ABSTRACT NAD kinase is the sole NADP+ biosynthetic enzyme. Despite the great significance of NADP+, to date no mitochondrial NAD kinase has been identified in human, and the source of human

mitochondrial NADP+ remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely

represents the missing source of human mitochondrial NADP+. The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria

of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists

MITOCHONDRIAL DGTP IS TIGHTLY BOUND TO RESPIRATORY COMPLEX I THROUGH THE NDUFA10 SUBUNIT Article Open access 23 June 2022 YEAST HOMOLOGS OF HUMAN MCUR1 REGULATE MITOCHONDRIAL PROLINE

METABOLISM Article Open access 25 September 2020 INTRODUCTION NAD+, NADP+ and their reduced forms (NADH and NADPH) function as cofactor involved in oxidative–reductive reactions1,2. NAD+ and

NADH mainly function in catabolic reactions, whereas NADP+ and NADPH are involved in anabolic reactions and defence against oxidative stress3. NAD+ and NADP+ are also known to function as

substrates in several reactions1,2,4,5,6,7. NAD kinase (NADK; EC 2.7.1.23) is the sole NADP+-biosynthetic enzyme known to catalyse phosphorylation of NAD+ to yield NADP+, using ATP or

inorganic polyphosphate (poly(P)) as the phosphoryl donor1, and has a vital role in the cell. Poly(P) is a linear polymer of orthophosphate residues linked by energy-rich phosphoanhydride

bonds; it has been found in bacteria, fungi and tissues of higher plants and animals, including humans8,9. Some bacterial and archaeal NADKs utilize both poly(P) and ATP as a phosphoryl

donor, although all characterized eukaryotic NADKs use ATP, not poly(P)10. Moreover, some bacterial and eukaryotic NADKs also catalyse the phosphorylation of NADH by utilizing ATP to yield

NADPH (NADH kinase (NADHK; EC 2.7.1.86) reaction)10. NADKs that have higher NADHK activity than NADK activity have also been identified, and in some cases classified as NADHK. So far, only

mitochondrial Pos5 of the yeast _Saccharomyces cerevisiae_ and peroxisomal NADK3 of the plant _Arabidopsis thaliana_ have been experimentally defined as NADHK3,11,12,13. In the case of

yeast, in addition to the gene (_POS5_) encoding mitochondrial Pos5, _S. cerevisiae_ has other two NADK genes (_UTR1_ and _YEF1_) encoding cytosolic Utr1 and Yef1 (refs 3,14,15). The NADK

triple mutant (_utr1 yef1 pos5_) is lethal11,16,17, emphasizing the significance of intracellular NADP(H) biosynthesis. The phenotypes of the _POS5_ mutant (_pos5_), which are probably

caused by decreased mitochondrial NADPH, indicate that Pos5 is responsible for mitochondrial NADP(H) synthesis and again emphasizes the importance of mitochondrial NADP(H)3,11,12,17.

Moreover, in addition to the gene (_NADK3_) encoding peroxisomal NADK3, _NADK1_ and _NADK2_ have been identified as NADK genes in _A. thaliana_13,18. The products of these genes, NADK1 and

NADK2, are localized in the cytosol and chloroplast, respectively19. In pea leaf, NADP+ enters the plant mitochondria20; thus, the NADP+ transporter, which is localized in the inner membrane

of mitochondria, is responsible for the supply of plant mitochondrial NADP+. In phylogenetic tree analysis, the plant NADK3 homologues, including _A. thaliana_ NADK3, are distinguishable

from NADK homologues, including Pos5 and Utr1 (ref. 21). However, the structural features of NADK3 homologues have not been reported. In contrast to yeasts and plants, only a single gene

encoding NADK has been found in the human genome; the product of this gene, human NADK, is localized to the cytosol22,23. No mitochondrial NADK has been found in human. Pollak _et al._23

stated that given the vital role of at least two NADK isoforms in yeast, one would certainly expect more than one mammalian NADK isoform; furthermore, any additional NADK isoforms in mammals

should have primary structures substantially different from virtually all previously identified enzymes, at least according to the currently available information1. An influx of NAD+

through human and mammal mitochondrial membrane has been reported24,25, but the source of human mitochondrial NADP+ has remained elusive despite its great significance. In this study, we

identified C5orf33, a human protein of unknown function, as an NADK3 homologue, based on homology search using the primary structure of NADK3 of _A. thaliana_ as a query. The C5orf33 protein

is a novel human mitochondrial NADK that is responsible for the missing source of mitochondrial NADP+ in human cells. RESULTS C5ORF33 IS A CANDIDATE HUMAN MITOCHONDRIAL NADK _A. thaliana_

