1 Nicotinamide adenine dinucleotide is transported into mammalian mitochondria 2 3 Antonio Davila, Jr. 1,2*, Ling Liu3*, Karthikeyani Chellappa1, Philip Redpath4, Eiko Nakamaru-Ogiso5, Lauren M. 4 Paolella1, Zhigang Zhang6, Marie E. Migaud4,7, Joshua D. Rabinowitz3, and Joseph A. Baur1 5 6 1Department of Physiology, Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, 7 University of Pennsylvania, Philadelphia, PA, USA 8 2PARC, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 9 3Lewis-Sigler Institute for Integrative Genomics, Department of Chemistry, Princeton University, Princeton NJ 10 4School of Pharmacy, Queen’s University Belfast, Belfast, BT9 7BL, Northern Ireland, UK 11 5Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, 12 Philadelphia, PA, USA 13 6College of Veterinary Medicine, Northeast Agricultural University, Harbin, China 14 7Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA (present location). 15 * These authors contributed equally to this work. 16 17 18 19 20 21 Keywords: NAD, NADH, nicotinamide, niacin, mononucleotide, redox ratio, mitochondrial biogenesis, Krebs cycle, 22 metabolic syndrome 23 24 25 26 27 28 29 30 31 32 33 34 To whom correspondence should be addressed: 35 36 Dr. Joseph A. Baur 37 Associate Professor of Physiology 38 3400 Civic Center Blvd, SCTR 12-114 39 Philadelphia, PA 19066, USA 40 Tel.: (215) 746-4585 41 Fax: (215) 898-5408 42 email: [email protected] 43 44 45 46

1

47 Abstract 48 49 Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has

50 alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide

51 (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human

52 mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while

53 NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative

54 phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and

55 ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments

56 with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that

57 NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane

58 is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH)

59 transporter.

60

61

2

62 Introduction

63

64 Nicotinamide adenine dinucleotide (NAD) is an essential reduction-oxidation (redox) cofactor as well as a

65 cosubstrate for a growing list of enzymes. Within the mitochondria, NAD accepts electrons from a variety of

66 sources and transfers them to complex I of the electron transport chain, ultimately resulting in the generation of

67 ATP. In addition, NAD serves as a cosubstrate for mitochondrial sirtuins and NAD glycohydrolases (1).

68 Mitochondrial NAD levels vary in a circadian fashion and can directly influence fuel selection (2), as well as

69 determine cell survival under stress (3). Despite these observations, the mechanisms responsible for generating

70 and maintaining the mitochondrial NAD pool remain incompletely understood.

71

72 NAD can be synthesized de novo from tryptophan or via the Preiss-Handler pathway from nicotinic acid, but

73 recycling of the nicotinamide generated by continuous enzymatic cleavage of NAD within the body requires the

74 NAD salvage pathway. This consists of two enzymes: Nicotinamide phosphoribosyltransferase (Nampt), which

75 produces nicotinamide mononucleotide (NMN) in what is considered the rate-limiting step (4), and Nicotinamide

76 mononucleotide adenylyltransferases (NMNATs), which convert NMN to NAD. Three isoforms of NMNAT have

77 been reported, with NMNAT1 localized to the nucleus, NMNAT2 to the Golgi apparatus and neuronal axons, and

78 NMNAT3 to the mitochondria, providing the first evidence that mitochondria contain some of the machinery to

79 maintain their own NAD pool (5). Nampt is primarily nuclear and cytosolic, however, a small portion co-purifies

80 with mitochondria from liver (3). Thus, it was suggested that mitochondria contain a complete NAD salvage

81 pathway and might recycle their own nicotinamide or take it up from the cytosol. Subsequently, Pittelli and

82 colleagues failed to detect Nampt in mitochondria purified from HeLa cells and presented immunofluorescence

83 evidence that it was excluded from the mitochondrial matrix (6). Accordingly, it was proposed that cytosolic NMN

84 is taken up into mitochondria and converted to NAD via NMNAT3 to generate the mitochondrial NAD pool (7).

85 However, Felici et al reported that the full-length transcript for NMNAT3 is not expressed in HEK293 cells, nor in a

86 variety of mammalian tissues, and that instead the endogenous gene produces two splice variants, one of which 3

87 produces a cytosolic protein, and the other of which produces a mitochondrial protein involved in NAD cleavage

88 rather than synthesis (FKSG76) (8). Interestingly, mice lacking NMNAT3 were reported to have defects primarily

89 in erythrocytes, which lack mitochondria, and to have normal NAD levels in heart, muscle, and liver (9) and normal

90 mitochondrial NAD content in multiple tissues (10). Felici et al went on to show that providing intact NAD, but not

91 any metabolic precursor, restores the mitochondrial NAD pool in cells where it was depleted by overexpression of

92 FKSG76. They concluded that mitochondria do not synthesize NAD at all, but rather take it up intact from the

93 cytosol, which in turn, can take up NAD from the extracellular space. This interpretation is at odds with recent

94 findings which show that NAD and NMN must first undergo extracellular degradation to nicotinamide, nicotinic

95 acid, or nicotinamide riboside in order to be taken up into cells (8,11). Moreover, while yeast and plant

96 mitochondria are known to contain NAD transporters, no mammalian counterparts have been described. Thus,

97 the source of mitochondrial NAD remains to be firmly established.

98

99 Here we present evidence that mitochondria directly import NAD. Consistent with previous reports of NMNAT

100 activity in mitochondrial lysates, we find that isolated mitochondria can synthesize NAD from NMN, but not from

101 nicotinamide. However, the majority of this activity is dependent on NMNAT1, which is not mitochondrial, and

102 results in the production of NAD outside of the organelles, rather than filling of the matrix. Using intact myotubes,

103 we demonstrate that isotopically labeled nicotinamide riboside, which is converted to NMN by nicotinamide

104 riboside kinases (NRKs) (12), contributes directly to the mitochondrial NAD pool without shuttling through an

105 intermediary step as nicotinamide. Substituting labeled nicotinic acid riboside, which generates NAD via cytosolic

106 NAD synthase (bypassing NMN), also results in labeling of mitochondrial NAD, suggesting that fully formed NAD,

107 rather than NMN, is transported.

108

109 Results

110

111 Isolated mitochondria synthesize NAD from NMN, but not from nicotinamide 4

112

113 We initially tested whether NAD levels would increase over time in isolated mitochondria incubated with NAD

114 precursors. In the absence of exogenous metabolizable substrates (state 1, as defined by (19,20)), warming

115 mitochondria isolated from murine skeletal muscle resulted in a rapid loss of NAD(H) content (data not shown).

