Mitochondrial NAD kinase Pos5 is required for CoQ biosynthesis in yeasts

Shogo Nishihara; Ikuhisa Nishida; Yasuhiro Matsuo; Tomohiro Kaino; Makoto Kawamukai

doi:10.1371/journal.pone.0346295

. 2026 Apr 2;21(4):e0346295. doi: 10.1371/journal.pone.0346295

Mitochondrial NAD kinase Pos5 is required for CoQ biosynthesis in yeasts

Shogo Nishihara ¹, Ikuhisa Nishida ², Yasuhiro Matsuo ^1,^3,⁴, Tomohiro Kaino ^1,^3,^4,^*, Makoto Kawamukai ^3,^*

Editor: Junzheng Yang⁵

PMCID: PMC13046142 PMID: 41926382

Abstract

Coenzyme Q (CoQ) is an essential component of the electron transport chain, and ten genes involved in CoQ biosynthesis have been identified in Schizosaccharomyces pombe. To gain further insight into CoQ biosynthesis, we screened the Bioneer gene-deletion library and found that the Δpos5 strain produced only 0.2-fold of the wild-type CoQ₁₀ level. Pos5 shares homology with Saccharomyces cerevisiae Pos5 (ScPos5), a mitochondrial NADH (or NAD⁺) kinase that generates NADPH (or NADP⁺). Heterologous expression of ScPOS5 in the S. pombe Δpos5 strain recovered CoQ content to 0.9-fold of the wild-type level, indicating functional conservation of Pos5 between the two yeasts. Consistently, CoQ₆ level in ΔScpos5 was decreased to 0.2-fold of that in the wild-type strain. The Δpos5 strain exhibited several phenotypes characteristic of CoQ-deficient S. pombe, including inability to grow on non-fermentable carbon sources, hypersensitivity to oxidative stress, and high sulfide production. Among CoQ biosynthetic enzymes, Coq6 monooxygenase is thought to utilize NADPH. Supplementation with VA or PHB partially restored CoQ production in the Δpos5 strain, while overexpression of coq6 had negligible effect. These findings suggest that Pos5 is required for the earlier step of CoQ biosynthesis.

Introduction

CoQ biosynthetic pathway

Coenzyme Q (CoQ), also known as ubiquinone, is an essential component of the respiratory chain required for energy production. CoQ cycles between reduced [CoQH₂] and oxidized [CoQ] forms [1]. This is a redox property important for electron transfer during respiration and for functioning as an antioxidant. Eukaryotes and bacteria belonging to the phylum Pseudomonadota synthesize CoQ endogenously, with species-specific variations in side chain length; for example, Homo sapiens and Schizosaccharomyces pombe produce CoQ₁₀, where the number of isoprene units is ten, Saccharomyces cerevisiae produces CoQ₆, and Escherichia coli produces CoQ₈ [2,3]. CoQ biosynthesis comprises mainly three stages: benzoquinone ring formation, isoprene side chain synthesis, and modification of the prenylated quinone ring [4]. The precursor of the side chain is synthesized from isopentenyl diphosphate and farnesyl diphosphate by polyprenyl diphosphate synthase [5]. Then, it is transferred to p-hydroxybenzoic acid (PHB) or p-aminobenzoic acid by p-hydroxybenzoate–polyprenyl diphosphate transferase (Coq2 or Ppt1) [6,7]. In eukaryotes, PHB is derived from tyrosine or other amino acids. The quinone ring of prenylated PHB then undergoes modifications, including methylations (Coq3 and Coq5), decarboxylation (Coq4), and hydroxylations (Coq6 and Coq7), to generate mature CoQ [2,8,9]. These reaction enzymes are encoded by nine genes in S. cerevisiae (COQ1-COQ9) and ten genes in S. pombe (dps1, dlp1, ppt1, and coq3-coq9) [10–14]. Those genes were utilized for CoQ₁₀ bioproduction in S. pombe [15]. In addition, benzoic acid inhibits the synthesis of CoQ [11], protein kinase A (Pka1) controls the level of CoQ [16], and regulatory factors such as Coq11 and Coq12 have recently been identified in S. pombe, suggesting that further more unknown factors are involved in regulating CoQ biosynthesis [17].

Among the deletion mutants that showed a lower CoQ₁₀ level in S. pombe, we selected the pos5 mutants for further analysis. Although Pos5 has been extensively studied in S. cerevisiae, very little is known about its function in S. pombe. In S. cerevisiae, Pos5 is a unique mitochondrial nicotinamide adenine dinucleotide NAD(H) kinase that generates NADPH or NADP⁺ from NADH or NAD⁺ [18–22]. Because of its polarity, mitochondrial NADP(H) is synthesized from NAD(H) via mitochondrial NAD(H) kinase, as no mitochondrial transporter has been identified in yeast. In mitochondria, NADP⁺ is essential for several processes, including the TCA cycle, amino acid biosynthesis, glutathione reduction, and Fe-S cluster biogenesis [19–22]. However, the relevance of NAD(H) kinase activity in CoQ biosynthesis has not been documented in any organism. Therefore, in this study, we focused on elucidating the role of Pos5 involves in CoQ synthesis.

Materials and methods

Yeast and E. coli strains, and growth media

Yeasts and E. coli strains used in this study are listed in Table 1. Yeast standard media and genetic manipulation methods have been described previously [23]. S. pombe strains were grown in complete YES medium (0.5% yeast extract (OXOID), 3% glucose, supplemented with 225 mg/mL adenine sulfate, 225 mg/mL leucine, 225 mg/mL uracil, 225 mg/mL histidine, and 225 mg/mL lysine hydrochloride). A non-fermentable carbon source medium, YEGES, containing 0.5% yeast extract, 2% glycerol, 1% ethanol, supplemented with 225 mg/mL adenine sulfate, 225 mg/mL leucine, 225 mg/mL uracil, 225 mg/mL histidine, and 225 mg/mL lysine hydrochloride, was used. PM medium comprised 0.3% potassium hydrogen phthalate, 0.56% sodium phosphate, 0.5% ammonium chloride, 2% glucose, and standard vitamins, minerals, and salts. PMGALU medium contained 0.375% glutamate as the nitrogen source instead of ammonium chloride and was supplemented with adenine sulfate, leucine, and uracil in PM. S. cerevisiae strains were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose). Synthetic defined (SD) medium (2% glucose and 6.7 g/L yeast nitrogen base without amino acids (BD Biosciences), containing 19 mg/L adenine sulfate; 76 mg/L each of arginine, histidine, lysine hydrochloride, methionine, uracil, and tryptophan; and 395 mg/L leucine). SD without glucose with glycerol (SD-C+glycerol) medium contained 3% glycerol and 6.7 g/L yeast nitrogen base without amino acids and the same amount of above amino acids, uracil, and adenine sulfate. SC medium consisted of 2% glucose and 6.7 g/L yeast nitrogen base without amino acids, supplemented with 19 mg/L adenine sulfate; 76 mg/L each of alanine, arginine, asparagine, aspartate, cysteine, glutamine, glutamate, glycine, isoleucine, histidine, L-inositol, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, uracil, valine; 7.6 mg/L p-aminobenzoic acid; and 395 mg/L leucine).

Table 1. Strain list.

Strain	Genotype	Resource
S. pombe
PR109	h^- leu1–32 ura4-D18	Lab stock
PR110	h ⁺ leu1–32 ura4-D18	Lab stock
KH2 (OG1)	h ⁺ leu1–32 ura4-D18 ppt1::kanMX6	Hayashi K. et al., 2014
KH6 (PC976)	h ⁺ leu1–32 ura4-D18 coq6::kanMX6	Hayashi K. et al., 2014
LJ1030	h ⁺ leu1–32 ura4-D18 dps1::kanMX6	Zhang M. et al., 2008
NSP7	h ⁺ leu1–32:leu1-pJK148P41nmt1-ScPOS5 ura4-D18 pos5::kanMX6	This study
NSP11	h ⁺ leu1–32:leu1-pJK148P41nmt1-pos5 ura4-D18 pos5::kanMX6	This study
NSP12	h ⁺ leu1–32:leu1-pJK148P41nmt1-MTS36UTR1 ura4-D18 pos5::kanMX6	This study
NSP13	h ⁺ leu1–32:leu1-pJK148P41nmt1-UTR1 ura4-D18 pos5::kanMX6	This study
NSP15	h ⁺ leu1–32:leu1-pJK148P41nmt1-ΔMTS83pos5 ura4-D18 pos5::kanMX6	This study
NSP16	h^- leu1–32 ura4-D18 pos5-GFP(S65T)-kanMX6	This study
NSP23	h ⁺ leu1–32:leu1-pJK148Pnmt1 ura4-D18	This study
NSP25	h ⁺ leu1-32:pJK148-Pnmt1-coq6 ura4-D18 pos5::kanMX6	This study
NSP26	h ⁺ leu1–32:leu1-pJK148Pnmt1 ura4-D18 pos5::kanMX6	This study
NSP27	h ⁺ leu1–32:leu1-pJK148Pnmt1 ura4-D18 coq6::kanMX6	This study
NSP28	h ⁺ leu1–32:leu1-pJK148Pnmt1-coq6 ura4-D18 coq6::kanMX6	This study
NSP60	h ⁺ leu1–32:leu1-pJK148Pnmt1-atd1 ura4-D18 pos5::kanMX6	This study
RM3	h ⁺ leu1–32 ura4-D18 cyc1::kanMX6	Miki R. et al., 2008
RYP7	h ⁺ leu1–32 ura4-D18 pos5::kanMX6	This study

