A novel NADH kinase is the mitochondrial source of NADPH in Saccharomyces cerevisiae

Abstract

Mitochondria require NADPH for anti-oxidant protection and for specific biosynthetic pathways. However, the sources of mitochondrial NADPH and the mechanisms of maintaining mitochondrial redox balance are not well understood. We show here that in Saccharomyces cerevisiae, mitochondrial NADPH is largely provided by the product of the POS5 gene. We identified POS5 in a S.cerevisiae genetic screen for hyperoxia-sensitive mutants, or cells that cannot survive in 100% oxygen. POS5 encodes a protein that is homologous to NAD(+) and NADH kinases, and we show here that recombinant Pos5p has NADH kinase activity. Pos5p is localized to the mitochondrial matrix of yeast and appears to be important for several NADPH-requiring processes in the mitochondria, including resistance to a broad range of oxidative stress conditions, arginine biosynthesis and mitochondrial iron homeostasis. Pos5p represents the first member of the NAD(H) kinase family that has been identified as an important anti-oxidant factor and key source of the cellular reductant NADPH.

Fig. 1. Comparison of oxidative stress sensitivity of pos5Δ and other mutants with anti-oxidant function. The indicated yeast strains were tested for growth by plating 5 µl of solution at 2.0, 0.2 and 0.02 OD₆₀₀ units onto YPD plates. –O₂ = growth in anaerobic culture jars; +air = aerobic growth; +100% O₂ = growth in chambers flushed with 100% O₂; +H₂O₂ = aerobic growth on YPD plates containing 2 mM H₂O₂; +PQ = aerobic growth on YPD plates containing 1 mM paraquat. Strains utilized: wild-type, BY4741; pos5Δ, CO205; sod1Δ, LJ284; sod2Δ, yap1Δ, pos9Δ, idp1Δ, zwf1Δ and gnd1Δ were obtained from ResGen as kanMX4 deletions in parental strain BY4741.

Fig. 2. Pos5p is a mitochondrial matrix protein that is needed for proper mitochondrial function. (A) The indicated strains were tested for growth by plating cell dilutions as in Figure 1 onto YPD (–O₂, +air, +100% O₂) or YPG (glycerol) plates in air. –O₂, +air, +100% O₂ = same as in Figure 1. Strains utilized: wild-type, BY4741; pos5Δ, CO205; pos5Δ + vector, strain CO205 transformed with vector pAA1; pos5Δ + POS5-GFP, strain CO205 transformed with plasmid pCO101. (B) Strain BY4741 transformed with pCO101 (Pos5–GFP) or pAA1 (control) was prepared for fluorescence microscopy as described in Materials and methods. Merged = merged images of DAPI and GFP fluorescence. (C) Cell lysates were prepared from strain BY4741 transformed with the Pos5–GFP plasmid pCO101 (P) or the vector control pAA1 (V), and 140 µg of total cell protein was fractionated into post-mitochondrial supernatant (PMS) and crude mitochondria (Mito) (left). Where indicated, mitochondria (25 µg of protein) were fractionated further into intermembrane space (IMS) and matrix components (right). All samples were subject to SDS–PAGE and immunoblotting using antibodies directed against either GFP (marking Pos5p), cytochrome b₂ (mitochondrial IMS) or Mas2 (mitochondrial matrix). (D) The indicated strains were tested for growth as in (A). Strains utilized: wild-type, BY4741; pos5Δ, BY4741 pos5Δ::kanMX4; coq1Δ, CO217; pos5Δ coq1Δ, CO200.

Fig. 3. Pos5p yeast homologs are not required for protection from hyperoxia or growth on a non-fermentable carbon source. (A) The amino acid sequences of S.cerevisiae Pos5p, Utr1p and Yel041p and human PPNK (accession No. NP_075394) were aligned using Clustal_W 1.5. Identical residues are highlighted in black, and similar residues are outlined in gray. (B) The indicated strains were tested for growth as in Figures 1 and 2A. Strains utilized: wild-type, BY4741; pos5Δ, CO205; utr1Δ, BY4742 utr1Δ::kanMX4; yel041wΔ, BY4741 yel041wΔ::kanMX4.