116 With the addition of substrate (pyruvate/malate, state 2) and ADP (state 3), the rate of NAD loss was

117 progressively slowed, and co-incubation with NMN, but not nicotinamide or nicotinic acid was found to maintain

118 NAD levels near the starting value (Figure 1a). To discern whether increased NAD content in the presence of NMN

119 truly reflected new synthesis, rather than slowed degradation, we held mitochondria in state 2 for 30 minutes to

120 establish a reduced NAD content, then added ADP (state 3) with or without NMN. Supplementation with NMN

121 restored mitochondrial NAD content in a time and concentration-dependent manner (Figure 1b-d). Synthesis of

122 NAD from NMN also appears to be at least partially dependent on membrane potential or ATP production, as

123 addition of the uncoupler FCCP or the complex I inhibitor rotenone significantly attenuated the rate of NAD

124 appearance (Figure 1e). Consistently, addition of ATP instead of ADP was sufficient to promote NAD synthesis,

125 suggesting that residual ATP present in the mitochondria after isolation was insufficient to support the NMNAT

126 reaction (Figure 1f).

127

128 In contrast to incubation with NMN, incubation of NAD-deficient mitochondria with nicotinamide did not affect

129 NAD concentration (Figure 2a). This was true whether or not exogenous phosphoribosylpyrophosphate (PRPP,

130 the second substrate for the Nampt reaction) was supplied. Because the localization of Nampt to mitochondria

131 was described in organelles derived from the liver, we also repeated this experiment with liver-derived

132 mitochondria. Similar to muscle-derived mitochondria, liver-derived organelles synthesized NAD readily from

133 NMN, but were incapable of utilizing nicotinamide to a measureable degree, whether or not PRPP was provided

134 (Figure 2b). To further investigate the involvement of NAD salvage enzymes, we employed specific inhibitors of

135 Nampt (FK866) and NMNAT (Gallotannin). As expected, Gallotannin reduced NAD synthesis from NMN (Figure 2c),

136 although the incomplete effect was puzzling, given that NMNAT3 is the most Gallotannin-sensitive isoform and is 5

137 expected to be completely inhibited at this dose (5). On the other hand, addition of FK866 had no effect, arguing

138 against the possibility that NMN breaks down to nicotinamide prior to incorporation into NAD.

139

140 Matrix NAD is not restored by NMN treatment in isolated mitochondria.

141

142 Given that a decline in matrix NAD content will eventually limit respiratory capacity, we next tested whether NMN

143 treatment could restore the respiratory capacity of mitochondria that had been held in state 2 for an extended

144 period. Despite increasing NAD, NMN treatment did not lead to recovery of state 3 respiration in isolated

145 mitochondria (Figure 3a). This suggested two possible interpretations: 1) that another form of mitochondrial

146 damage unrelated to NAD content limited respiration, or 2) that the newly synthesized NAD was not localized in

147 the matrix where it would be able to participate in mitochondrial metabolism. To test the latter possibility, we

148 pelleted mitochondria after NMN treatment and compared the NAD contents of the pellet and supernatant to the

149 whole mixture. Surprisingly, the increase in NAD was almost exclusively outside of the organelles, with no rescue

150 of matrix NAD content after NMN treatment (Figure 3b). We next considered the possibility that mitochondria

151 are sparingly permeable to NAD directly. While low concentrations of NAD failed to have a major impact on matrix

152 NAD content, high (5-10 mM) external NAD led to an appreciable increase. Notably, this concentration is far in

153 excess of whole cell or tissue NAD concentration (~300-1000 μM), but is only slightly above our estimates for NAD

154 concentration in the mitochondrial matrix (3-4 mM, based on the approximation that 1 mg of mitochondrial

155 protein corresponds to ~1uL of matrix volume (21)). Thus, high external concentrations may be required to create

156 a gradient that favors import. Consistent with these findings, 10 mM external NAD prevented the loss of matrix

157 NAD content over time in mitochondria held in state 2, and significantly slowed the decline in respiratory capacity

158 (Figure 3c-d).

159

160 Cytosolic NMN contributes to mitochondrial NAD

161 6

162 To test the behavior of mitochondria in intact cells with physiologically relevant cytosolic concentrations of NAD

163 and NMN, we next employed an isotopic labeling approach. Nicotinamide riboside (NR) is taken up by cells and

164 converted to NMN by nicotinamide riboside kinases (NRKs) (11,12). We treated intact C2C12 myotubes with NR

165 that had been isotopically labeled on both the nicotinamide ring and the ribose moieties, such that its

166 incorporation into NMN and subsequent conversion to NAD would result in retention of both heavy isotopes,

167 whereas degradation of NR by polynucleotide phosphorylase or enzymatic consumption of NAD to generate

168 nicotinamide would separate the labels (Figure 4a). We detected a high proportion of doubly labeled NMN and

169 NAD in mitochondria isolated from the myotubes, unequivocally demonstrating that cytosolic NMN contributes to

170 mitochondrial NAD without an intermediated step involving degradation to nicotinamide (Figure 4 b-c). The

171 slightly more rapid appearance of doubly-labeled NAD in intact whole cell lysates as compared to isolated

172 mitochondria is suggestive that at least some NAD synthesis is occurring outside of the organelles. Since NRK is

173 not present in mitochondria, NMN must be produced in the cytosol, but these data do not allow us to distinguish

174 whether mitochondrial NAD arises from conversion of imported NMN or from direct uptake of cytosolic NAD.

175

176 To discern whether mitochondria have the ability to directly import intact NAD, rather than relying on synthesis

177 from imported NMN, we performed isotopic labeling experiments using nicotinic acid riboside (NAR, Figure 5 –

178 figure supplement 1). The final step in conversion of NAR to NAD requires the cytosolic enzyme NAD synthase

179 (NADS, Figure 4a). Thus, NAD synthesis from this precursor should occur only in the cytosol, and should leave

180 NMN unlabeled. In contrast to this expectation, we found that feeding labeled NAR resulted in nearly equivalent

181 labeling of the total and mitochondrial pools of both NAD and NMN (Figure 5 a-b). We considered several

182 possibilities to explain the observed NMN labeling: 1) The labeled NAR could have been contaminated with

183 labeled NR, resulting in direct production of both NAMN and NMN, 2) Given the much higher concentration of

184 NAD in cells, non-enzymatic degradation of a small amount of labeled NAD during extraction could account for a

185 substantial portion of the NMN signal, and 3) NMN could be generated from NAD through enzymatic processes

186 such as reverse flux through NMNATs or degradation of NADH by the mitochondrial Nudix hydrolase Nudt13 and 7

187 oxidation of the resulting NMNH (22,23). To be sure the parent NAR contained no detectable NR contamination

188 we tested it against NR standards; a spike of as little as 0.01nM NR into 1uM M NAR was robustly detected,

189 whereas no signal was present in the NAR alone, thereby excluding the first possibility that labeled NMN arose

190 from contaminating NR (Figure 5c). Notably, our data also suggest that nicotinic acid-containing nucleotides are

191 not able to enter the mitochondria at all. While NAR was almost undetected by our techniques, we observed a

192 dramatic exclusion of NAMN and NAAD from the mitochondrial fractions (Figure 5d). Next we examined the

193 possibility that nonenzymatic degradation of NAD might contribute to the NMN signal. By varying the extraction

194 conditions, we were able to confirm that spontaneous hydrolysis of NAD is a substantial source of contamination

195 of the NMN pool when metabolites are suspended in aqueous solutions (Figure 5e,f).