NADK3 homologues are distinguishable from NADK homologues on the phylogenetic tree21, although there has been no report of the features of the primary structure of NADK3. To obtain more

information, we performed a BLASTP26 search using the primary structure of NADK3 as a query against all organisms in KEGG (Kyoto Encyclopedia of Genes and Genomes website,

http://www.genome.jp/kegg/); 1,127 proteins were detected as NADK3 homologues (_E_-values, e-179 to 9.9) with _E_-values less than 10. Human C5orf33 protein was unexpectedly detected as the

134th homologue (_E_-value, 9e-06), whereas human NADK was detected as the 833rd homologue (_E_-value, 0.25). When BLASTP search was conducted using the primary structure of human NADK as a

query, human C5orf33 protein was not included in the detected human NADK homologues (total 1,854 proteins; _E_-values ranging from 0.0 to 6.1) as reported previously1, although the C5orf33

protein contains an NADK motif (Pfam, PF01513 (ref. 27)) (Fig. 1a). NADK3 was detected as the 1,817th homologue (_E_-value, 0.49) among human NADK homologues. BLASTP analysis showed that the

primary structure of human C5orf33 protein (442 amino-acid residues) is 25% identical (37% similar) to that of NADK3 (317 residues). Moreover, using Mitoprot, we identified a

mitochondrial-targeting sequence in the N-terminus of the primary structure of the C5orf33 protein28 (Fig. 1a). These features of the primary structure suggested that the C5orf33 protein is

a novel candidate human mitochondrial NADK. _IN VIVO_ ASSAY OF NADK ACTIVITY USING _S. CEREVISIAE_ Two complementary DNAs corresponding to mRNAs from the human _C5orf33_ gene, transcript

variant 1 (accession no. NM_001085411.1) and transcript variant 2 (accession no. NM_153013.3), are deposited in the NCBI database. Transcript variant 1 encodes full-length C5orf33 protein

(442 residues) (Fig. 1a; Supplementary Fig. S1). Transcript variant 2 encodes the C5orf33 protein lacking N-terminal 163 amino acids (Δ163C5orf33 protein, 279 residues) (Fig. 1a). Note that

variant 2 also is encompassed by Δ100C5orf33 protein (342 residues; Fig. 1a). The NADK activity of C5orf33, Δ62C5orf33, Δ100C5orf33 and Δ163C5orf33 proteins (Fig. 1a) were examined using the

_S. cerevisiae_ NADK triple mutant _utr1 yef1 pos5_. Δ62C5orf33 protein lacks the 62-residue predicted mitochondrial-targeting sequence. As shown in Fig. 2a, MK1598 cells (an NADK triple

mutant having YCplac33-_UTR1_) carrying pRS415 alone were inviable on synthetic complete (SC)+5-fluoroorotic acid (FOA) due to the loss of YCplac33-_UTR1_ and the lethality of the resultant

_utr1 yef1 pos5_ cell11. However, MK1598 cells carrying an additional _S. cerevisiae_ mitochondrial NADHK gene (_POS5_) on pRS415 (pMK2127) were viable on SC+FOA, although YCplac33-_UTR1_

was dropped (Fig. 2a), indicating that the NADHK activity of Pos5 rescued the lethality of the _utr1 yef1 pos5_ cells. MK1598 cells carrying the gene encoding either the full-length C5orf33

protein (pMK3269) or the Δ62C5orf33 protein (pMK3270) were viable on SC+FOA medium, but those carrying the _Δ100C5orf33_ gene (pMK3243) or the _Δ163C5orf33_ gene (pMK3244) were not,

indicating that the C5orf33 and Δ62C5orf33 proteins exhibit NADK or NADHK activity. _IN VITRO_ NAD(H)K ACTIVITIES OF PURIFIED C5ORF33 To confirm the NADK or NADHK activity of C5orf33 protein

_in vitro_, we expressed recombinant C5orf33 and Δ62C5orf33 proteins in _Escherichia coli_ Rosetta-gami(DE3)pLysS as described in Methods, Supplementary Methods and Supplementary Table S1.