Fig. 4. Recombinant Pos5p is an NADH kinase. (A) SDS– polyacrylamide gel from recombinant Pos5p purification procedures. std, molecular weight standards; lane 1, uninduced cells; lane 2, induced cells; lane 3, sonication supernatant; lane 4, MES/urea extract; lane 5, Tris/urea extract; lane 6, DEAE flow-through; lane 7, 15 µg of refolded Pos5p (see Materials and methods). (B) and (C) NADH and NAD⁺ kinase activities were assayed as described in Materials and methods using either (B) purified recombinant Pos5p (left hand histogram for both NADH and NAD⁺) and chicken liver NAD⁺ kinase (Sigma; right hand histogram for both NADH and NAD⁺) or (C) crude mitochondria obtained from cells grown to mid-log phase in YPD. Results are presented either in the form of enzyme units (B) where one unit = 1.0 µmol NADPH or NADP⁺ produced per min, or as a percentage of the total activity of wild-type mitochondria (C). Yeast strains utilized in (C) are the same as in Figure 3B. The reported values in (B) and (C) are the mean of three independent experiments; error bars are standard deviations.

Fig. 5. Mitochondrial iron homeostasis defects in pos5Δ. (A) The indicated strains were grown to mid-log phase and the iron content measured in mitochondria and post-mitochondrial supernatant fractions by atomic absorption spectroscopy. (B) and (C) Cell lysates were prepared for the indicated yeast strains and tested for succinate dehydrogenase (B) and aconitase (C) activity. One unit of enzyme activity is equivalent to 1.0 nmol of substrate converted/min/mg protein. Substrates were dichlorophenol indophenol (SDH) and cis-aconitate (aconitase). In (A–C), the reported values are the mean of three independent experiments; error bars are standard deviations. (D) The indicated strains were tested for growth as in Figure 2A. Strains utilized: wild-type, BY4741; pos5Δ, CO205; isa2Δ, LJ102; fet3Δ, BY4741 fet3Δ::kanMX4; pos5Δ fet3Δ, CO207.

Fig. 6. Amino acid auxotrophies of pos5Δ and zwf1Δ strains. The indicated strains were tested for growth by spotting 10 µl of solution at 2.0, 0.2 and 0.02 OD₆₀₀ units onto complete SD plates with arginine, or SD plates lacking methionine or arginine. Strains utilized: wild- type, BY4742; pos5Δ, BY4742 pos5Δ::kanMX4; zwf1Δ, BY4742 zwf1Δ::kanMX4.

Fig. 7. Proposed model for the generation of NADPH in the cytoplasm and mitochondria of yeast. In the cytosol, NAD⁺ may be phosphorylated to NADP⁺ by an NAD⁺ kinase (e.g. S.cerevisiae Utr1p). The resultant NADP⁺ is then converted to NADPH by enzymes of the pentose phosphate pathway (PPP). NADPH-requiring reductases (e.g. TRX and GSH reductases) can then use NADPH as a cofactor, thereby regenerating NADP⁺ for the PPP. In the mitochondria, a different pathway exists for the primary generation of NADPH. The NADH generated in the tricarboxylic acid (TCA) cycle apparently can be phosphorylated by the NADH kinase Pos5p, generating NADPH. Following consumption of NADPH via anti-oxidant reductases, an NADP⁺ phosphatase presumably regenerates NAD⁺ for recycling back into the TCA cycle. NADP⁺-IDHm (Idp1p) may also contribute to NADPH regeneration, although to a minimal extent in S.cerevisiae. Arrows shown in bold indicate enzymatic reactions that are critical for oxidative stress resistance.