196

197 Cytosolic NAD(H) is imported into the mitochondria

198

199 To overcome contamination of the NMN signal by NAD breakdown, we employed two strategies: manipulation of

200 the size of the NMN pool enzymatically, and altering the metabolite extraction protocol to avoid suspension in

201 water. In the first, we used a CRISPR-based system to target each of the three NMNAT isoforms with two

202 independent guide RNAs in C2C12 myoblasts (Supplementary File 1). All cell lines differentiated into myotubes

203 with no apparent differences in size or structure at the end of the week-long differentiation protocol. Loss of

204 NMNAT1 protein expression was verified by western blot, while we were unable to reliably detect NMNAT2 or

205 NMNAT3 using available antibodies. However, reduction of mRNA expression and loss of wild type DNA sequence

206 at the target sites were observed in the cell lines (Figure 6h; Figure 6 -figure supplement 2; Supplementary File 2)

207 and data not shown). Myotube NAD content was significantly reduced in the two lines targeting NMNAT1, as

208 compared to controls or lines targeting the other isoforms (Figure 6a). There were no significant differences in

209 mitochondrial NAD content in freshly isolated organelles (Figure 6b, ice). However, organelles from the NMNAT1-

210 targeted lines showed increased susceptibility to NAD depletion by holding in state 2, and limited ability to

211 synthesize NAD from NMN (Figure 6b), supporting the model that the majority of NMNAT activity in mitochondrial 8

212 preparations arises from contaminating NMNAT1, rather than matrix-localized NMNAT3. This conclusion was

213 further supported by proteinase K treatment of mitochondrial preparations. Limited digestion removes proteins in

214 the supernatant or on the outer mitochondrial membrane, while leaving matrix proteins intact. Accordingly,

215 proteinase K reduced conversion of NMN to NAD without affecting respiratory capacity (Figure 6 – figure

216 supplement 2). In addition to reduced NAD concentration, myotubes with NMNAT1 targeted had dramatically

217 increased NMN content in whole-cell lysates, consistent with a major role for this isoform in NAD synthesis from

218 NMN (Figure 6c). Rescuing NMNAT1-targeted cells with human NMNAT1, which does not contain the targeted

219 sequence, attenuated the increase in NMN concentration, and enhanced NAD synthesis from NMN by isolated

220 mitochondria (Figure 6 – figure supplement 3). In contrast, targeting NMNAT2 or NMNAT3 did not lead to obvious

221 changes in pyridine nucleotide distribution (Figure 6d). As hoped, treatment of NMNAT1 targeted cells with NAR

222 led to a large discrepancy in the fractional labeling detected for the total NMN and NAD pools (Figure 6e). This is

223 consistent with dilution of any NMN signal that resulted from NAD hydrolysis into the larger pool of unlabeled

224 NMN, but also consistent with the possibility that flux through NMNAT1 contributed to some labeling of NMN in

225 wild type cells. In either case, the higher labeling of NAD as compared to NMN allowed us to resolve the source of

226 mitochondrial nucleotides. The fractional labeling of mitochondrial pyridine nucleotide pools clearly approached

227 that of NAD and far exceeded that of (whole cell) NMN (Figure 6f,g). This labeling pattern can only be explained if

228 NAD is taken up directly by mitochondria in NMNAT1-targeted cells. Thus, our findings indicate that mitochondrial

229 pyridine nucleotides originate from imported NAD (or NADH), rather than import of cytosolic NMN.

230

231 In a parallel approach, we were able to demonstrate that by injecting methanolic extracts directly into the LC-MS

232 without a drying/concentration step, we were able to completely avoid the artifactual hydrolysis of NAD to NMN

233 (Figure 7a,b). Repeating the NAR treatment using this method revealed very low NMN levels in the whole cell

234 lysates with almost no detectable labeling, whereas the fractional labeling of NAD was consistent with that in

235 previous experiments (Figure 7c). Mitochondria isolated from these cells contained labeled NAD, confirming that

236 they import fully synthesized NAD from the cytosol. NMN in mitochondria was also labeled, and we speculate that 9

237 this reflects degradation of a small proportion NAD during the isolation process. Similarly, treating the human cell

238 lines HEK293 and HL-60 with doubly-labeled NAR resulted in the appearance of doubly-labeled NAD within the

239 mitochondria in the absence of detectable NMN (Figure 7d). Taken together, our experiments confirm that

240 despite the lack of any recognized transporter, mammalian mitochondria, like their yeast and plant counterparts,

241 are capable of importing NAD(H).

242

243 Discussion

244

245 Mammalian mitochondria lack obvious homologues of the NAD transporters found in yeast and plant

246 mitochondria, raising the question of how they are able to obtain the cofactor. Evidence has been presented in

247 support of direct NAD uptake (8,24), or intramitochondrial synthesis from nicotinamide (3,25) or NMN (7). Our

248 current results support the model that direct uptake of intact NAD contributes to the mitochondrial NAD pool.

249 However, we note that we cannot exclude further contributions from intramitochondrial NAD synthesis.

250

251 Yang et al. showed that a portion of Nampt co-purifies with mitochondria from liver, suggesting the model that

252 mitochondria contain an intact NAD salvage pathway, and take up nicotinamide, rather than NMN or NAD (3). This

253 proposal is consistent with earlier work by Grunicke and coworkers showing that 14C-labeled nicotinamide

254 incubated with isolated mitochondria is incorporated into both NMN and NAD (25). However, it is possible that an

255 exchange reaction catalyzed by NAD glycohydrolases (or sirtuins), rather than net biosynthesis could have been

256 responsible for the labeling observed in these experiments (26). Moreover, we were not able to observe net NAD

257 synthesis when isolated mitochondria were supplied with nicotinamide, with or without exogenous PRPP.

258 Importantly, we cannot exclude that PRPP might need to be generated within the mitochondrial matrix, or that

259 mitochondrial Nampt activity might be present in certain cell types or under certain stresses. However, the

260 present data do not support the ability of mitochondria to synthesize NAD autonomously from nicotinamide, and

10

261 we note that neither Nampt nor PRPP synthetase has been reported as a mitochondrial protein in the recently

262 updated MitoCarta2.0 database (27).