C5orf33 was expressed as an insoluble protein, whereas Δ62C5orf33 protein was partially soluble: ~28% of Δ62C5orf33 protein was soluble and 72% was insoluble (Supplementary Methods). We

purified the soluble Δ62C5orf33 protein using TALON column chromatography; the resulting product is referred to hereafter as ‘purified C5orf33 protein’ (Fig. 2b). SDS–PAGE analysis showed

that the molecular mass of the purified C5orf33 protein was 43 kDa, in agreement with the predicted mass (45 kDa; His-tag (1.4 kDa) plus Δ62C5orf33 protein (43.3 kDa)). We assayed the NADK

activity of C5orf33 protein using ATP and poly(P) (metaphosphate) as a phosphoryl donor; by thin layer chromatography (TLC) analysis, we detected the formation of NADP+ from NAD+ and of ADP

from ATP (Fig. 2c), showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors. The kinetic properties of C5orf33 protein were determined by assay of NADK

activity using the continuous method (see Methods), and compared with those of human NADK29 (Table 1). Both _K_m (for NAD+) and _V_max of C5orf33 protein (0.022±0.001 mM and 0.091±0.001 U 

mg−1) were significantly lower than those of human NADK (1.07 mM and 18.5 U mg−1)29 (Table 1). However, at the physiological concentrations of NAD+ found in human mitochondria and cytosol,

the NADK activity of C5orf33 protein would be comparable to that of human NADK, as described in the Discussion (Fig. 2d). With regard to the substrate specificity of C5orf33 protein, the

NADHK activity of C5orf33 protein was only 10% of its NADK activity (Table 1), although the primary structure of C5orf33 protein is similar to that of NADHK (NADK3 (ref. 13)). ATP-dependent

nicotinic acid adenine dinucleotide (NAAD) kinase activity of C5orf33 protein was not detected by TLC. NAAD phosphate (NAADP), the phosphorylated NAAD, is a calcium-mobilizing second

messenger in human6,7. Although NADKs (Utr1 and Yef1) of _S. cerevisiae_ catalyse NAADP synthesis by phosphorylation of NAAD16, human NADK has no NAAD phosphorylation activity22.

Furthermore, TLC analysis detected no phosphorylation of either ADP-ribose, adenosine or 5′-AMP by C5orf33 protein. Sphingosine kinase, diacylglycerol kinase and 6-phosphofructokinase

activities of purified C5orf33 protein were assayed by monitoring formation of ADP, as described in Supplementary Methods. Although formation of ADP was observed in the presence of NAD+ even

in the presence of vehicle alone (16% (v/v) dimethylformamide or 10% (v/v) ethanol), formation of ADP in the presence of sphingosine, diacylglycerol (1,2-dioleoyl-_sn_-glycerol) and

fructose 6-phosphate was not, indicating that purified C5orf33 protein exhibits no sphingosine kinase, diacylglycerol kinase or 6-phosphofructokinase activity, although these kinases have

been suggested to be weakly related to NADK with respect to structure and function30. On the other hand, as phosphoryl donors, C5orf33 protein could use ATP, other nucleotide triphosphates

and poly(P) (metaphosphate, hexametaphosphate and tetrapolyphosphate), but neither ADP, trimetaphosphate, phosphoenolpyruvate nor phosphocreatine (Table 1). It should be noted that C5orf33

protein exhibits a poly(P)-dependent NADK activity comparable to that of its ATP-dependent activity, as it has been postulated that only a subset of bacterial or archaeal NADKs exhibit high

poly(P)-dependent NADK activity, whereas eukaryotic NADKs do not10. Moreover, C5orf33 protein exhibited a saturation curve with ATP (Supplementary Fig. S2), whereas human NADK exhibited

sigmoidal kinetics with ATP29. Although human NADK is inhibited by NADH and NADPH, but not NADP+ (ref. 29), the NADK activity of C5orf33 protein was inhibited by NADH, NADPH and in

particular NADP+ (Table 1). MITOCHONDRIAL LOCALIZATION OF C5ORF33 PROTEIN C5orf33 protein was predicted to be localized in mitochondria because of the presence of a mitochondrial-targeting

sequence (Fig. 1a). An _in vivo_ assay of C5orf33 protein localization in _S. cerevisiae_ indicated that the C5orf33 protein is localized in mitochondria of _S. cerevisiae_, whereas

Δ62C5orf33 protein that lacks the mitochondrial-targeting sequence is not (Supplementary Fig. S3). To confirm the localization of C5orf33 protein in mitochondria of human cells, HEK293A

cells were transiently transfected with a plasmid expressing C terminally FLAG-fused C5orf33 protein or Δ62C5orf33 protein, and observed by fluorescence microscopy after immunostaining (Fig.