Bioneer disruptant (Ver. 4)
Δleu1	h ⁺ ade6-M216 leu1–32 ura4-D18 leu1::kanMX4	Kim D.U. et al., 2010
Δpos5	h ⁺ ade6-M216 leu1–32 ura4-D18 pos5::kanMX4	Kim D.U. et al., 2010
Δarg11	h ⁺ ade6-M216 leu1–32 ura4-D18 arg11::kanMX4	Kim D.U. et al., 2010

S. cerevisiae
BY4741	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	Lab stock
MK1601	MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pos5::kanMX4	Kawai S.
Δcoq2	MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 coq2::kanMX4	Multiple-System Atrophy Research Collaboration. 2013

E. coli
DH5α	F^- Φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK^-, mK⁺) phoA supE44 λ^- thi-1 gyrA96 relA1	Lab stock

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Construction of S. pombe strains

The oligonucleotide primers used in this study are listed in S1 Table. S. pombe pos5 on the chromosome was disrupted by replacing pos5 with a selectable marker as previously described [24]. The 1.6-kb kanMX6 module was amplified using flanking sequences corresponding to the 5’ and 3’ ends of pos5. Resistant colonies were selected on YES plates containing 100 mg/L G418, and pos5 disruption was verified using colony PCR. DNA fragments of 500–600 bp corresponding to the 5’ or 3’ regions of the gene were amplified by PCR using pos5del-A and pos5del-B or pos5del-C and pos5del-D primer pairs (S1 Table). The amplicons were fused to the ends of the kanMX6 module using PCR. The PR110 strain was transformed with the resulting pos5::kanMX6 fragments to obtain the pos5 disruptant. The chromosomal deletion of pos5 was confirmed by PCR using the nb2 and pos5del-check primers. The obtained strain was designated as RYP7 (Δpos5). Pos5-GFP-tagged strain was constructed using the recombinant PCR approach described in a previous study [24]. The pFA6a-GFP(S65T)-kanMX6 plasmid [24] was used as the template DNA, and the resulting PCR products carried the GFP-kanMX6 cassette in the 3’ region downstream of pos5. The oligonucleotides pos5-TAGW, pos5-TAGX, pos5-TAGY, and pos5-TAGZ were used to construct the pos5-GFP-kanMX6 strain. The resulting pos5-GFP-kanMX6 cassette was introduced into the PR109 strain, and the transformants carrying the GFP-fused pos5 were verified by colony PCR [25]. The S. cerevisiae Δpos5 strain (ΔScpos5; MK1601) was provided by Shigeyuki Kawai (Ishikawa Prefectural University).

Plasmid construction

The plasmids used in this study were constructed by a method described previously (S2 Table) [10]. Each gene encoding NAD⁺/NADH kinase was PCR amplified using the S. pombe PR110 genome and the S. cerevisiae BY4741 genome as templates, with primers containing restriction sites. The amplified fragments were digested using restriction endonucleases and then inserted into the appropriate sites of the pREP41, pJK148-P_nmt1 or pJK148-P41_nmt1 vector by ligation. pREP41-pos5 was constructed by inserting the PCR product amplified using pos5(SalI)-F and pos5(BamHI)-R primers into the SalI and BamHI sites of pREP41. pREP41-coq6 was constructed by inserting the fragment digested from pREP1-coq6 by SalI and SmaI into the same sites of pREP41 [10]. Further, the other plasmids pREP41-ScPOS5 and UTR1 were also constructed similarly. To construct mitochondrial NAD kinase, mitochondrial-targeting sequence of coq3 from S. pombe was fused to the UTR1 sequence from S. cerevisiae. The mitochondrial transit peptide in S. cerevisiae Pos5p was 62 amino acids from the N-terminus, and its homologous position is 83 amino acids in S. pombe Pos5. Thus, the primers were designed to anneal at 298 bp from the 5’-terminus of Pos5. pJK148-Pnmt1 was constructed from pJK148 and pREP3X. The Pnmt1-MCS-Tnmt1 region was amplified and inserted into KpnI and SacI sites of pJK148. pJK148-P41nmt1 was constructed from pJK148 and pREP41X. The P41nmt1-MCS-Tnmt1 region was amplified and inserted into KpnI and SacI sites of pJK148. pJK148-P41nmt1-pos5 was constructed by inserting the pos5 insert fragment digested from pREP41-pos5 into the SalI and BamHI sites of pJK148-P41nmt1. The other plasmids pJK148-P41nmt1-ScPOS5, UTR1, Spcoq3MTS36UTR1, ΔMTS83pos5, and pJK148-Pnmt1-coq6 were constructed similarly. To examine the cellular localization of Pos5, GFP fusion was generated by inserting pos5 into the pSLF272L-GFP(S65A) vector [26,27]. pSLF272L-pos5-GFP(S65A) was constructed by inserting the PCR product amplified using the pos5-GFP(XhoI)-F and pos5-GFP(NotI)-R3 primers into XhoI and NotI sites of pSLF272L-GFP(S65A). The genes amplified by PCR were verified using DNA sequencing.

CoQ extraction and measurement

Yeast precultures were inoculated into large-volume media and incubated for the indicated times. Unless otherwise specified, strains were grown at 30°C in 55 mL of liquid media (with or without specific supplements), starting from an initial density of 1 × 10⁵ cells/mL, and cultured for 48 or 72 hours. Cell numbers were counted using a Sysmex CDA-1000B (Sysmex, Tokyo, Japan), and the OD₆₀₀ was measured using a Shimadzu UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan). Cells were harvested, and CoQ was extracted using the autoclave method as described previously [10]. Prior to extraction, 5 µg of CoQ₆ was added to each sample as an internal standard. Crude CoQ samples were separated by normal-phase thin-layer chromatography using a Kieselgel 60 F₂₅₄ plate (Merck Millipore, MA, USA). The TLC was developed with benzene as the solvent. After development, the TLC plate was visualized under UV illumination, and the bands corresponding to CoQ₆ and CoQ₁₀ were excised and extracted with hexane/isopropanol (1:1, v/v). The sample solvents were evaporated, and the dried solids were dissolved in ethanol. Purified CoQ samples were analyzed using high-performance liquid chromatography on a Shimadzu HPLC Class VP series instrument (Shimadzu). A reversed-phase YMC-Pack ODS-A column (A-312–3 AA12S03-1506PT, 150 × 6 mm, 3-μm particle size, 120 Å, YMC, Kyoto, Japan) was used. The mobile phase consisted of ethanol at a flow rate of 1.0 mL/min. CoQ₆, and CoQ₁₀ were detected by UV absorption at 275 nm.

Isolation of mitochondria

Yeast cells were pre-cultured for 24 hours in 100 mL of YES medium and then inoculated into 3 L of YES medium. After incubation for 16–20 hours, cells were harvested at OD₆₀₀ = 1. Mitochondria were isolated according to a previously described method [27] with slight modifications. In the current experiment, we incubated the pellet with 100 mM Tris-SO₄ and 10 mM DTT for 30 minutes at 30°C. To improve the yield of mitochondria, the pellet obtained by the initial homogenization and centrifugation was resuspended in a buffer containing 0.6 M mannitol, 20 mM HEPES-KOH, 0.5 mM EDTA, and 1 mM PMSF, and further homogenized 15 times.