263

264 The mitochondrial localization of NMNAT3 strongly suggests that the organelles might be capable of taking up and

265 using NMN from the cytosol when required. In agreement with previous studies detecting NMNAT activity in

266 mitochondrial lysates (5,28-30), we demonstrate that isolated mitochondria synthesize NAD from NMN. However,

267 the use of intact organelles in our experiments allowed us to discern that the vast majority of, if not all NAD

268 generated from NMN by isolated mitochondria ends up outside the matrix. Moreover, the bulk of this activity is

269 lost when mitochondria are isolated from myotubes lacking the nuclear isoform NMNAT1, suggesting that it arises

270 from small amounts of contamination in the mitochondrial preparations. Since NMNAT1 has been localized

271 exclusively to the nucleus (5,31), our observations most likely reflect nuclear contamination rather than

272 association of NMNAT1 with the mitochondrial outer membrane. It is tempting to speculate that this might also

273 account for the observation of Felici et al. that their mitochondrial lysates contained NMNAT activity that was not

274 attributable to any transcript of the Nmnat3 gene (8). Therefore, our data on isolated mitochondria do not

275 provide direct evidence for the ability of NMN import to contribute to mitochondrial NAD.

276

277 While mammalian mitochondria are generally considered to be impermeable to pyridine nucleotides (32,33), at

278 least two studies have previously reported evidence for uptake of NAD. Rustin et al. reported that direct addition

279 of NAD restored mitochondrial NAD levels and respiration rate in digitonin-permeabilized human cells that had

280 reduced NAD content due to extended culture without medium changes (24), although it is not clear that a rapid

281 breakdown and resynthesis could be completely excluded in these experiments. Felici et al. reported evidence

282 that in HEK293 cells, brain, skeletal muscle, and kidney, the full length transcript described for NMNAT3 does not

283 exist, and that instead, two splice variants are detectable, encoding a cytosolic protein and the mitochondrial

284 protein FKSG76, neither of which is translated at detectable levels (8). This observation is underscored by the lack

285 of obvious phenotypes in most tissues of mice lacking the NMNAT3 gene, with the exception of erythrocytes, 11

286 which have cytosolic NMNAT3 and no mitochondria (9). Interestingly, overexpression of FKSG76 depletes NAD

287 from the mitochondria of intact cells. These depleted mitochondrial NAD levels can be rescued by exogenous

288 NAD, but not by any precursor, leading the authors to propose that intact NAD crosses the plasma membrane and

289 subsequently enters the mitochondria directly. Notably, the NAD precursors provided by Felici et al. should all be

290 incorporated into the nucleocytosolic pool of NAD, and thus would be available to replenish mitochondrial NAD

291 via direct transport. The lack of rescue of FKSG76-depleted mitochondrial NAD levels after precursor treatment

292 therefore implies that either import of precursors or NAD synthesis from them is too slow to compete with the

293 degradation mechanism, whereas direct NAD influx is rapid, or that other aspects of NAD synthesis are impaired

294 in these cells. In addition, it has been reported by Nikiforov et al. that pyridine nucleotides are not transported

295 across cell membranes efficiently and are instead broken down to the corresponding nucleosides or further

296 before being taken up (7). This model is distinctly at odds with the finding of Felici et al. that extracellular NAD but

297 not nicotinamide riboside is able to restore mitochondrial NAD in cells overexpressing FKSG76.

298

299 Our studies using isotopically labeled NR and NAR unequivocally demonstrate that the mitochondrial NAD pool

300 can be established through direct import of NAD (or NADH). Using doubly labeled NR resulted in nearly equivalent

301 labeling of NMN and NAD in whole cell lysates or mitochondria. The existence of doubly labeled NAD within the

302 mitochondria in this experiment proves that mitochondrial NAD synthesis does not require nicotinamide import

303 (as this would only carry a single label), but does not distinguish whether NMN or NAD was transported. To

304 accomplish this, we differentially labeled NAD and NMN by providing doubly labeled NAR. NAR is converted to

305 NAD in the cytosol via NAMN and NAAD, and therefore should not label the NMN pool. Our initial experiment was

306 compromised by hydrolysis of a minority of the NAD to NMN during extraction, but when this technical hurdle

307 was overcome, either by CRISPR targeting of NMNAT1 to enlarge the NMN pool or by directly injecting methanolic

308 extracts into the LC-MS to avoid hydrolysis, the expected pattern of NAD labeling without NMN labeling was

309 obtained. Under these conditions, the mitochondrial pool of NAD was also labeled, demonstrating that it

310 originated from imported cytosolic NAD, rather than NMN. Although it remains technically possible that cytosolic 12

311 NADH could be converted to NMNH by Nudix hydrolase activity, then rapidly imported and converted to

312 mitochondrial NADH without equilibrating with NMN, we were unable to detect labeling of NMNH in NAR-treated

313 myotubes, and thus view this as a remote possibility.

314

315 Therefore, our results indicate that mammalian mitochondria contain an NAD or NADH transporter. While we are

316 not the first to suggest that mitochondria can take up NAD(H), the identity of the putative transporter in

317 mammalian mitochondria has never been elucidated and its existence continues to be debated. A number of

318 proteins have been identified that allow NAD to cross membranes (34,35), but none of these have been shown to

319 act in mitochondria (36-38). The known member of the mitochondrial solute carrier family that transports NAD,

320 SLC25A17, has been localized exclusively to peroxisomes, where it functions to exchange NAD, FAD and free CoA

321 for adenosine 3ʹ,5ʹ-diphosphate, FMN and AMP (37,39). In yeast and plants, nucleoside deoxyribosyltransferases

322 transport NAD across the mitochondrial inner membrane from the cytosol by exchanging AMP and GMP or more

323 slowly by uniport (37,40). However, candidate mammalian NAD transporters identified based on sequence

324 homology have proven to have alternative targets (e.g., the mitochondrial folate carrier) (3,38,41). Recently, the

325 plant mitochondrial NAD transporter, AtNDT2, was targeted and expressed in the mitochondrial membrane of

326 human HEK293 cells, which resulted in the redistribution of cellular NAD into mitochondria (29). Surprisingly, this

327 led to a slower proliferation, a significant reduction oxidative respiration and a dramatic loss of cellular ATP, which

328 was attributed to a metabolic shift from oxidative phosphorylation to glycolysis (29). These results were

329 interpreted to suggest that a mitochondrial NAD transporter is unlikely to exist in human cells. Nonetheless, our

330 findings support the ability of mammalian mitochondria to import NAD and suggest that the toxicity of AtNDT2

331 may be more related to its specific kinetics or regulation than to a generalizable effect of NAD transport.

332 Importantly, our findings do not exclude the possibility that NMN import and synthesis via NMNAT3 also

333 contribute to the mitochondrial NAD pool. Indeed, Cambronne et al. recently employed a fluorescent biosensor to

334 demonstrate that mitochondrial NAD levels are sensitive to depletion of either NMNAT3 (mitochondrial) or

335 NMNAT2 (Golgi/cytosolic), implying that both NMN and NAD import contribute to the mitochondrial NAD pool 13

336 (42). This observation suggests that a mitochondrial transporter for NMN may also await discovery. Alternatively,

337 it is possible that NMNAT3 could function primarily to reverse NAD(H) hydrolysis or could work in combination

338 with enzymes such as Nudt13 that generate NMN(H) (22).