3). This observation demonstrated that the C5orf33 protein is localized in mitochondria, whereas Δ62C5orf33 protein is not, confirming that C5orf33 protein is a mitochondrial NADK, and that

its N-terminal 62 residues contain a functional mitochondrial-targeting sequence. Moreover, we separated the mitochondrial and cytosolic fractions of siRNA-transfected HEK293A cells and

confirmed using anti-C5orf33 antibody that C5orf33 protein was localized in the mitochondrial fraction but not in the cytosolic fraction as below (Fig. 5e). WIDESPREAD EXPRESSION OF C5ORF33

MRNA To determine whether _C5orf33_ gene is transcribed in human tissue under physiological conditions, tissue-specific transcription of mRNAs encoding C5orf33 protein, human NADK and

glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were examined by absolute qPCR. The gene encoding GAPDH was selected as a housekeeping gene31,32. For absolute qPCR, standard curves were

created using synthesized mRNAs of the genes encoding C5orf33 proteins, human NADK and GAPDH. As shown in Fig. 4, the presence of mRNAs encoding C5orf33 protein, human NADK and GAPDH were

confirmed in all examined tissues. The relative levels of transcripts of the GAPDH gene in testes, small intestine, colon, stomach and placenta were in agreement with those reported32,

guaranteeing the quality of the RNAs at least in these tissues. The mRNA levels of C5orf33 protein and human NADK were only a few per cent of the levels of GAPDH (for example, only 2.1%

(human _NADK_ gene) and 7.8% (_C5orf33_ gene) in brain) (Fig. 4a). However, levels of C5orf33 mRNA were higher than those of the human _NADK_ gene in all tissues examined except for colon,

spleen and skeletal muscle (Fig. 4b). In particular, C5orf33 mRNA levels were significantly higher than human NADK mRNA levels in heart, liver, testes and small intestine. The mRNA levels of

both genes were low in skeletal muscle, possibly reflecting the virtual absence of both the pentose phosphate and the fatty acid synthetic pathways in this tissue22. C5ORF33 AFFECTS

MITOCHONDRIAL REACTIVE OXYGEN SPECIES (ROS) GENERATION To obtain information regarding the physiological role of C5orf33 protein, we knocked down the human _C5orf33_ gene by siRNA

transfection of HEK293A cells. Of the six siRNA duplexes we tested (Supplementary Table S2), transfection with siRNA#2 resulted in maximum C5orf33 knockdown (~70% reduction) and siRNA#2 was

selected for subsequent knockdown experiments (Supplementary Methods and Fig. 5a). At 2 or 3 days after transfection with siRNA#2, we observed substantial knockdown at the mRNA level (Fig.

5a). At 3 days after transfection with either siRNA#2 or MISSION siRNA Universal Negative Control #1 (control siRNA), C5orf33 mRNA levels were higher than those in the corresponding samples

that had been incubated for 1 or 2 days after transfection. We also made observations at the protein level, using anti-C5orf33 antibody. We detected obvious knockdown of C5orf33 protein

levels at 2 days and especially 3 days after transfection, but not after a 1-day incubation (Fig. 5b). In accordance with the relative increase in C5orf33 transcript level 3 days after

transfection, the absolute level of C5orf33 protein was highest at this time point. The anti-C5orf33 antibody was specific for purified C5orf33 protein, and did not detect purified human

NADK, guaranteeing that the signal we detected accurately represented C5orf33 protein rather than a cross-reaction with NADK (Supplementary Fig. S4). By contrast, the anti-human NADK

antibody was nonspecific, detecting both C5orf33 protein and human NADK (Supplementary Fig. S4). Next, we evaluated the effect of the C5orf33 knockdown. Mitochondrial morphology was not

affected by knockdown of C5orf33 for 3 days after transfection (Supplementary Fig. S5). During this period, cell viability was also unaffected by knockdown, even in the presence of menadione

(Supplementary Fig. S5, Fig. 5c). However, intracellular ROS levels were obviously increased 3 days after knockdown of C5orf33, but not 1 and 2 days, either in the absence or presence of

menadione, although ROS levels were higher following menadione treatment (Supplementary Fig. S5, Fig. 5d). Menadione is cytotoxic to various cell lines, including HEK293 (refs 33,34), and

induces apoptosis of cultured cells via elevation of peroxide and superoxide radical levels35. Moreover, we separated the mitochondrial and cytosolic fractions of HEK293A cells that had been

incubated for 3 days after transfections with siRNA#2 and control siRNA, and again confirmed the mitochondrial localization of C5orf33 protein as well as the knockdown of C5orf33 protein

level in mitochondrial fraction of siRNA#2-transfected cells, using anti-C5orf33 antibody (Fig. 5e). NADK activity of the mitochondrial fraction was decreased to 47% upon knockdown of