Measurement of NADP(H)

The concentrations of NADP⁺ and NADPH in isolated yeast mitochondria extracts were determined using an enzymatic cycling assay according to a method reported previously [21,28,29]. Briefly, 50 µL of each sample was mixed with an equal volume of either 0.1 N HCl (for NADP⁺ measurement) or 0.1 N KOH (for NADPH measurement), followed by incubation at 85°C for 3 min. Subsequently, the treated extracts and the corresponding NADP⁺ or NADPH standards were added to a reaction mixture to a final volume of 200 µL containing 100 mM HEPES-KOH (pH 8.0), 0.5 mM EDTA, 2.5 mM glucose-6-phosphate (G6P), 1.66 mM phenazine ethosulfate, and 0.42 mM MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The reaction was initiated by the addition of 0.5 U of G6P dehydrogenase, and absorbance at 570 nm (A₅₇₀) was measured using a Corona SH-9000Lab microplate reader (Hitachi, Tokyo, Japan).

Mitochondrial staining and fluorescence microscopy

Mitochondria were stained using the MitoTracker Red FM dye (Invitrogen, Thermo Fisher Scientific, Inc). Cells were suspended in PMU medium and incubated with 50 nM MitoTracker Red FM at room temperature for 1 hour. Imaging was performed at 1000x magnification using a BX2-FL-2 fluorescence microscope (Olympus). GFP(S65A) fluorescence was observed at an excitation wavelength of 485 nm. Fluorescent images were obtained using a DP74-SET-A digital camera (Olympus) connected to the microscope and processed using cellSens ver.2.2 (Olympus).

Data and statistical analyses

Data from control and experimental samples were compared using the two-sample t-tests in Microsoft Excel (WA, USA). p-values <0.05 were considered statistically significant. Data from control and experimental samples were compared using one-way ANOVA with a post hoc test (Dunnett’s test) performed with EZR (Jichi Medical University, Tochigi, Japan) [30]. EZR is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander designed to add statistical functions frequently used in biostatistics.

Results

The S. pombe Δpos5 strain exhibits a phenotype similar to that of the CoQ-deficient strain

We have previously investigated the genes involved in CoQ biosynthesis of S. pombe using a Bioneer gene-deletion library and obtained approximately 40 individual gene-deleted strains with a CoQ₁₀ content lower than that of the wild-type strain [17]. In this study, we selected a Δpos5 strain from these strains for further analysis because it exhibited respiration deficiency, similar to CoQ-deficient strains, in addition to low CoQ₁₀ production. We independently constructed a Δpos5 strain to ensure that the phenotype observed in the Δpos5 strain from Bioneer Corp. is the same as our construct. CoQ levels in the Δpos5 strain of our construct were decreased to 0.2-fold of those in the wild-type strain (Fig 1A and 1B) as in the originally screened Bioneer Δpos5 strain (Fig 1C and 1D). Subsequently, we examined the phenotypes previously observed in CoQ-deficient strains of S. pombe, which exhibit respiratory deficiency, growth delay in minimal media, H₂O₂ sensitivity, and enhanced H₂S production [13,31]. The Δpos5 strain failed to grow on YEGES medium containing glycerol and ethanol as non-fermentable carbon sources and showed retarded growth on YES containing hydrogen peroxide as well as on minimal medium (Fig 1E). Supplementation with arginine partially restored Δpos5 growth (S1 Fig) as observed previously [32]. This is due to the requirement of NADPH for Arg11-catalyzed reaction in arginine biosynthesis. When grown on YES containing CuSO₄, Δpos5 colonies also developed a brown coloration, similar to the Δdps1 strain, which is completely defective in CoQ₁₀ synthesis (Fig 1E). In addition, Δpos5 cells showed a round morphology, which is often seen in the mutants related to sexual differentiation [33]. The phenotype was reverted to the normal rod shape upon expression of pos5 or mitochondrially targeted UTR1, which encodes a cytosolic NADK responsible for NADP(H) synthesis in S. cerevisiae, resembling the morphology of wild-type cells (S2 Fig). To determine whether the reduced CoQ level in the Δpos5 strain is simply a consequence of defective respiration, we next compared the CoQ content of the Δpos5 strain with that of a cytochrome c-deficient respiration mutant (Δcyc1) (Fig 1F and 1G). The Δcyc1 strain did not show a marked decrease in CoQ levels, suggesting that respiratory deficiency alone does not account for the low CoQ level in the Δpos5 strain. These results indicate that Pos5 is specifically important in CoQ biosynthesis in S. pombe.

Fig 1 — A, B: CoQ₁₀ levels of wild-type and *Δpos5* strains. Cells were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per culture volume (B). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Student’s t-test) versus wild-type strain. C, D: CoQ₁₀ levels of the *Δleu1* and *Δpos5* strains obtained from Bioneer Corp. Cells were cultured in YES medium for 48 hours. Bars indicate the CoQ₁₀ content per cell (C) and per volume (D). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Student’s t-test) versus *Δleu1* strain. E: Wild-type, *Δdps1*, and *Δpos5* strains were serially diluted (1:5) from 1 x 10⁷ cells/mL and spotted onto YES, YEGES (2% glycerol and 1% ethanol), YES + 2, 3 mM H₂O₂, YES + 0.5 mM CuSO₄, and PMLU media. Plates were incubated at 30°C for 3–7 days (YES: 3 days, YEGES, YES + H₂O₂, YES + CuSO₄: 5 days, PMLU: 7 days). The *Δdps1* strain, which is CoQ-deficient, was included for comparison. F & G: Comparison of CoQ levels between *Δpos5* and *Δcyc1* strains. Wild-type, *Δcyc1*, and *Δpos5* strains were cultured in YES medium for 48 hours. Bars indicate CoQ₁₀ content per cell (F) and per volume (G). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Student’s t-test) versus the wild-type strain. NS: no significant difference.

Pos5 functions as an NAD(H) kinase

S. pombe pos5 gene is predicted to encode a mitochondrial NAD(H) kinase because the Pos5 protein shares 37% identity with S. cerevisiae Pos5, a well-characterized mitochondrial NADH kinase (Fig 2A) [20,34]. To verify the functional similarity of SpPos5 and ScPos5, we constructed the Δpos5 + pJK148-P41nmt1-ScPOS5 strain (NSP7), in which ScPos5 was integrated at the chromosomal leu1 locus of the S. pombe Δpos5 strain, and measured its CoQ content. CoQ levels in the NSP7 strain were recovered to 0.9-fold of that observed in the Δpos5 + pos5 strain (NSP11) (Fig 2B and 2C), indicating functional similarity of these two proteins.

Fig 2 — A: Sequence alignment of the Pos5 amino acid sequences from *S. pombe* (L972) and *S. cerevisiae* (S288C). Alignment was performed using ClustalW and visualized with the boxshade server. Conserved NAD kinase regions (I and II) are indicated by blue box. Motif I (GGDG) is part of the ATP-binding site, and Motif II represents a nucleotide-binding site. B, C: Restoration of CoQ₁₀ level in the *Δpos5* strain by *ScPOS5* overexpression. Wild-type+vector (NSP23), *Δpos5* + vector (NSP26), *Δpos5*+*pos5* (NSP11), and *Δpos5 + ScPOS5* (NSP7) strains were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (B) and per volume (C). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus *Δpos5* + vector strain.

In the S. cerevisiae Δpos5 strain, mitochondrial NADP(H) levels are decreased [21]. To determine whether the S. pombe Δpos5 strain also influences mitochondrial NADP(H), we isolated mitochondria from the S. pombe Δpos5 strain and quantified NADP(H) content as described in materials and methods. Mitochondrial NADP⁺ level and total NADP(H) level in Δpos5 were decreased to 0.6- and 0.8-fold, respectively, of those in the wild-type strain (Fig 3A and 3B), supporting that S. pombe pos5 encodes NADP(H) kinase.

Fig 3 — A, B: Wild-type and *Δpos5* strains were cultured in 3 L YES liquid medium and harvested at mid-log phase. Cell pellets were treated with DTT and Zymolyase for cell wall degradation. Spheroplasts were homogenized and centrifuged to obtain mitochondrial fractions. Protein concentrations in mitochondria-enriched fractions were quantified using the Bradford method. Mitochondrial NADP(H) concentrations were measured enzymatically using glucose-6-phosphate dehydrogenase. A: Quantification of mitochondrial NADP⁺ and NADPH in wild-type and *Δpos5* strains. B: Total NADP(H) levels presented.

ScPos5p is involved in CoQ biosynthesis in S. cerevisiae

The S. cerevisiae Δpos5 (ΔScpos5) strain has previously been reported to exhibit respiratory deficiency, hydrogen peroxide sensitivity, and arginine auxotrophy [19]. We confirmed these phenotypes (Fig 4A). However, since the role of ScPos5 in CoQ biosynthesis has never been documented, we quantified CoQ levels in a ΔScpos5 strain. CoQ₆ levels in ΔScpos5 were decreased to 0.2-fold of those in the wild-type strain (Fig 4B and 4C), which is similar to the CoQ deficiency observed in the S. pombe Δpos5 mutant (Fig 1). Thus, ScPos5 is also involved in CoQ biosynthesis in S. cerevisiae to a similar extent as observed in the S. pombe Δpos5 strain.