339

340 In summary, we show that mammalian mitochondria are capable of directly importing NAD (or NADH). This

341 finding strongly suggests the existence of an undiscovered transporter in mammalian mitochondria.

342

343 Materials and Methods

344

Key Resources Table Reagent type (species) or Source or Designation Identifiers Additional information resource reference C2C12; ATCC CRL- myotubes; Mouse myoblast, 1772 cell line (Mus musculus) myoblasts; ATCC mycoplasma negative Human leukemia, authenticated by STR ATCC CCL- profiling, mycoplasma cell line (Homo sapiens) HL-60 ATCC 240 negative Human embryonic kidney, authenticated by STR Gift from Morris profiling, mycoplasma cell line (Homo sapiens) 293; HEK293 Birnbaum’s lab negative strain, strain background The Jackson (Mus musculus) C57BL/6 mice Laboratory 000664 C57BL/6J LentiCRISPR v2 recombinant DNA reagent (Lentiviral vector) Addgene 52961 psPAX2 (Lentiviral recombinant DNA reagent packaging plasmid) Addgene 12260 pMD2.G (Lentiviral recombinant DNA reagent envelope expressing plasmid) Addgene 12259 pLX304 DNASU plasmid recombinant DNA reagent (Gateway repository NMNAT1 Clone ID: HsCD00434593 14

Lentiviral vector) chemical compound, drug ATP Sigma-Aldrich A2383 Protease from Bacillus chemical compound, drug Protease Sigma-Aldrich P5380 lichenformis chemical compound, drug ADP Sigma-Aldrich A2754 chemical compound, drug Pyruvate Sigma-Aldrich P2255 chemical compound, drug Malate Sigma-Aldrich M1000 N3501 chemical compound, drug B-NMN Sigma-Aldrich 100MG chemical compound, drug PRPP Sigma-Aldrich P8296 chemical compound, drug FCCP Sigma-Aldrich C2920-10MG chemical compound, drug Oligomycin Sigma-Aldrich O4876-5MG Protease inhibitor chemical compound, drug cocktail (Sigma); Protease inhibitor cocktail PI Sigma-Aldrich P8340 solution Alcohol A3263- chemical compound, drug dehydrogenase Sigma-Aldrich 150KU D5540- chemical compound, drug Diaphorase Sigma-Aldrich 500UN chemical compound, drug Resazurin Sigma-Aldrich R7017 Flavin chemical compound, drug mononucleotide Sigma-Aldrich F6750 Nicotinamide; chemical compound, drug NAM Sigma-Aldrich 72345 chemical compound, drug Hexadinitrine Sigma-Aldrich 107689 Nicotinic acid; chemical compound, drug NA Sigma-Aldrich N4126 chemical compound, drug Perchloric Acid Sigma-Aldrich 244252 NAD; Nicotinamide chemical compound, drug adenine 1011279650 dinucleotide Roche 01 Proteinase K; 0311588700 chemical compound, drug ProtK Roche 1 Protease chemical compound, drug inhibitor 1169749800 Complete protease inhibitor cocktail (Roche) Roche 1 cocktail tablets Bovine serum albumin chemical compound, drug 0311705700 Fraction V, heat shock, fatty BSA Roche 1 acid free ThermoFisher chemical compound, drug Puromycin Scientific A111380-03 10 mg/mL stock ThermoFisher chemical compound, drug Blasticidin Scientific R21001

15

Enzo Life ALX-270-418- chemical compound, drug Gallotannin Sciences G001 Novo-Nordisk 100 units/mL; recombinant chemical compound, drug Insulin Novolin N U-100 DNA origin chemical compound, drug Fugene 6 Promega E2691 NR; isotope- labeled chemical compound, drug nicotinamide riboside PMID: 27508874 NAR; isotope- chemical compound, drug labeled nicotinic acid riboside this paper Micro BCA commercial assay or kit Protein Assay Thermo Fisher Kit Scientific 23235 SuperSignal Thermo Fisher commercial assay or kit West femto kit Scientific 34095 Gift from Lee anti-NMNAT1 Kraus, Zhang et antibody (rabbit al. JBC 2009 polyclonal) PMID: 19478080 anti-VDAC antibody (rabbit monoclonal) Abcam ab154856 [EPR10852(B)] anti-B-actin HRP antibody (mouse monoclonal) Abcam ab20272 [mAbcam 8226] Amersham ECL anti-rabbit antibody Secondary GE Healthcare IgG, HRP-linked whole Ab antibody Life Sciences NA934 (from donkey) Amersham ECL anti-mouse antibody Secondary GE Healthcare IgG, HRP-linked whole Ab antibody Life Sciences NA931 (from sheep) 345

346 Mitochondrial isolation from skeletal muscle and liver

347 Male C57BL/6 mice were euthanized by cervical dislocation, and their gastrocnemius and quadriceps muscles

348 were dissected and placed immediately in ice-cold muscle homogenization buffer (100 mM KCl, 50 mM Tris-HCl

349 (pH 7.4), 5 mM MgCl2, 1 mM EDTA (pH 8.0) and 1.8 mM ATP) at pH 7.2. The entire procedure was performed at

350 4°C. The fat and connective tissues were removed and the muscle tissue was chopped into small pieces. The

351 chopped muscle was incubated for 2 minutes in protease medium (60U of protease from Bacillus lichenformis

352 (Sigma) per mL of homogenization buffer), washed twice with homogenization buffer, and transferred to an ice- 16

353 cold Teflon Potter Elvehjem homogenizer containing homogenization buffer. The muscle was homogenized using

354 a motor-driven homogenizer for 10 minutes at 150 rpm. A small aliquot of the homogenate was then removed

355 and stored at -80°C for further analysis. The volume of the remaining homogenate was doubled with

356 homogenization buffer and centrifuged at 720 x g for 5 minutes at 4°C. The pellet was resuspended in

357 homogenization buffer and centrifuged for an additional 5 minutes at 720 x g. The supernatants were combined

358 and centrifuged at 10,000 x g for 20 minutes at 4°C. The supernatant was discarded and the pellet was

359 resuspended in homogenization buffer and further centrifuged for 10 minutes at 10,000 x g. The final

360 mitochondrial pellet was resuspended in resuspension buffer (225 mM sucrose, 44 mM KH2PO4, 12.5 mM

361 Mg(OAc)2, and 6mM EDTA; pH 7.4) and maintained on ice. Mitochondrial protein content was quantified using

362 the Micro BCA Protein Assay Kit (Thermo Scientific).