C5orf33 (Fig. 5f). These observations indicate that C5orf33 protein, that is, human mitochondrial NADK is involved in the regulation of the generation of mitochondrial ROS. DISCUSSION Only a

single gene encoding NADK has been previously reported to exist in the human genome; the product of this gene, human NADK, is localized in cytosol22,23. Until now, no mitochondrial NADK had

been identified in human. Thus, the source of human mitochondrial NADP(H) has been elusive despite its great significance. In this study, it was revealed that the C5orf33 protein is a novel

human mitochondrial NADK that is responsible for the missing source of mitochondrial NADP+ in human cells. In HEK293 human cells, the concentration of NAD+ in whole cells and mitochondria

are 0.37 and 0.25 mM, respectively36. Given that mitochondria occupy 15% of the volume of a neuron37, we estimated the cytosolic concentration of NAD+ to be 0.39 mM. On the other hand, the

concentration of ATP, which complexed with magnesium, is estimated to be 2 and 8 mM in mitochondria and cytosol of HeLa cell, respectively38. From a saturation curve determined in the

presence of 2 mM ATP (the physiological concentration within mitochondria)38, we estimated the NADK activity of C5orf33 protein at 0.25 mM NAD+ to be 3.5 U per 1 μmol of C5orf33 protein

(Fig. 2d). In the same manner, the activity of human NADK at 0.39 mM NAD+ was estimated to be 12 U per 1 μmol of human NADK subunit in the presence of 8 mM ATP (cytosolic concentration of

ATP)38 (Fig. 2d). Moreover, C5orf33 mRNA levels were higher than those of human NADK in almost all tissues (Fig. 4). Collectively, although the _V_max of C5orf33 protein is significantly

lower than that of human NADK (Table 1), under physiological conditions the NADK activity of the C5orf33 protein would be comparable to that of human NADK. BLASTP analyses using each of the

primary structures of human C5orf33 protein and human NADK as a query in eukaryotic protein databases detected 107 C5orf33 homologues and 316 human NADK homologues, all of which contain the

NADK motif. Phylogenetic trees constructed based on the structures of the 107 proteins as described in Supplementary Methods gave three clades: (i) animal and protist proteins including the

human C5orf33 protein (total 65 proteins; _E_-values from 0.0 to 0.074); (ii) plant proteins including NADK3 (18 proteins; _E_-values from 8e−11 to 0.049); and (iii) other proteins from

several eukaryotes (24 proteins; _E_-values from 0.017 to 5.4) (Supplementary Fig. S6). These clades were, respectively, designated as (i) C5orf33 homologues, (ii) plant NADK3 homologues and

(iii) NADK homologues. The 24 other proteins from several eukaryotes were found in 316 human NADK homologues, gave much lower _E_-values ranging from 0.0 to 4e−51, and hence were designated

as NADK homologues. Predicted mitochondrial-targeting sequences were detected in the primary structures of 38 of 65 C5orf33 homologues, and 4 of 18 plant NADK3 homologues, but not in 24

NADK homologues (Supplementary Fig. S6). Given that eukaryotes consist of animals (for example, vertebrates and insects), protists, plants and fungi, we concluded that animals and protists,

but not plants and fungi, have C5orf33 homologues. C5orf33 homologues were found in almost all animals and many protists; 48 of 52 animals and 13 of 29 protists. Although 42 of 52 animals

and 5 of 29 protists possess both C5orf33 and NADK homologues, some carry only either a C5orf33 homologue or NADK homologue; one protist (_Naegleria gruberi_) has none (Supplementary Table

S3). It is noteworthy that six animals (for example, pig and horse) and eight protists (for example, those in genus _Plasmodium_) possess the C5orf33 homologue as sole NADP+ biosynthetic

enzyme. We performed multiple alignment of the primary structures of human C5orf33 protein, plant NADK3 and nine NADKs (Supplementary Fig. S7). The GGDG motif39, NE/D short motif40, and the

Asp, Thr and Tyr residues (corresponding to Asp-189, Thr-200 and Tyr-202 of Ppnk (NADK of _Mycobacterium tuberculosis_) and taking part in the creation of NAD+-binding site) in the

NADK-conserved region40,41 are completely conserved in the primary structures of the nine NADKs. In the structure of the C5orf33 protein, although the GGDG motif and NE/D short motif were

completely conserved, the Asp, Thr and Tyr residues were not, corresponding instead to Ser-306, Lys-317 and Trp-319. We also found an additional region, termed the C5orf33 additional region,

in the structure of the C5orf33 protein. In the sequence of NADK3, the GGDG motif and NE/D short motif are conserved; the Asp, Thr and Tyr residues were Ser-209, Thr-220 and Ala-222; and

the NADK3 additional region, which does not correspond to the C5orf33 additional region, is found. In the aligned structures of 65 C5orf33 homologues, the GGDG motif was conserved in 58 of

65 structures; the NE/D short motif was completely conserved (that is, in 65 of 65 structures) (Supplementary Fig. S7). With regard to the Ser-306, Lys-317 and Trp-319 of the human C5orf33

protein, the Ser-306 was conserved as Ser/Thr, the Trp-319 was completely as Trp, although the Lys-317 was not conserved and corresponded to Lys/Ser/Thr/Arg. In the aligned structures of