Fig 4 — A: *S. cerevisiae* wild-type, *Δcoq2*, and *Δpos5* strains were serially diluted (1:10) from an initial OD₆₀₀ = 2, spotted onto the indicated media, and incubated at 30°C for several days (SC, SD (glucose), SD (glucose)+H₂O₂, SD (glucose) without arginine: 3 days. SD (glycerol): 6 days). The *Δcoq2* strain was included as a representative CoQ-deficient strain. B, C: CoQ₆ quantification in the *S. cerevisiae* Δ*pos5* strain. Wild-type and *Δpos5* strains were cultured in YPD medium for 48 hours, starting from an initial OD₆₀₀ = 0.02. Diamonds (◆) show cell number. Bars indicate CoQ₆ content per cell (B) and per volume (C). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Student’s t-test) versus wild-type strain.

Localization of NAD(H) kinase to mitochondria is required for CoQ biosynthesis

We next investigated the localization of the Pos5-GFP strain, in which Pos5-GFP was expressed from the pos5 locus. The Pos5-GFP signal did not show the expected mitochondrial localization pattern (S3A Fig). In addition, the Pos5-GFP strain failed to maintain normal CoQ production and showed a CoQ level similar to the Δpos5 strain (S3B and S3C Fig), indicating a loss of Pos5 function by tagging GFP which probably interfered Pos5 function. Therefore, we constructed a pSLF272L-Pos5-GFP(S65A) plasmid to express Pos5-GFP exogenously and examined Pos5 localization by introducing it into the wild-type strain. The Pos5-GFP fluorescence overlapped with the MitoTracker Red FM signal (Fig 5), indicating that Pos5 localizes to mitochondria as a mitochondrial NAD(H) kinase. It also indicates Pos5-GFP retains partial functionality, because multicopy Pos5-GFP but not a single copy of that is functional.

Fig 5 — Wild-type harboring pSLF272L-GFP or pSLF272L-pos5-GFP cells were incubated in PMU medium containing 0.1 μM thiamine for 8 hours. Cells were collected at mid-log phase, stained with MitoTracker Red for 1 hour, washed, and observed by fluorescent microscopy. White bars indicate a scale of 10 μm.

Given the phenotypes of the pos5 strain are specific for mitochondrial function, the mitochondrial localization of Pos5 NAD(H) kinase is thought to be essential for CoQ synthesis as shown in S. cerevisiae [20]. To directly examine the significance of mitochondrial localization of NAD(H) kinase in CoQ biosynthesis, we constructed pJK148-P41nmt1-ΔMTS83pos5, pJK148-P41nmt1-UTR1, and pJK148-P41nmt1-MTS36UTR1 plasmids. The ΔMTS83pos5 construct is designed to express a Pos5 protein lacking the N-terminal 83 amino acids. The UTR1 construct expresses a cytosolic NAD(H) kinase from S. cerevisiae. The MTS36UTR1 construct is designed to express a fusion protein comprising the N-terminal 36 amino acids of SpCoq3 fused to S. cerevisiae Utr1p. These constructs were introduced into the Δpos5 strains to generate Δpos5 + ΔMTS83pos5 (NSP15), Δpos5 + UTR1 (NSP13), and Δpos5 + MTS36UTR1 (NSP12) strains. CoQ quantification showed that only MTS36UTR1 restored CoQ production, whereas neither ΔMTS83pos5 nor UTR1 could recover CoQ levels in the Δpos5 strain (Fig 6A and 6B). Consistently, a mitochondrial-targeted Utr1 restored CoQ levels in the Δpos5 strain (S4 Fig) and Utr1-GFP fusion (mito-UTR1-GFP) localized correctly to mitochondria and restored CoQ levels in such a strain (S5 Fig). Thus, these results demonstrate that cytosolic NAD(H) kinase from S. cerevisiae can replace the function of mitochondrial NAD(H) kinase when it is expressed in mitochondria, indicating that the localization of NAD(H) kinase to mitochondria is critical for CoQ biosynthesis.

Fig 6 — A, B: Wild-type+vector (NSP23), *Δpos5* + vector (NSP26), *Δpos5*+*pos5* (NSP11), *Δpos5* + *ΔMTS83pos5* (NSP15), *Δpos5* + *UTR1* (NSP13), and *Δpos5* + *MTS36UTR1* (NSP12) strains were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per volume (B). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the *Δpos5* + vector strain. NS: no significant difference versus the *Δpos5* + vector strain.

Vanillic acid and PHB partially restored CoQ content in the Δpos5 strain

Among the reactions in CoQ biosynthesis, Coq6 catalyzes C5-hydroxylation of the quinone precursor and requires reducing equivalents from NAD(P)H, through ferredoxin and ferredoxin reductase [35–37]. Ferredoxin is reduced by ferredoxin reductase utilizing NAD(P)H to provide electrons to Coq6 reaction in S. cerevisiae. In S. pombe, the ferredoxin reductase Arh1 utilizes both NADPH and NADH [38]. Based on these observations, we hypothesized Coq6 activity may be impaired in the Δpos5 strain. To test this, we overexpressed coq6 in the Δpos5 strain and measured CoQ content. However, CoQ levels in the Δpos5 strain overexpressing coq6 were comparable to that in the Δpos5 strain integrating a vector (Fig 7A and 7B).

Fig 7 — Wild-type strain integrating the vector (NSP23), *Δpos5* integrating the vector (NSP26), *Δpos5* expressing *pos5* (NSP11), and *Δpos5* expressing *coq6* (NSP25) were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per volume (B). Error bars indicate the S.D. of three independent measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the *Δpos5* + vector strain. NS: no significant difference with the *Δpos5* + vector strain.

In contrast, the Δcoq6 strain expressing coq6 on the chromosome clearly restored the CoQ level (S6 Fig). Because exogenous vanillic acid (VA) is known to restore CoQ levels in the Δcoq6 strain (Fig 8A) [17], we added VA to the Δpos5 strain. VA clearly increased CoQ levels in the Δcoq6 strain (Fig 8B and 8C). Although statistical difference was not observed, the addition of VA tended to increase CoQ levels in the Δpos5 strain (Fig 8D and 8E). These results suggest that Coq6 is not fully functional in the Δpos5 strain, but that impaired Coq6 activity is not the sole reason for the decreased CoQ content.

Fig 8 — A: Schematic of the CoQ biosynthetic pathway in *S. pombe* and the quinone precursors used. PHB is the substrate for the early steps of CoQ biosynthesis. VA bypasses the reactions catalyzed by Coq6 and Coq3. -R indicates the decaprenyl moiety. B, C, D & E: Effect of VA to CoQ levels in *Δcoq6* and *Δpos5* strains. B, C: Wild-type and *Δcoq6* strains were cultured in YES and YES + VA (0.5 or 5 mM) medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (B) and per volume (C). Error bars indicate the S.D. of three measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the *Δcoq6* strain. NS: no significant difference with the *Δcoq6* strain. ND: not detected. D, E: Wild-type and *Δpos5* strains were cultured in YES and YES + VA (0.5 or 5 mM) for 48 hours. Bars indicate CoQ₁₀ content per cell (D) and per volume (E). Error bars indicate the S.D. of three measurements. **: p < 0.01; *: p < 0.05 statistical significance in CoQ levels (Dunnett’s test) versus the *Δpos5* strain. NS: no significant difference with the *Δpos5* strain. F & G: Effect of PHB to CoQ levels in the *Δpos5* strain. Wild-type and *Δpos5* strains were cultured in YES and YES + PHB (0.5 mM) medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (F) and per volume (G). Error bars indicate the S.D. of three measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the *Δpos5* strain.

We next examined the effect of p-hydroxybenzoate (PHB), a quinone precursor, on CoQ production in the Δpos5 strain. PHB is condensed with decaprenyl diphosphate by Ppt1 to synthesize decaprenyl-PHB, which subsequently undergoes modifications to generate CoQ₁₀ [39]. Supplementation of 0.5 mM PHB partially increased CoQ levels in the Δpos5 strain compared to the untreated condition (Fig 8F and 8G), suggesting that NADP(H) availability affects a reaction upstream of CoQ biosynthesis.