363

364 For isolation of liver mitochondria, the liver was placed into ice-cold mitochondrial isolation buffer [MIB; 225 mM

365 immediately after dissection, and

366 chopped into small pieces. The liver was homogenized by 10 strokes in an ice-cold Teflon Potter Elvehjem

367 homogenizer containing MIB. The homogenate was centrifuged at 750

368 supernatant was centrifuged for a further 5 minutes at 750 g. The mitochondrial fraction was recovered by

369 centrifugation of the supernatant for 20 minutes at 5000 g. The pellet was resuspended in homogenization buffer,

370 and re-centrifuged at 5000 g for a further 10 minutes. The final mitochondrial pellet was resuspended in MIB

371 lacking BSA for protein determination as described (Thermo Scientific).

372

373 Mitochondrial treatments and extraction

374 For all experiments, purified mitochondria containing 100ug of total protein were resuspended in ice-cold or pre-

375 warmed MirO5 respiration buffer (Oroboros) containing the indicated compounds at a final concentration of

376 1ug/uL. Pyruvate, Malate, ADP, β-NMN, PRPP, FCCP and Oligomycin were purchased from Sigma. NAD and NADH

377 were from Roche. Gallotannin was from Enzo Life Sciences. For timed incubation experiments, the mitochondrial 17

378 suspensions were maintained at 37°C in a shaking heat block with the tube caps opened. Proteinase K treatments

379 (0.25 or 0.5 mg/mL, Roche) were performed for 30 minutes on ice with 1.1 mg/mL mitochondrial protein, then

380 protease inhibitor cocktail (1:100, Sigma P8340) was added prior to warming. For NAD and NADH determination

381 from the mitochondrial suspension, 50μg of mitochondrial protein were transferred to tubes containing 10% (v/v)

382 of either Perchloric Acid (Sigma) or KOH (Sigma) to achieve final concentrations of 0.6M or 0.1M, respectively.

383 The mitochondrial lysates were vortexed vigorously, then centrifuged at max speed for 10 minutes and the

384 supernatant was collected for analysis and maintained on ice or stored at -70°C. Prior to storage or analysis, the

385 KOH lysate were incubated at 55°C for 10 minutes to degrade any residual NAD, then cooled on ice for 5 minutes.

386

387 NAD-NADH cycling assay

388 An enzyme-based cycling assay was used to determine NAD in experiments without tracers (Figure 1-3; Figure

389 6a,b). Immediately prior to analysis, mitochondrial lysates were diluted 1:10 in ice-cold phosphate buffer (pH 8). 5

390 uL of this dilution was then subjected to an enzymatic cycling assay in a 100 μL total volume as described

391 previously (13). Briefly, NAD standards or diluted mitochondrial extracts were added to a cycling mixture

392 consisting of 2% ethanol, 100 μg/mL alcohol dehydrogenase, 10 μg/mL diaphorase, 20 μM resazurin, 10 μM flavin

393 mononucleotide, 10 mM nicotinamide, 0.1% BSA in 100 mM phosphate buffer, pH 8.0. The cycling reaction was

394 incubated at room temperature, and the appearance of resorufin (generated during each oxidation-reduction

395 cycle) was measured by fluorescence excitation at 544 nm and emission at 590 nm.

396

397 Cell culture

398 C2C12 myoblasts and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented

399 with 4.5g/L D-Glucose, 2mM Glutamine, 10% FBS and antibiotics. C2C12s were purchased from ATCC and HEK293s

400 were a gift from Morris Birnbaum’s lab. Care was taken to maintain these cells within the log phase of growth and

401 to avoid allowing them to become confluent. For differentiation into myotubes, C2C12s were grown to

18

402 confluence, washed once with DPBS (Gibco) and the media was replaced with DMEM containing 2% Horse serum

403 (Gibco) overnight. Following this, the media was replaced every day for 7 days with DMEM containing 2% Horse

404 serum and 1uM insulin (modified from (14)). HL-60 cells were grown in cultured in Iscove’s Modified Dulbecco’s

405 Medium (IMDM) supplemented with 20% FBS and antibiotics. HL-60 cells were purchased from ATCC. Care was

406 taken to maintain these suspension cells at a concentration below 106 cells per mL. Human cell lines (HEK293 and

407 HL-60) were authenticated by STR profiling and all three cells lines tested negative for mycoplasma.

408

409 Generation of CRISPR cell lines

410 The CRISPR/Cas9 system was used to target each of the three individual isoforms of NMNAT in C2C12 cells. For

411 each isoform, two separate guide RNA sequences (gRNA) were targeted toward the 3’ end of the coding region

412 and were designed using the CRISPR design tool (http://crispr.mit.edu). A sequence from the ROSA 26 genes (R26)

413 was used as a control. The gRNA sequences are listed in Supplementary File 1. The gRNAs were cloned into the

414 LentiCRISPR v2 vector backbone (Addgene, #52961) between Esp3I sites downstream of the hU6 promoter.

415 Lentivirus was produced by co-transfection of the lentiviral transfer vector with the pMD2-G envelope and psPAX2

416 packaging vectors into 293 cells using Fugene 6 transfection reagent (Promega). The media was changed 24 hours

417 following transfection. The virus-containing supernatant was collected 48 hours post-transfection and filtered

418 through a 0.22μm syringe filter to eliminate cells. C2C12 myoblasts were infected with virus in media containing

419 8μg/mL hexadinethrine (Sigma) in a dropwise manner with gentle swirling. 24 hours following infection, the virus

420 was removed and the cells were selected in 1.5 μg/mL Puromycin (Gibco). Rescue of NMNAT1 targeted cells was

421 accomplished by introduction of a lentiviral vector pLX304 (clone ID:HsCD00434593, DNASU, Arizona) expressing

422 the human form of NMNAT1 (not targeted by the mouse-specific gRNA used to delete the gene). To generate

423 active virus, the vector was co-transfected with the pMD2-G envelope and psPAX2 packaging vectors into 293

424 cells using Fugene 6 transfection reagent (Promega). Supernatant containing lentivirus was filtered through a

425 0.22μm syringe filter, and used to infect cells in the presence of 8μg/mL hexadinethrine (Sigma). After 24 hours,

426 the virus was removed and the cells were selected in 3ug/mL Blasticidin (Invitrogen). 19

427

428 Western blot analysis

429 Whole cell or mitochondrial lysates from differentiated C2C12 cells were prepared with RIPA lysis buffer (50 mM

430 Tris-HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 0.5 mM EDTA, 150 mM sodium chloride) supplemented

431 with protease inhibitor cocktail (Roche). Forty μg of lysate, 20 μg of mitochondrial pellet, or 10 μg of supernatant

432 were run on a 10% gel (Bio-Rad) and transferred to PVDF membrane (Immobilon). The membrane was probed

433 with rabbit polyclonal anti-NMNAT1 (1:500 dilution) as previously described (13) or anti-VDAC (Abcam) followed

434 by secondary antibody incubation. Immunoblots were developed using SuperSignal West femto kit (Thermo

435 Fisher Scientific) on a Bio-Rad imaging system. Blots were then stripped and re-probed with HRP-conjugated β-

436 actin antibody (Abcam).