C5orf33 homologues, we found two conserved sequences, motif 1 and motif 2. Motif 1 is PX-GXN(T/S)DP; motif 2 is S-G-X(C/M), where X and hyphen (-) mean hydrophobic (Ile, Phe, Val, Leu and

Met) and any amino-acid residues, respectively. Of the aligned 65 sequences, 50 possessed both motifs; of the remaining 15 sequences, 3 contained motif 1 alone, 8 contained motif 2 alone and

4 contained neither. The sequences corresponding to the C5orf33 additional region were also found in 62 of the 65 aligned sequences. In the aligned structures of 18 plant NADK3 homologues,

the GGDG motif, the NE/D short motif and motif 1 were completely conserved (in 18 of 18 sequences); motif 2 was almost completely conserved (in 17 of 18 sequences). The Ser-209, Thr-220 and

Ala-222 residues of NADK3, corresponding to Asp-189, Thr-200 and Tyr-202 of Ppnk, were conserved as Ser, Thr/Ser and Ala residues. Sequences corresponding to the NADK3 additional region were

found in 17 of the 18 sequences (Supplementary Fig. S7). Taken together, these structural features may contribute to the unique properties of human C5orf33 protein, that is, high capacity

to utilize poly(P), as well as low _K_m and low _V_max, and inhibition by NADP+ and of plant NADK3, that is, low _K_m and high _V_max for NADH13, possibly by using a different set of

active-site side chains. METHODS PLASMIDS The plasmids and primers used in this study are listed in Supplementary Tables S4 and S5. Briefly, pMK2127 is pRS415 carrying the _POS5_ promoter

(Ppos5) driving _POS5_ (ref. 11). Genes encoding C5orf33 (full-length) and its truncations (Fig. 1a) were inserted into downstream Ppos5 by replacing _POS5_ of pMK2127 with these genes.

Genes encoding C5orf33 and its deleted proteins were also inserted into _BamH_I/_Sma_I site of pQE-80L (Qiagen), yielding pMK3271, pMK3272 and pMK3241, respectively. These proteins were

expressed as His (MRGSHHHHHHGS; 1.4 kDa)-tagged proteins to their N-termini. For expression of C terminally FLAG-tagged human C5orf33 and Δ62C5orf33 proteins in HEK293A cells, cDNAs of human

C5orf33 and Δ62C5orf33 were inserted into pFLAG-CMV-5a (Sigma), yielding pMK3602 and pMK3603, respectively. For absolute qPCR, DNA sequences corresponding to cDNA of human GAPDH (1,310 bp;

1,008 bp of _GAPDH_ gene, 5′-flanking 102 bp and 3′-flanking 200 bp), human NADK (3,032 bp; 1,341 bp of human _NADK_ gene, 5′- 42 bp, 3′- 1,649 bp), and C5orf33 variant 1 (3,852 bp; 1,324 bp

of _C5orf33_ gene and 3′- 2,528 bp) were inserted into pBluescript II SK (+), yielding pMK3420, pMK3421 and pMK3422, respectively. Accurate synthesis of all the constructed plasmids was

confirmed by DNA sequencing. YEAST STRAINS AND CULTIVATIONS For the cultivation of yeast, standard yeast media (yeast extract/peptone/dextrose, SC and synthetic dextrose media) were used42.

If required, amino acids were added to synthetic dextrose medium. Liquid medium were solidified using 2% agar. Yeast cells were cultured aerobically at 30 °C. For plasmid shuffling, solid SC

medium containing 0.1% FOA (SC+FOA medium) was used. FOA is toxic for the cells containing _URA3_ (ref. 43). _S. cerevisiae_ strains in BY4742 background (_MAT_α _leu2_Δ_0 lys2_Δ_0 ura3_Δ_0

his3_Δ_1_) were used. _S. cerevisiae_ MK1598 cell is an NADK triple mutant carrying YCplac33-_UTR_1 (BY4742 _utr1_Δ::_kanMX4 yef1_Δ::_HIS3 pos5_Δ::_hphMX4_ YCplac33-_UTR1_) and is lethal on

SC+FOA medium11,16. _IN VITRO_ ASSAYS NADK activity of purified C5orf33 protein was assayed at 37 °C by the modified continuous method as described previously29. Briefly, NADPH formation

was continuously measured at _A_340 in a reaction mixture (1 ml) containing 5 mM NAD+, 5 mM ATP, 5 mM glucose 6-phosphate, 0.5 U glucose 6-phosphate dehydrogenase (Sigma, St Louis, MD), 5 mM