We then tested overexpression of the atd1 gene, which encodes a potential enzyme that converts p-hydroxybenzaldehyde to PHB, in the Δpos5 strain to see any effect on CoQ biosynthesis. The result showed slight increased CoQ levels in such a strain comparing with the one without the atd1 expression (S7 Fig), but the difference was not statistically significant.

Discussion

In this study, we showed that the mitochondrial NAD(H) kinase Pos5 is required for proper CoQ biosynthesis in both S. pombe and S. cerevisiae. In the Δpos5 strains of both species, CoQ levels were reduced to approximately 20% of the wild-type levels, indicated that Pos5 is important but not essential for CoQ biosynthesis. This observation indicates that the role of Pos5 in CoQ biosynthesis is different from the indispensable CoQ biosynthesis genes such as dps1, dlp1, and coq2 to coq9, which are involved in the synthesis of prenyl tail and modification of the quinone ring precursor in S. pombe. Previous genetic screening in the S. pombe mutant have identified coq11 and coq12 as nonessential but functionally important for CoQ production [17]. Thus, pos5, coq11, and coq12 are categorized as the factors that significantly affect CoQ levels without being absolutely required for CoQ synthesis. Because CoQ is indispensable for human survival, individuals who harbor mutations reducing CoQ production to ~20% of normal levels suffer severe damage in muscle, brain, and kidney tissues [9]. Therefore, identifying genes that are involved in CoQ biosynthesis is critical for understanding human genetic disorders associated with CoQ levels. Given that humans possess a mitochondrial NAD(H) kinase [40], exploring its relevance in CoQ biosynthesis is important for future research.

Pos5 is a mitochondrial NAD(H) kinase. This has been shown in S. cerevisiae Pos5 by in vitro assays demonstrating that purified Pos5 phosphorylates NAD⁺ and NADH, with considerably higher NADH kinase activity [20,34]. Introduction of the S. cerevisiae POS5 gene in the S. pombe pos5 mutant restored CoQ production, supporting the idea that Pos5 is also an NAD(H) kinase. In S. cerevisiae, wild-type mitochondria contain approximately four times as much NADPH as the pos5 mutant mitochondria and 2.5 times as much NADP⁺ [21]. In contrast, in the S. pombe Δpos5 strain, we observed a reduction in total NADP(H) levels, with NADP⁺ showing the most pronounced reduction. This may be due to the species difference.

The pos5 deletion mutant exhibited several phenotypes, including respiratory deficiency, sensitivity to hydrogen peroxide, growth delay on minimal media, requirement of arginine for growth, elevated H₂S production, a rounded cell morphology and reduced CoQ levels. The S. cerevisiae Δpos5 strain showed similar phenotypes except for H₂S production [20,22]. In S. pombe, excessive H₂S is produced caused by non-functionality of the sulfide quinone reductase (Hmt2) [41], which oxidizes sulfide using CoQ. Because S. cerevisiae lacks a similar enzyme, sulfide accumulation is not enhanced by CoQ deficiency. Mitochondrial NADP(H) produced by the NAD(H) kinase Pos5 is essential for maintaining the electron transfer system, presumed by the instability of Fe-S cluster proteins within the complex II and III components [22]. When we tested CoQ levels in a respiration-deficient mutant (the Δcyc1 strain), the CoQ levels were not drastically decreased. While we cannot rule out a possibility that Pos5 deficiency indirectly affects CoQ levels via impaired Fe-S cluster biogenesis, deficiency of respiration itself is not a cause of lower CoQ levels in the Δpos5 strain. Our observation that PHB restores CoQ biosynthesis implies that the core biosynthetic machinery downstream of PHB is functional.

We showed that the expression of a cytosolic NAD kinase in mitochondria restored CoQ levels in S. pombe Δpos5, indicating that a sufficient mitochondrial NADPH pool is necessary for CoQ biosynthesis. Because Coq6 uses reducing equivalents of NADPH via ferredoxin and ferredoxin reductase [37,42], we initially hypothesized that Coq6 activity is limiting in the Δpos5 strain. However, overexpression of coq6 in the Δpos5 strain did not restore the CoQ level. By contrast, the addition of the quinone precursor analog VA and PHB partially increased the CoQ level in Δpos5. This suggests that the primary defect in the Δpos5 strain lies in the synthesis of the quinone precursor. In the quinone precursor synthesis pathway in S. pombe, the aldehyde dehydrogenase Atd1 is thought to catalyze the conversion of p-hydroxybenzaldehyde to PHB with NADH or NADPH reduction. Although overexpression of atd1 in the Δpos5 strain did not clearly increase the CoQ levels, we observed a slight positive effect. Based on our findings, we propose that NADPH availability affects quinone precursor synthesis.

In conclusion, we found that the mitochondrial NAD(H) kinase Pos5 is critical in CoQ biosynthesis in both budding and fission yeasts. Our results suggest that the requirement for NADPH lies in the synthesis of the precursor of CoQ biosynthesis, although more detailed analysis is necessary to define the specific reaction(s) that depend on mitochondrial NADPH.

Supporting information

S1 Fig. The S. pombe Δpos5 strain exhibits arginine auxotrophy.

Wild-type, Δarg11, and Δpos5 strains were serially diluted (1:5) from 1 x 10⁷ cells/mL and spotted onto YES, PMGALU, and PMGALU+0.4 mg/mL arginine media. Plates were incubated at 30°C for 4 days. The Δarg11 strain, an arginine auxotroph, was included for comparison.

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S2 Fig. Morphological analysis of the Δpos5 strain expressing NADK.

Wild-type+vector (NSP23), Δpos5 + vector (NSP26), Δpos5+pos5 (NSP11), Δpos5 + ΔMTS83pos5 (NSP15), Δpos5 + UTR1 (NSP13), and Δpos5 + MTS36UTR1 (NSP12) strains were grown at 30°C in PMLU to the mid-logarithmic phase. The cells were resuspended in PMLU and observed using a BX2-FL-2 microscope (Olympus). The scale bars indicate 10 µm.

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pone.0346295.s002.tiff^{(2.1MB, tiff)}

S3 Fig. Genomic Pos5 tagged with GFP does not localize properly to mitochondria and shows reduced CoQ levels.

A: Localization analysis of the Pos5-GFP strain. Pos5-GFP cells were collected at mid-log phase and stained with MitoTracker Red for 1 hour. After washing, the cells were examined using fluorescence microscopy. B, C: CoQ₁₀ quantification of the Pos5-GFP strain. Wild-type, Δpos5, and Pos5-GFP strains were cultured in YES for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (B) and per volume (C). Error bars indicate the S.D. of three measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the Δpos5 strain.

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S4 Fig. Restoration of CoQ in the S. pombe Δpos5 strain by expression of mitochondrially localized S. cerevisiae Utr1 on the plasmid.

A, B: Wild-type and Δpos5 strains harboring pREP41, pREP41-pos5, pREP41-UTR1, or pREP41-MTS36UTR1 were cultured in PMU medium for 72 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per volume (B). Error bars indicate the S.D. of two measurements.

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pone.0346295.s004.tiff^{(2.1MB, tiff)}

S5 Fig. Restoration of CoQ in the S. pombe Δpos5 strain by expression of mitochondrially localized S. cerevisiae Utr1 tagged with GFP.

A: Fluorescent microscopy of the Δpos5 strain expressing mitochondrial or cytosolic NAD⁺/NADH kinase tagging with GFP at the C-terminus. Wild-type and Δpos5 strains harboring pSLF272L-GFP(S65A) or pSLF272L-UTR1-GFP(S65A) were grown at 30°C in 10 mL PMU, while Δpos5 strains harboring pSLF272L-pos5-GFP(S65A) and pSLF272L-MTS36UTR1-GFP(S65A) were grown at 30°C in 10 mL PMU + 0.1 μM thiamine. Cells were collected at 8 hours after inoculation from 5 x 10⁵ cells/mL and stained with MitoTracker Red. The scale bar indicates 10 μm. B & C: Wild-type and Δpos5 strains harboring pSLF272L-GFP(S65A), pSLF272L-pos5-GFP(S65A), pSLF272L-UTR1-GFP(S65A), or pSLF272L-MTS36UTR1-GFP(S65A) were cultured in PMU medium for 72 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (B) and per volume (C). Error bars indicate the S.D. of three measurements. *: p < 0.05; statistical significance in CoQ levels (Dunnett’s test) versus the Δpos5 strain expressing GFP. NS: no significant difference. ND: not detected. D: Plasmid map of pSLF272L-pos5-GFP(S65A), pSLF272L-UTR1-GFP(S65A), and pSLF272L-MTS36UTR1-GFP(S65A). The vector pSLF272L contains Pnmt41, GFP(S65A), and Tnmt1.