437

438 Synthesis of tracers

439 We designed double isotope-labeled nicotinamide riboside (NR) and nicotinic acid riboside (NAR) tracers, with a

440 single 13C and a single deuterium on the nicotinamide and ribose moieties, respectively (Figure 4a). Direct

441 incorporation of the intact tracer into NAD yields double-labeled NAD, whereas breakdown and resynthesis by the

442 salvage pathway of any cell yields single-labeled NAD (Figure 4a). The synthesis of the labelled NR was reported

443 previously (15). The synthesis of the 2H, 13C NAR was accomplished as follows: 13C-Nicotinamide was hydrolysed

444 under basic aqueous conditions to generate 13C-nicotinic acid, which following silylation was coupled to the 2D-

445 tetraacetylated riboside under Vorbruggen conditions to yield the triacetylated 2H, 13C-NAR. Standard

o 2 13 446 deprotection conditions employing NH3g-MeOH at -20 C for 4 days were employed to the generate H, C NAR.

447 2H, 13C NAR was isolated as a mixture of / anomers present in a 15:85 ratio, which could not be successfully

448 separated. This / distribution proves reproducible, and is not observed for the non-labelled NAR (1H NMR, 13C

449 NMR, MS, HRMS). The 1H NMR spectra of labeled and unlabeled NAR are provided in Figure 5 – figure

20

12 13 1 2 450 supplement 1. ESI-MS m/z 258.0926 (M+H); Exact mass calculated for ( C12 C1 H13 H1N1O6; M+H) 258.0917;

451 found 258.0926.

452

453 Isotopic labeling of cells

454 For the tracer studies, C2C12 myotubes, HEK293, or HL-60 cells were treated with double-isotope labeled 0.1mM

455 nicotinamide riboside (NR) or nicotinic acid riboside (NAR) in complete culture medium for 4 hours before

456 extracting. The cells were then rapidly harvested using trypsin and media containing the label, were washed with

457 ice-cold isolation buffer, and then 10% of the volume was removed and re-pelleted. To this pellet, 200uL of 80:20

458 methanol:water (pre-chilled on dry ice) was added, vortexed vigorously and maintained on dry ice until processing

459 as described below.

460

461 Mitochondria were isolated from the remaining 90% of the cells by a method modified from Trounce et al (16).

462 Briefly, the cells which were pelleted and resuspended in a mitochondrial isolation buffer (H-buffer) consisting of

463 210mM mannitol, 70mM sucrose, 1mM EGTA, 5mM HEPES, 0.5% BSA, pH 7.2. The cells were physically sheared

464 in an ice-cold glass-glass dounce homogenizer then centrifuged at low-speed (720 x g for 10 minutes, 4°C). The

465 supernatant containing the mitochondria was transferred to a separate tube, and pellet underwent a second

466 round of homogenization and centrifugation. The supernatants were combined and further purified for the

467 removal of cell debris through additional rounds of low speed spins. The resultant supernatant was subjected to

468 two rounds of high-speed centrifugation (10,000 x g for 30 minutes total, 4°C). The resultant pellets of purified

469 mitochondria were dissolved in cold resuspension buffer (225mM sucrose, 44mM KH2PO4, 12.5mM Mg-acetate,

470 and 6mM EDTA; pH 7.4) and briefly spun (10,000 x g for 2 minutes, 4°C) in order to remove the mannitol from

471 which interfered with the mass-spectrometry measurement.

472

473 Metabolism was quenched and metabolites were extracted by aspirating the wash buffer and immediately adding

474 500uL 80% methanol (pre-chilled on dry ice). After 30 min of incubation on dry ice, the resulting mixture was 21

475 centrifuged at 10,000 g for 5 min. The alcoholic supernatants were then subjected to LC-MS directly (Figure 7) or

476 evaporated under nitrogen and resuspended in 200uL water. Because mitochondrial fractions were prepared

477 from 9 times as much starting material as whole cell lysates, the ion counts obtained from mitochondria were

478 divided by 9 to facilitate a rough comparison. However, this method underestimates the mitochondrial

479 contribution as there is some loss of material during isolation of the organelles. Quantification of NAM and NMN

480 was performed by adding standard compounds to the solution.

481

482 In each experiment on isolated mitochondria, a single isolation was performed and the mitochondria subdivided

483 into the indicated treatment groups. For experiments in unmodified cells, all experimental dishes were split from

484 the same parental dish prior to the experiment. In the case of genetically modified cell lines, all of the cells to be

485 compared were generated simultaneously from the same parent line, and were handled equivalently throughout

486 the study.

487

488 LC-MS Instrumentation and method development

489 Nicotinamide, NMN, NR, and NAD+, NAMN, NAR and NAAD+ were analyzed within 24 hours by reversed-phase ion

490 pairing chromatography coupled with positive-mode electrospray-ionization on a Q Exactive hybrid quadrupole-

491 orbitrap mass spectrometer (Thermo); Liquid chromatography separation was achieved on a Poroshell 120 Bonus-

492 RP column (2.1 mm ×150 mm, 2.7 μm particle size, Agilent). The total run time is 25 min, with a flow rate of 50

493 μL/min from 0 min to 12 min and 200μL/min from 12 min to 25 min. Solvent A is 98: 2 water: acetonitrile with 10

494 mM ammonium acetate and 0.1 % acetic acid; solvent B is acetonitrile. The gradient is 0-70 % B in 12 min (17). All

495 isotope labeling patterns were corrected for natural abundance using AccuCor with correction matrices calculated

496 based on the chemical formula and the mass of the metabolite (18).

497

498 Statistics

22

499 Results are expressed as mean ± standard error of the mean of biological replicates. Biological replicates are

500 individually treated samples (cells or isolated mitochondria) whereas technical replicates are repeat assays of the

501 same biological replicate. No outliers were excluded from the presented data. No formal power analyses were

502 used to design experiments. Sample sizes were chosen based on prior experience with similar assays.

503 Comparisons between two groups were performed using Students t test to establish reported p-values. All

504 statistical analyses were performed using Prism 6 (GraphPad Software, Inc).

505

506 Acknowledgements

507

508 We thank all members of the Baur lab for constructive feedback and suggestions. NMNAT1 antibody was a kind

509 gift from L. Kraus. This work was supported by grants from the National Institutes of Health (R01-DK098656 and

510 R01-AG043483 to J.A.B.; K12-GM081259 to A.D.J.). We thank the University of Pennsylvania Diabetes Research

511 Center (DRC) for the use of the Metabolomics Core (P30-DK19525).

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633 42. Cambronne, X. A., Stewart, M. L., Kim, D., Jones-Brunette, A. M., Morgan, R. K., Farrens, D. L., 634 Cohen, M. S., and Goodman, R. H. (2016) Biosensor reveals multiple sources for mitochondrial 635 NAD(+). Science 352, 1474-1477 636

637

27

638 Figure legends 639 640 Figure 1. Mitochondria synthesize NAD from nicotinamide mononucleotide. (A) Mitochondria isolated from

641 murine skeletal muscle were maintained for 30 min at 37°C with shaking in respiratory state 3 (MirO5 respiration

642 buffer containing 10mM Pyruvate, 5uM Malate, 12.5mM ADP) supplemented with 0.5mM NAM, NA, or NMN.