MgCl2 (40 mM MgCl2 in the case of the determination of _K_m and _V_max for ATP, 20 mM MgCl2 in that of _K_m and _V_max for NAD+), 100 mM Tris-HCl (pH 8.0) and appropriate concentration of

purified protein. NADK activity was also assayed by a stop method as described previously29. When NADK activity was analysed by TLC, 5 μl of the reaction mixture containing 5 mM NAD+, 5 mM

MgCl2, 100 mM Tris–HCl (pH 8.0), 4.8 μg of purified C5orf33 protein and 5 mM ATP or 2.4 mg ml−1 metaphosphate was incubated overnight at 37 °C. Metaphosphate was used at 2.4 mg ml−1, as 2.4 

mg ml−1 sodium tetrapolyphosphate is 5 mM. Authentic compounds and the incubated reaction mixture were spotted onto a TLC glass plate silica gel 60F254 (Merck), dried, developed with a

solvent system of isobutyrate—500 mM NH4OH (5:3 v/v)44 and detected by exposure to UV light at 254 nm. The inhibitory effects of NADP+, NADH and NADPH on NADK assay were investigated by a

stop method as described previously29. The NADHK activity was assayed as described previously29 in a reaction mixture (1 ml) containing 2 mM NADH, 5 mM ATP, 5 mM MgCl2, 100 mM Tris–HCl (pH

8.0) and purified protein. NADK activity of C5orf33 protein was determined by a stop method to compare it with the NADHK activity of the enzyme. NADK activity of mitochondrial fraction was

also assayed by the stop method in the reaction mixture as above (40 μl; 5 mM NAD+, 5 mM ATP, 5 mM MgCl2, 100 mM Tris–HCl (pH 8.0) and mitochondrial preparations (1.4–35 μg protein))

followed by cycling assay of NADP+ as described45. In each experiment, the assays were linear with respect to both times (0, 1 and 2 h) and concentrations of mitochondrial preparations (at

least two points). One unit of enzyme activity was defined as 1 μmol NADP+ or NADPH produced in 1 min at 37 °C; specific activity was expressed in U mg−1 protein, unless otherwise stated.

Kinetic values were calculated by fitting the data to the appropriate Michaelis–Menten equations using KaleidaGraph software (Synergy Software). EXPRESSION AND PURIFICATION OF C5ORF33

PROTEIN To express C5orf33 and Δ62C5orf33 proteins, MK3274 and MK3275 cells (_E. coli_ Rosetta-gami(DE3)pLysS (Novagen) cells carrying pMK3271 and pMK3272, respectively) were precultured

overnight in 700 ml LB medium (350 ml per 500 ml Sakaguchi flask) supplemented with 100 μg ml−1 ampicillin and 34 μg ml−1 chloramphenicol. The cells collected from the cultures (~75 or 700 

ml culture) were inoculated in 1.5 or 13.5 l LB medium (1.5 per 2 l Sakaguchi flask) supplemented with the same antibiotics, and aerobically cultured at 37 °C, until _A_600 reached 0.50–1.0.

Isopropyl-β-D-thiogalactopyranoside was then added to obtain a final concentration of 25 μM and cell cultivation was continued further at 16 °C under aerobic conditions for 5 days. The

conditions for expression were determined as described in Supplementary Methods and Supplementary Table S1. The crude extract was prepared as described29. The Δ62C5orf33 protein was purified

as described in Supplementary Methods and the purified protein was regarded as purified C5orf33 protein. TRANSFECTION AND INDIRECT IMMUNOFLUORESCENCE MICROSCOPY HEK293A cells cultivated in

Dulbecco’s modification of Eagle’s minimal essential medium containing 10% fetal bovine serum (DMEMS) supplemented with penicillin (50 μg ml−1) and streptomycin (50 μg ml−1) on coverslips at

37 °C were transfected with pMK3602 or pMK3603 by Lipofectamine 2000 (Invitrogen), and further cultivated for 2 days at 37 °C. The transfected HEK293A cells were stained with MitoTracker

Red CMXRos (500 nM in fresh DMEMS; Molecular Probes) for 45 min, washed with PBS, fixed with 3.7% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked using PBS

containing 3% bovine serum albumin (BSA). The primary antibody was rabbit anti-FLAG (Sigma) and the secondary antibody was AlexaFluor 488-conjugated anti-rabbit IgG (Invitrogen). Images were

acquired using an inverted FV1000-D IX81 (Olympus, Tokyo, Japan) microscope. ABSOLUTE QPCR Human total RNAs were purchased from Clontech and Agilent Technologies. The synthesized mRNAs of