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pone.0346295.s005.tiff^{(2.1MB, tiff)}

S6 Fig. Genomic integration of pJK148-Pnmt1-coq6 restores CoQ production in the Δcoq6 strain.

A, B: Wild-type strains integrated with the vector, Δcoq6 strains integrated with the vector, and Δcoq6 strains integrated with pJK148-Pnmt1-coq6 were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per volume (B). Error bars indicate the S.D. of three measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus the Δcoq6 strain integrated with the vector. ND: not detected.

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pone.0346295.s006.tiff^{(2.1MB, tiff)}

S7 Fig. Over-expression of atd1 in Δpos5.

A, B: Wild-type+vector (NSP23), Δpos5 + vector (NSP26), Δpos5+pos5 (NSP11), and Δpos5 + atd1 (NSP60) strains were cultured in YES medium for 48 hours. Diamonds (◆) show cell number. Bars indicate CoQ₁₀ content per cell (A) and per volume (B). Error bars indicate the S.D. of three measurements. **: p < 0.01; statistical significance in CoQ levels (Dunnett’s test) versus Δpos5 strain integrated with vector. NS: no significant difference.

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pone.0346295.s007.tiff^{(2.1MB, tiff)}

S1 Table. Primer list.

(XLSX)

pone.0346295.s008.xlsx^{(12.6KB, xlsx)}

S2 Table. Plasmid list.

(XLSX)

pone.0346295.s009.xlsx^{(10.8KB, xlsx)}

Acknowledgments

We thank Dr. T. Ogawa (Shimane University) for his help in NADPH and NADP⁺ measurements, Dr. S. Kawai (Ishikawa Prefectural University) for providing an S. cerevisiae pos5 strain, and Dr. Y. Tamura (Yamagata University) for his advice on the isolation of mitochondria from S. pombe. We thank R. Yanai for constructing the pos5 deletion strain and H. Sumi for her assistance.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was partly supported by grant-in-aid funding from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (#17H03806, #21H02117 and # 24K08715 to M. K.; #18K05393 to T. K.; #18K14377 to I. N.); the Mishima Kaiun Memorial Foundation to I. N.; the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (#957613) to M. K.; we also thank the Faculty of Life and Environmental Sciences at Shimane University for the financial support for publishing this report. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0346295.r001

Decision Letter 0

Junzheng Yang

9 Dec 2025

Dear Dr. Kawamukai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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“This work was partly supported by grant-in-aid funding from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (#17H03806, #21H02117 and # 24K08715 to M. K.; #18K05393 to T. K.; #18K14377 to I. N.); the Mishima Kaiun Memorial Foundation to I. N.; the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry (#957613) to M. K.; we also thank the Faculty of Life and Environmental Sciences at Shimane University for the financial support for publishing this report.”

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1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

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Reviewer #1: Yes

Reviewer #2: Yes

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**********

Reviewer #1: 1. The finding that the chromosomally integrated Pos5-GFP fusion did not localize correctly and was non-functional is critical but not fully explored. Was this due to the tag interfering with the mitochondrial targeting signal (MTS) or protein folding? This result should be discussed, as it serves as an important cautionary note for tagging strategies and confirms the necessity of the MTS.

2. The manuscript uses Student's t-test for all pairwise comparisons. When multiple strains or conditions are compared to a single control (e.g., *Δpos5+vector* in Fig 6), a one-way ANOVA with a post-hoc test (e.g., Tukey's) is more statistically appropriate. The authors should re-analyze their data using the correct statistical model or clearly justify the use of multiple t-tests.

3. The partial rescue by PHB is intriguing. The authors suggest it affects a reaction "upstream of CoQ biosynthesis." Since PHB is the direct substrate for Ppt1 (Coq2), does this imply that the Δpos5 strain has reduced PHB availability? Is the synthesis of PHB from tyrosine (or its mitochondrial import) potentially NADPH-dependent? This point warrants deeper discussion in the Results or Discussion section.

4. The use of "Orange" and "Green" bars in the graphs, with descriptions in the figure legends, is non-standard and can be confusing. A clearer approach would be to use direct labels on the graphs or within the figure panels themselves (e.g., "CoQ10 / cell" and "CoQ10 / vol"). Additionally, the y-axes for cell number and CoQ content should be more distinctly separated or represented in a dual-axis graph with clear labeling.

5. The authors correctly note that Pos5 is important but not essential, unlike core coq genes. However, they should more explicitly discuss whether the CoQ deficiency is a direct consequence of low NADPH for biosynthesis or an indirect effect of general mitochondrial dysfunction (e.g., impaired Fe-S cluster biogenesis, which is also Pos5-dependent). The data with the Δcyc1 mutant argue against a general respiratory defect, but other mitochondrial processes could be involved.

Reviewer #2: This is a well-conducted and clearly presented study that establishes a novel and important role for the mitochondrial NAD kinase Pos5 in coenzyme Q (CoQ) biosynthesis in both Schizosaccharomyces pombe and Saccharomyces cerevisiae. The authors provide compelling genetic and biochemical evidence showing that pos5 deletion mutants in both yeasts exhibit a significant (~80%) reduction in CoQ levels, accompanied by characteristic CoQ-deficient phenotypes. The demonstration of functional conservation through heterologous complementation, the requirement for mitochondrial localization of the NAD kinase activity, and the partial rescue by quinone precursors are particularly strong aspects of the work. The findings are novel and have potential implications for understanding human disorders related to CoQ deficiency.

Major Points:

(1)The authors conclude that Pos5/NADPH is required for an "earlier step" in CoQ biosynthesis, based on the partial rescue by PHB and vanillic acid (VA) and the lack of effect from coq6 overexpression. This is a reasonable hypothesis. However, to strengthen this conclusion, it would be informative to directly measure the levels of early intermediates (e.g., decaprenyl-PHB) in the Δpos5 mutant compared to wild-type and perhaps a coq6 mutant. This could more precisely pinpoint the bottleneck.

(2)Figure 3 shows a decrease in total mitochondrial NADP(H) in the S. pombe Δpos5 strain. The claim that "NADP+ showed the most pronounced reduction" is made in the text (Page 29), but the graphical presentation in Fig. 3A (stacked bars) makes it difficult for the reader to independently assess the relative changes in NADP+ vs. NADPH. Presenting these as separate bar graphs or including the numerical values in a supplementary table would enhance clarity and support the statement regarding species-specific differences with S. cerevisiae.

(3)The discussion proposes Atd1 as a potential NADPH-dependent link in the quinone precursor pathway (Page 30). This is an interesting speculation. Could the authors provide any preliminary data or genetic interaction (e.g., double mutant analysis, overexpression of atd1 in Δpos5) to test this hypothesis? If not, the text should more clearly frame this as a suggested model for future investigation.

(4) Several figures (e.g., Figs 1, 2, 4, 6-8) use a dual Y-axis format (cell number and CoQ content). While informative, the graphs are somewhat crowded. Ensuring high resolution and clear differentiation of data series in the final version is essential.

Figure 5 (localization) lacks a scale bar in the provided image. This must be added.

The labels "Orange bars" and "Green bars" in the figure legends refer to colors not visible in the grayscale PDF. Please use patterns (hatching, shading) or direct labeling (e.g., "left axis: CoQ10 per 10^9 cells; right axis: CoQ10 per 50 mL culture") to ensure accessibility.

(5) The manuscript states the use of Student's t-test. For all multi-group comparisons (e.g., Fig. 6, 7, 8), please confirm that the appropriate statistical test (e.g., ANOVA with post-hoc test) was applied where applicable, and specify which groups are being compared when significance is indicated.

Minor Points:

Abstract: The phrase "inability to grown on non-fermentable carbon sources" should be corrected to "inability to grow".

Page 9, Abstract & Page 19, Results: "CoQ10 level in ΔScpos5 were decreased..." should be "was decreased" or "levels were decreased".

Page 12: "Synthetic defied (SD) medium" should be "Synthetic defined (SD) medium".

Page 15: "...primers containing restriction sites from the S. pombe PR110 genome and the S. cerevisiae BY4741 genome as the template." The phrasing is slightly ambiguous. Consider: "...using the S. pombe PR110 genome and the S. cerevisiae BY4741 genome as templates, with primers containing restriction sites."

Page 19: "CoQ levels in the Δpos5 strain of our construct were decreased to 0.2-fold of thoes in the wild-type..." should be "those".

Page 30: "...sulfide accumulation is not enhanced CoQ deficiency." Should likely be "is not enhanced by CoQ deficiency."

