension is shown in blue. The color-coding of the amino acids are: A197, pink; L237, cyan; P220, orange; and S232, purple. These locations are the sites of the parental mutant, Coq8-A197V and the three revertants, Rev-AL (L237P), Rev-BL (P220S), and Rev-CL (S232N). Rev-DL and Rev-EL also contained S232N. The predicted 32 amino acid mitochondrial targeting sequence of Coq8 [42] has been removed from the model to allow for the accurate depiction of the mature polypeptide. A inset, The predicted distances between the A197 and each of the amino acid substitutions present in Rev-AL, Rev-BL, and Rev-CL are shown on the structure following its rotation of 90°counterclockwise. B, Yeast Coq8 is depicted in the same orientation and color-coding as for the previously published ADCK3 [13]. C, Multiple sequence alignment and depiction of the locations of the mutations present in each of the yeast revertants. Secondary structure as predicted in the model of Coq8 is depicted above the yeast Coq8 sequence. A197V is present within the coq8-3 parental mutant, and is present in each of the revertants. In addition, L237P occurs in Rev-AL, the P220S in Rev-BL, and the S232N is present in Rev-CL, Rev-DL and Rev-EL. The alignment included the designated Coq8 homologs from S. cerevisiae, S. pombe, E. coli, A. thaliana, C. elegans, M. musculus, and H. sapiens. The amino acid alignment was built using MUSCLE and visualized using EsPript [43].

Fig 5. In YPD liquid medium, biosynthesis of Q₆ in Rev-CL is nearly comparable to WT, while Rev-AL produces substantially lower levels of Q₆ compared to WT, and in Rev-BL Q₆ is not detected.

A, Levels of unlabeled Q₆ (¹²C-Q₆) and B, de novo synthesized ¹³C₆-Q₆ (¹³C₆-Q₆) in each strain were determined at the designated time points after labeling with ¹³C₆-4HB in YPD medium. Unlabeled Q₆ and de novo labeled ¹³C₆-Q₆ were not detected in coq8Δ, NP-183A or in Rev-BL at any of the time points; this is denoted as “ND” at the 5 h time point for simplicity. C and D, The expanded scales show the levels of unlabeled and de novo ¹³C₆-Q₆ present in the Rev-AL strain. Error bars, S.D. of n = 3 biological replicates (unpaired Student’s t test between all strains compared to WT, with statistical significance represented by: *p<0.05, **p<0.005, ***p<0.0005). Results are shown for three independent biological replicate experiments.

Fig 6. Rev-CL synthesizes late-stage intermediate DMQ₆, while Rev-AL and Rev-BL accumulate the early-stage intermediate, HHB.

A, Levels of unlabeled ¹²C-DMQ₆; B, de novo synthesized ¹³C₆-DMQ₆; C, unlabeled ¹²C-HHB; and D, de novo synthesized ¹³C₆-HHB were determined in WT, coq8Δ, NP-183A, Rev-AL, Rev-BL, and Rev-CL at the designated time points after labeling with ¹³C₆-4HB in YPD medium. Error bars, S.D. of n = 3 biological replicates (unpaired Student’s t test between all strains compared to WT, with statistical significance represented by: *p<0.05, **p<0.005, ***p<0.0005). Panel A and B show non-detectable values for the lipids analyzed for coq8Δ, NP-183A, Rev AL, and Rev BL for all time points, indicated as “ND” for the first time point for simplicity. Panels C and D show non-detectable levels of HHB in WT. Results are shown for three independent biological replicate experiments.

Fig 7. Rev-CL and Rev-AL are able to synthesize Q₆ in YPG liquid medium, and Rev-CL produces comparable levels of total Q₆ (¹²C-Q₆ + ¹³C₆-Q₆) as WT.

A, Levels of unlabeled Q₆ (¹²C-Q₆) and B, de novo synthesized ¹³C₆-Q₆ (¹³C₆-Q₆) in WT, coq8Δ, NP-183A, Rev AL, Rev BL, and Rev CL were determined after labeling with ¹³C₆-4HB for 5 hours in YPD or YPG liquid medium. C, Total Q₆ (¹²C-Q₆ + ¹³C₆-Q₆) content is shown for all labeled strains in both YPD and YPG medium. Error bars, S.D. of n = 3 biological replicates (unpaired Student’s t test between all strains compared to WT in the same type of medium, with statistical significance represented by: *p<0.05, **p<0.005, ***p<0.0005). “ND” indicates lipids levels that were non-detectable in the indicated strains. Results are shown for three independent biological replicate experiments.