GAPDH, human NADK, and C5orf33 variant 1 (Supplementary Fig. S1) were prepared using the _in vitro_ Transcription T7 kit (Takara, Otsu, Japan) using as templates, respectively, pMK3420,

pMK3421, and pMK3422 linearized by _Xho_I. The molar concentration of the synthesized mRNAs was calculated from _A_260, and the calculated molecular mass of the synthesized mRNAs (GAPDH, 413

 kDa; human NADK, 943 kDa; C5orf33 variant 1, 1,193 kDa). RNA was isolated from siRNA-transfected HEK293A cells using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan). cDNAs were

prepared from RNA and quantitated by absolute qPCR using LineGene (BioFlux, Tokyo, Japan) and SYBR Green Real-time PCR Master Mix (Toyobo) as described in Supplementary Methods. SIRNA

TRANSFECTION The siRNA duplexes (Sigma) for knockdown of _C5orf33_ gene are listed in Supplementary Table S2. MISSION siRNA Universal Negative Control #1 (control siRNA; Sigma) was used as

siRNA duplex for a negative control. Other details were described in Supplementary Methods. Cell viability of the transfected HEK293A cells was measured using

3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or calcein AM (Molecular Probes) and intracellular ROS of the cells were detected as described23,33,46 and in

Supplementary Methods. WESTERN BLOTTING Proteins were separated by SDS–PAGE47 and analysed using anti-C5orf33 (Abnova, rabbit polyclonal, 1:2,500 dilution or 1:312,500 as in Supplementary

Fig. S4), anti-cytochrome C oxidase subunit II (EPR3314) (CoxII; Abcam, rabbit monoclonal, 1:10,000), anti-GAPDH (6C5) (loading control; Abcam, mouse monoclonal, 1:500), anti-human NADK

(Abnova, mouse monoclonal, 1:500 as in Supplementary Fig. S4) antibodies, which were diluted in Can Get Signal solution 1 (Toyobo). Horseradish peroxide–conjugated anti-rabbit IgG

(HRP–anti-rabbit IgG; Amersham Biosciences) and HRP–anti-mouse IgG (Amersham Biosciences) were diluted in Can Get Signal solution 2 (Toyobo) at dilutions of 1:2,000 and 1:10,000,

respectively. MagicMark XP Western Standard (Invitrogen) or Precision Plus Protein Standard (Bio-Rad) was used as the standard. For proteins in lysates of transfected HEK293A cells, cells in

4 × 0.6 ml transfection-mixture were collected by treatment with trypsin/EDTA solution (Nacalai Tesque), suspended and boiled for 10 min in 48 μl 1 × SDS buffer. Signal intensities were

estimated using ImageQuant TL. PREPARATION OF MITOCHONDRIA From transfected HEK293A cells, we prepared mitochondria using the Mitochondria Isolation Kit for Cultured Cells (MitoSciences).

Details are described in Supplementary Methods. OTHER ANALYTICAL METHODS Protein concentration of the crude extract was determined by the Bradford method48, and BSA was used for a standard.

The concentration of purified C5orf33 protein was determined using _A_280. A solution containing purified C5orf33 protein with _A_280 of 1.0 was calculated to contain 0.9 mg ml−1 from the

molar extinction coefficient (51,255), and the calculated molecular mass (46,353 Da) of the C5orf33 protein using ExPASy ( http://br.expasy.org/tools/protparam.html). The molecular mass of

the enzyme was estimated by gel filtration chromatography as described previously29. The phylogenetic tree was constructed as described in Supplementary Methods. ADDITIONAL INFORMATION HOW

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ACKNOWLEDGEMENTS This work was partially supported by the Funding Program for Next Generation World-Leading Researchers (to S.K.) and by Grant-in-Aid for Young Scientists (B) from the Japan

Society for the Promotion of Science (to S.K.). We thank Dr Teruo Kawada, Dr Nobuyuki Takahashi and Mr Tomoya Sakamoto, Graduate School of Agriculture, Kyoto University, for providing us

with HEK293A cells and for technical assistance with cultivation and immunostaining of these cells. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Food and Biological Science,

Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan Kazuto Ohashi, Shigeyuki Kawai & Kousaku Murata

Authors * Kazuto Ohashi View author publications You can also search for this author inPubMed Google Scholar * Shigeyuki Kawai View author publications You can also search for this author

inPubMed Google Scholar * Kousaku Murata View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.O. and S.K. designed and performed experiments.

K.O., S.K. and K.M. wrote the manuscript. S.K. and K.M. reviewed/edited the manuscript. CORRESPONDING AUTHOR Correspondence to Kousaku Murata. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures S1-S7, Supplementary Tables S1-S5, Supplementary Methods and

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characterization of a human mitochondrial NAD kinase. _Nat Commun_ 3, 1248 (2012). https://doi.org/10.1038/ncomms2262 Download citation * Received: 16 March 2012 * Accepted: 06 November 2012

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