References: The reference list appears comprehensive. Please ensure all in-text citations have a corresponding entry and vice-versa.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2026 Apr 2;21(4):e0346295. doi: 10.1371/journal.pone.0346295.r002

Author response to Decision Letter 1

26 Jan 2026

Thank you for evaluating our work and careful checking of our manuscript. We appreciate the positive responses from two reviewers. We marked the amended parts in red characters in the revised manuscript. We also extended the morphological analysis in Figure S2.

Reviewer #1

1) The finding that the chromosomally integrated Pos5-GFP fusion did not localize correctly and was non-functional is critical but not fully explored. Was this due to the tag interfering with the mitochondrial targeting signal (MTS) or protein folding? This result should be discussed, as it serves as an important cautionary note for tagging strategies and confirms the necessity of the MTS.

Answer: The improper localization and non-functionality of the chromosomally integrated Pos5-GFP protein is a matter of our interest too. Our immunoblotting analysis detected the Pos5-GFP fusion protein in a correct size, without seeing free GFP. The fusion protein appeared to be synthesized properly rather than being degraded. Therefore, we think the C-terminal GFP tagging caused steric hindrance. The GFP tag likely interferes the protein’s folding that would prevent correct localization and proper enzymatic function. We have added this point in the result (P18, L9 and 14).

2) The manuscript uses Student's t-test for all pairwise comparisons. When multiple strains or conditions are compared to a single control (e.g., *Δpos5+vector* in Fig 6), a one-way ANOVA with a post-hoc test (e.g., Tukey's) is more statistically appropriate. The authors should re-analyze their data using the correct statistical model or clearly justify the use of multiple t-tests.

Answer: Thank you for this suggestion. We have re-analyzed the data using a one-way ANOVA followed by Dunnett’s test for multiple comparisons against the control strain, instead of Student’s t-test. We confirmed the statistical significance using this appropriate analysis (Figures 1, 2, 4, 6, 7, and 8; Supplementary figures 3, 4, 5, 6 and 7; their legends; the method (P12, L4)).

3) The partial rescue by PHB is intriguing. The authors suggest it affects a reaction "upstream of CoQ biosynthesis." Since PHB is the direct substrate for Ppt1 (Coq2), does this imply that the Δpos5 strain has reduced PHB availability? Is the synthesis of PHB from tyrosine (or its mitochondrial import) potentially NADPH-dependent? This point warrants deeper discussion in the Results or Discussion section.

Answer: Knowledge of the PHB biosynthetic pathway in S. pombe is still limited. Atd1 is a candidate enzyme that converts 4-hydroxybenzaldehyde to PHB. We tested overexpression of the atd1 gene in the Δpos5 strain to see any effect on CoQ synthesis. The result showed slight increased CoQ levels in such a stain comparing with the one without the atd1 expression (Fig. S7), but the difference was not statistically significant. We can not clearly conclude the involvement of NADPH in the Atd1 reaction. We have included this data and discussion in the revised manuscript (P21, L21; P25, L6).

4) The use of "Orange" and "Green" bars in the graphs, with descriptions in the figure legends, is non-standard and can be confusing. A clearer approach would be to use direct labels on the graphs or within the figure panels themselves (e.g., "CoQ10 / cell" and "CoQ10 / vol"). Additionally, the y-axes for cell number and CoQ content should be more distinctly separated or represented in a dual-axis graph with clear labeling.

Answer: Thank you for this suggestion. We revised the figures to improve readability following your suggestion. We separated the data into two distinct panels; one for CoQ content per cell and one for CoQ content per culture volume. We replaced the color-based description with grayscale and made distinct patterns to ensure the figures are easily readable (Figures 1, 2, 4, 6, 7 and 8; Supplementary figures 3, 4, 5, 6 and 7).

5) The authors correctly note that Pos5 is important but not essential, unlike core coq genes. However, they should more explicitly discuss whether the CoQ deficiency is a direct consequence of low NADPH for biosynthesis or an indirect effect of general mitochondrial dysfunction (e.g., impaired Fe-S cluster biogenesis, which is also Pos5-dependent). The data with the Δcyc1 mutant argue against a general respiratory defect, but other mitochondrial processes could be involved.

Answer: While we cannot rule out a possibility that Pos5 deficiency indirectly affects CoQ levels via general mitochondrial dysfunction (e.g., impaired Fe-S cluster biogenesis), our data argue against this being the primary cause. Specifically, if the defect was solely due to the dysfunction of Fe-S cluster-dependent enzymes (such as Coq6, which requires ferredoxin and Arh1), supplementation of the upstream precursor PHB in the ∆pos5 strain should not rescue CoQ levels. The observation that PHB restores CoQ biosynthesis implies that the core biosynthetic machinery downstream of PHB is functional, and that the primary bottleneck lies in the supply of the precursor. We added this point in the revised manuscript (P24, L15)

Reviewer #2

1) The authors conclude that Pos5/NADPH is required for an "earlier step" in CoQ biosynthesis, based on the partial rescue by PHB and vanillic acid (VA) and the lack of effect from coq6 overexpression. This is a reasonable hypothesis. However, to strengthen this conclusion, it would be informative to directly measure the levels of early intermediates (e.g., decaprenyl-PHB) in the Δpos5 mutant compared to wild-type and perhaps a coq6 mutant. This could more precisely pinpoint the bottleneck.

Answer: This is an important point. We attempted to detect early intermediates, such as 3-decaprenyl-4-hydroxybenzoate (4-HP10), in the Δpos5 strain using LC-MS/MS. However, the levels of these intermediates were below the limit of detection under our current experimental conditions. Further optimization of extraction and detection methods is required to profile these trace intermediates, which remains to be a subject for future investigation.

2) Figure 3 shows a decrease in total mitochondrial NADP(H) in the S. pombe Δpos5 strain. The claim that "NADP+ showed the most pronounced reduction" is made in the text (Page 29), but the graphical presentation in Fig. 3A (stacked bars) makes it difficult for the reader to independently assess the relative changes in NADP+ vs. NADPH. Presenting these as separate bar graphs or including the numerical values in a supplementary table would enhance clarity and support the statement regarding species-specific differences with S. cerevisiae.

Answer: As suggested, we have modified Figure 3 to present the total NADP(H) levels and the specific redox states (NADP+ and NADPH) in separate graphs to allow for easier comparison (New Fig. 3).

3) The discussion proposes Atd1 as a potential NADPH-dependent link in the quinone precursor pathway (Page 30). This is an interesting speculation. Could the authors provide any preliminary data or genetic interaction (e.g., double mutant analysis, overexpression of atd1 in Δpos5) to test this hypothesis? If not, the text should more clearly frame this as a suggested model for future investigation.

Answer: We performed the suggested experiment. Overexpression of atd1 in the Δpos5 strain slightly increased CoQ levels, although it was not statistical significance (p > 0.05). We have included these results in Supplementary figure S7 and added this result in the result (P21, L21)

4) Several figures (e.g., Figs 1, 2, 4, 6-8) use a dual Y-axis format (cell number and CoQ content). While informative, the graphs are somewhat crowded. Ensuring high resolution and clear differentiation of data series in the final version is essential.

Figure 5 (localization) lacks a scale bar in the provided image. This must be added.

Answer: Thank you for this suggestion. Please refer to our response to Reviewer #1, Point 4. We separated the graphs to resolve the crowding issue.

5) The manuscript states the use of Student's t-test. For all multi-group comparisons (e.g., Fig. 6, 7, 8), please confirm that the appropriate statistical test (e.g., ANOVA with post-hoc test) was applied where applicable, and specify which groups are being compared when significance is indicated.

Answer: Please refer to our response to Reviewer #1, Point 2. We re-analyzed the data using ANOVA followed by Dunnett’s test.

Minor Points:

Thank you for careful checking of our manuscript. We corrected according to your suggestion.

Abstract: The phrase "inability to grown on non-fermentable carbon sources" should be corrected to "inability to grow".

Answer: We corrected (P2, L13).

Page 9, Abstract & Page 19, Results: "CoQ10 level in ΔScpos5 were decreased..." should be "was decreased" or "levels were decreased".

Answer: We corrected (P2, L11; P20, L17).

Page 12: "Synthetic defied (SD) medium" should be "Synthetic defined (SD) medium".

Answer: We corrected (P5, L16).

Answer: We corrected (P8, L15).

Page 19: "CoQ levels in the Δpos5 strain of our construct were decreased to 0.2-fold of thoes in the wild-type..." should be "those".

Answer: We corrected (P17, L10).

Page 30: "...sulfide accumulation is not enhanced CoQ deficiency." Should likely be "is not enhanced by CoQ deficiency."