Fig 8. Assessment of Q₆ and Q₆-intermediates in strains following incubation on YPG plate medium reveals the presence of Q₆ in Rev-BL.

A, Levels of unlabeled ¹²C-CoQ₆; B, ¹²C-DMQ₆; and C, ¹²C-HHB in WT, coq8Δ, NP-183A, Rev AL, Rev BL, and Rev CL were determined on colonies that were cultured on YPG solid medium for two days. Error bars, S.D. of n = 3 biological replicates (unpaired Student’s t test between all strains compared to WT, with statistical significance set at p<0.05). “ND” indicates lipids levels that were undetectable in the indicated strains. Results are shown for three independent biological replicate experiments.

Fig 9. Growth in YPGal (a nonrepressive carbon source) reveals all three revertants are capable of de novo ¹³C₆-Q₆ production.

A, Levels of unlabeled ¹²C-Q₆ and de novo ¹³C₆-Q₆; B, unlabeled ¹²C-DMQ₆ and de novo ¹³C₆-DMQ₆; C, unlabeled ¹²C-HHB and de novo ¹³C₆-HHB in WT, coq8Δ, NP-183A, Rev-AL, Rev-BL, and Rev-CL were determined in cultures of yeast labeled for 5 hours with ¹³C₆-4HB in YPGal + 0.1% Dextrose liquid media. The expanded y axis in the panel A inset demonstrates the levels of Q₆ production in Rev-AL and Rev-BL. Error bars, S.D. of n = 3 biological replicates (unpaired Student’s t test between all strains compared to WT, with statistical significance represented by: *p<0.05, **p<0.005, ***p<0.0005). “ND” indicates lipids levels that were non-detectable and lower than background in the indicated strains. Results are shown for three independent biological replicate experiments.

Fig 10. Amounts of indicator Coq polypeptides in mitochondria isolated from Rev-CL are restored to near wild-type content.

The amounts of Coq4, Coq7, Coq8, and Coq9 polypeptides were determined by SDS-PAGE and immunoblotting. Samples were separated on 12% SDS-PAGE gels and then transferred to PVDF membranes for immunoblotting with antisera to the designated yeast Coq polypeptides. 25 μg of purified mitochondria was analyzed for each strain. Arrows indicate each antibody-detected protein in their respective blots; a coq null strain in each respective Coq polypeptide blot was used as a reference for the absence of the desired band of interest. Blots were performed at least two times each (see S4–S6 Figs). The Mdh1 blot serves to validate the samples used in all the blots; the samples that were loaded in the Mdh1 blot are those of the same preparation as used for the Coq4, Coq7, Coq8, and Coq9 blots.

Fig 11. Model for the restoration of Q₆ biosynthesis in three revertants.

A, Under conditions of Coq8 expression in wild-type yeast, benchmark values of Q₆ production are noted and the wild-type amounts of key Coq polypeptides (Coq4, Coq7, Coq8, and Coq9) are defined. B, the yeast coq8Δ mutant lacks Q₆, has a lower content of the Coq7 polypeptide, the Coq4, Coq8, and Coq9 polypeptides are not detectable, and the early intermediate HHB accumulates. C, the yeast mutant expressing the Coq8-A197V polypeptide lacks Q₆, has a lower content of the Coq7 polypeptide, the Coq4 and Coq9 polypeptides are not detectable, and the early intermediate HHB accumulates. D, The dominant Rev-CL revertant restores Q₆ production and the stability of the Coq4, Coq7 and Coq9 polypeptides. E, The recessive Rev-AL revertant has partially restored Q₆ biosynthesis. However, amounts of the Coq4 and Coq9 polypeptides are low and this results in greatly impaired synthesis of Q₆. F, The recessive Rev-BL revertant exhibits very low levels of Q₆ biosynthesis, and has dramatically decreased content of the Coq4 and Coq9 polypeptides, even though Coq8 and Coq7 polypeptides remain readily detectable.