Answer: We corrected (P24, L11).

References: The reference list appears comprehensive. Please ensure all in-text citations have a corresponding entry and vice-versa.

Answer: We checked all citations.

Attachment

Submitted filename: 20260123 Answers to reviews.doc

pone.0346295.s011.doc^{(49.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0346295.r003

Decision Letter 1

Junzheng Yang

10 Mar 2026

Dear Dr. Kawamukai,

Please submit your revised manuscript by Apr 24 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Junzheng Yang

Academic Editor

PLOS One

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

Reviewer #1: (No Response)

Reviewer #3: The manuscript is clearly written and well illustrated. The conclusions are supported by the results. The authors seem to have adressed most reviewers’ comments. I only have a few minor comments.

P3 L9 : the claim that « most prokaryotes synthesize CoQ endogenously » is incorrect, as CoQ biosynthesis is restricted to bacteria belonging to the phylum Pseudomonadota (doi : 10.1101/2025.09.17.676790). All other bacterial phylla, nor archea, do not produce CoQ.

P10 L18 : « OD600 = 1 point ». This seems strange. You can use « OD600 = 1 »

P10 L19 « Mitochondria were isolated according to a method described previously [27] with slight modification. ». The modifications should be indicated.

Fig 2A : red is used to display conserved residues between the two sequences. Therefore, the amino acids of two insertions which are found only in the S. cerevisiae sequence should be displayed in a different color than red.

Figure legends are long and repetitive (for example fig 1A-B, fig 2B-C, fig 6A-B, fig 7A-B). Many details could be placed in the methods section to shorten the figure legends.

P14 L3 : when first talking about UTR1, please explain the function of this protein.

P20 L14-16 : « Coq6 catalyzes C5-hydroxylation of the quinone precursor and requires reducing equivalents from NAD(P)H, through ferredoxin and ferredoxin reductase [35, 36]. » References 35 and 36 do not support this claim. The papers that show this are the following doi : 10.1016/j.chembiol.2011.07.008 and 10.1002/cbic.202300738

P20 L21 « However, CoQ levels of per cells and per volume » delete « of »

P21 L13 « clearly restored the CoQ a. » please correct to « clearly restored the CoQ level. »

**********

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

Reviewer #3: No

**********

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PLoS One. 2026 Apr 2;21(4):e0346295. doi: 10.1371/journal.pone.0346295.r004

Author response to Decision Letter 2

14 Mar 2026

The manuscript is clearly written and well illustrated. The conclusions are supported by the results. The authors seem to have addressed most reviewers’ comments. I only have a few minor comments.

Thank you for careful checking of our revised manuscript. Your comments help to improve the accuracy of the description. We answered the comments and explained the revised points.

Ans. As you pointed out, we changed this part to ‘’belonging to the phylum Pseudomonadota’ and added the reference

P10 L18 : « OD600 = 1 point ». This seems strange. You can use « OD600 = 1 »

Ans. Yes, we changed so (P10 L17).

P10 L19 « Mitochondria were isolated according to a method described previously [27] with slight modification. ». The modifications should be indicated.

Ans. We added the description of the modified point in P10, L19.

Ans. We changed according to your suggestion (New Figure2A).

Figure legends are long and repetitive (for example fig 1A-B, fig 2B-C, fig 6A-B, fig 7A-B). Many details could be placed in the methods section to shorten the figure legends.

Ans. We agreed. We delete some sentences and moved to M&M. (Fig 1A-B, Fig 2B-C, Fig 6A-B, Fig 7A-B).

P14 L3 : when first talking about UTR1, please explain the function of this protein.

Ans. We added the description (P14 L1).

Ans. You are right. We added the appropriate references (P20 L4).

P20 L21 « However, CoQ levels of per cells and per volume » delete « of »

Ans. We amended this part and also the other parts (P20 L16 and else)

P21 L13 « clearly restored the CoQ a. » please correct to « clearly restored the CoQ level. »

Ans. We amended this part (P20 L23).

Attachment

Submitted filename: renamed_7321a.docx

pone.0346295.s012.docx^{(16.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0346295.r005

Decision Letter 2

Junzheng Yang

17 Mar 2026

Mitochondrial NAD kinase Pos5 is required for CoQ biosynthesis in yeasts

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PLoS One. doi: 10.1371/journal.pone.0346295.r006

Acceptance letter

Junzheng Yang

PONE-D-25-62406R2

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. The S. pombe Δpos5 strain exhibits arginine auxotrophy.

(TIFF)

pone.0346295.s001.tiff^{(2.1MB, tiff)}

S2 Fig. Morphological analysis of the Δpos5 strain expressing NADK.

(TIFF)

pone.0346295.s002.tiff^{(2.1MB, tiff)}

S3 Fig. Genomic Pos5 tagged with GFP does not localize properly to mitochondria and shows reduced CoQ levels.

(TIFF)

pone.0346295.s003.tiff^{(2.1MB, tiff)}

S4 Fig. Restoration of CoQ in the S. pombe Δpos5 strain by expression of mitochondrially localized S. cerevisiae Utr1 on the plasmid.

(TIFF)

pone.0346295.s004.tiff^{(2.1MB, tiff)}

S5 Fig. Restoration of CoQ in the S. pombe Δpos5 strain by expression of mitochondrially localized S. cerevisiae Utr1 tagged with GFP.

(TIFF)

pone.0346295.s005.tiff^{(2.1MB, tiff)}

S6 Fig. Genomic integration of pJK148-Pnmt1-coq6 restores CoQ production in the Δcoq6 strain.

(TIFF)

pone.0346295.s006.tiff^{(2.1MB, tiff)}

S7 Fig. Over-expression of atd1 in Δpos5.

(TIFF)

pone.0346295.s007.tiff^{(2.1MB, tiff)}

S1 Table. Primer list.

(XLSX)

pone.0346295.s008.xlsx^{(12.6KB, xlsx)}

S2 Table. Plasmid list.

(XLSX)

pone.0346295.s009.xlsx^{(10.8KB, xlsx)}

Attachment

Submitted filename: 20260123 Answers to reviews.doc

pone.0346295.s011.doc^{(49.5KB, doc)}

Attachment

Submitted filename: renamed_7321a.docx

pone.0346295.s012.docx^{(16.8KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Mitochondrial NAD kinase Pos5 is required for CoQ biosynthesis in yeasts

Shogo Nishihara

Ikuhisa Nishida

Yasuhiro Matsuo

Tomohiro Kaino

Makoto Kawamukai

Roles

Abstract

Introduction

CoQ biosynthetic pathway

Materials and methods

Yeast and E. coli strains, and growth media

Table 1. Strain list.

Construction of S. pombe strains

Plasmid construction

CoQ extraction and measurement

Isolation of mitochondria

Measurement of NADP(H)

Mitochondrial staining and fluorescence microscopy

Data and statistical analyses

Results

The S. pombe Δpos5 strain exhibits a phenotype similar to that of the CoQ-deficient strain

Fig 1. The Δpos5 strain exhibits a phenotype similar to that of a CoQ-deficient strain.

Pos5 functions as an NAD(H) kinase

Fig 2. ScPOS5 overexpression restores CoQ levels in the S. pombe Δpos5 strain.

Fig 3. The Δpos5 strain exhibits decreased NADP(H) levels.

ScPos5p is involved in CoQ biosynthesis in S. cerevisiae

Fig 4. The S. cerevisiae Δpos5 strain exhibits a CoQ-deficient phenotype.

Localization of NAD(H) kinase to mitochondria is required for CoQ biosynthesis

Fig 5. Localization analysis of Pos5-GFP.

Fig 6. Budding yeast NAD kinase targeted to mitochondria restores CoQ10 levels in the Δpos5 strain.

Vanillic acid and PHB partially restored CoQ content in the Δpos5 strain

Fig 7. Overexpression of coq6 does not increase CoQ10 levels in the Δpos5 strain.

Fig 8. Addition of VA and PHB increase CoQ10 levels in the Δpos5 strain.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Junzheng Yang

Roles

Author response to Decision Letter 1

Decision Letter 1

Junzheng Yang

Roles

Author response to Decision Letter 2

Decision Letter 2

Junzheng Yang

Roles

Acceptance letter

Junzheng Yang

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 6. Budding yeast NAD kinase targeted to mitochondria restores CoQ₁₀ levels in the Δpos5 strain.

Fig 7. Overexpression of coq6 does not increase CoQ₁₀ levels in the Δpos5 strain.

Fig 8. Addition of VA and PHB increase CoQ₁₀ levels in the Δpos5 strain.