643 (N=2-4). (B) Mitochondria initially held for 30 min in state 2 (MirO5 respiration buffer containing 10mM Pyruvate,

644 5uM Malate; 37°C with shaking) were then supplemented with NMN alone or NMN + ADP and incubated for an

645 additional 30 minutes at 37°C. (N=2). (C) Time course of mitochondrial NAD levels before and after addition of

646 NMN or NMN + ADP. (N=2). (D) Isolated mitochondria were held in state 2 for 30 min before adding ADP to

647 stimulate state 3 respiration for 60 min in the presence of increasing amounts of NMN added concomitantly with

648 ADP. (N=2). (E) Isolated mitochondria were maintained in state 2 at 37°C with shaking for 30 minutes and then

649 transitioned to state 3 in the absence or presence of NMN (0.5mM), FCCP (4uM), or rotenone (ROT; 0.5uM) and

650 incubating for an additional 60 minutes at 37°C with shaking. (N=4). (F) Isolated mitochondria were held in state 2

651 for 30 minutes before being supplemented with NMN alone, NMN + ADP or NMN + ATP and incubated for an

652 additional 30 minutes at 37°C (N=2-4). The data shown are means ± SEM from two or more biological replicates,

653 each measured in technical duplicate and are representative of three independent experiments. (*, P < 0.05; **, P

654 < 0.001; ***, P < 0.0001; 2-tailed, unpaired Student’s t-test)

655

656 Figure 2. Isolated mitochondria do not produce NAD from nicotinamide. (A) Mitochondria isolated from

657 murine skeletal muscle were held in respiratory state 2 for 30 min at 37°C with shaking before addition of ADP

658 (state 3) and incubation for 60 min in the absence or presence of the precursors NMN, NAM, PRPP, or NAM and

659 PRPP (0.5mM). (N=2). Results are representative of two independent experiments. (B) Mitochondria isolated

660 from murine liver were held in state 2 at 37°C for 30 min with shaking before the addition of ADP (state 3) in the

661 presence or absence of 0.5mM NMN, NAM or NAM and PRPP. (N=2). (C) Muscle mitochondria were maintained

662 in state 2 for 30 min before the addition of ADP (state 3) and further incubated at 37°C for 60 min in the presence

28

663 or absence of NMN and inhibitors Gallotannin (100uM) or FK866 (10nM). (N=4). (*, P < 0.05; **, P < 0.005; ***, P

664 < 0.0001; 2-tailed, unpaired Student’s t-test)

665

666 Figure 3. NAD synthesized from NMN by isolated mitochondria remains outside of the organelles. (A) State 3

667 coupled mitochondrial oxygen consumption. Isolated skeletal muscle mitochondria were extracted directly (ice) or

668 maintained in respiratory state 2 (MirO5 respiration buffer containing 10mM Pyruvate, 5uM Malate) at 37°C with

669 shaking for 60 min before addition of 12.5mM ADP (state 3) with or without 0.5mM NMN and incubated an

670 additional 30 min at 37°C before being measured. (N=2-3). (B) Isolated skeletal muscle mitochondria were

671 maintained in state 2 at 37°C with shaking for 30 min before addition of ADP (state 3) with or without NMN and

672 incubated an additional 60 min. The mitochondrial suspension was then either lysed directly in 0.6M perchloric

673 acid (final concentration) or centrifuged at 10,000xg for 2 minutes at 4°C to collect the supernatant and

674 (subsequently washed) pellet which were then extracted with perchloric acid. (N=2). (C and D) Isolated skeletal

675 muscle mitochondria were maintained in state 2 at 37°C with shaking with or without 10mM NAD. At the

676 indicated time points, aliquots were removed from the pooled mitochondrial suspensions and centrifuged to

677 separate the pellet and supernatant (C), or analyzed for state 3 respiratory capacity using high-resolution

678 respirometry (D). (N=2). Results are expressed as mean ± SEM and are representative of three independent

679 experiments. (*, P < 0.05; **, P < 0.005; ***, P < 0.0001; unpaired Student’s t-test)

680

681 Figure 4. Nicotinamide riboside is incorporated intact into mitochondrial NAD. (A) Isotopically-labeled

682 nicotinamide riboside (NR) or nicotinic acid (NAR) was synthesized to contain a C-13 on the pyridine carboxyl

683 group and a deuterium on the ribose moiety (NAR labeling shown). (B) Fractional labeling of NAD in C2C12 whole

684 cell lysate and isolated mitochondria following 4 hours of incubation with double-labeled NR with or without the

685 NMNAT inhibitor, gallotannin (Gallo; 100uM). (No label group, N=1; NR 4hr group, N=4; NR + Gallo 4hr group,

686 N=3). (C) Fractional labeling of NMN found in C2C12 whole cell lysate and isolated mitochondria following 4 hours

687 of incubation with double-labeled NR with or without gallotannin (Gallo; 100uM). (No label group, N=1; NR 4hr 29

688 group, N=4; NR + Gallo 4hr group, N=3). Data shown are means ± SEM and are representative of 2 independent

689 experiments.

690

691 Figure 5. Nicotinic acid riboside is incorporated intact into mitochondrial NAD. (A) Fractional labeling of NAD in

692 C2C12 whole cell lysates and isolated mitochondria following 4 hours of incubation with doubly-labeled NAR.

693 (N=5). (B) Fractional labeling of NMN in C2C12 whole cell lysates and isolated mitochondria following 4 hours of

694 incubation with doubly-labeled NAR. (N=5). (C) Confirmation of the lack of NR contamination in NAR. 1uM NAR

695 was combined with increasing concentrations of NR (0-100nM) to demonstrate that NR is absent in the NAR and

696 readily detected by this methodology. (Single measurements). (D) Total ion counts for NAAD and NAMN in whole

697 cell lysates and mitochondrial isolates from differentiated C2C12 cells treated with isotopically-labeled NR or NAR

698 tracers for 4 hours. Results expressed as means ± SEM. (N=3). (E) Incubation of NAD at 37°C in water, but not 80%

699 methanol results in substantial degradation to NMN. Blue bars show unlabeled NMN resulting from degradation

700 from a 20uM NAD standard spike; Red bars indicate labeled NMN from spiked-in standard (1uM, dual labeled).

701 (N=3). (F) NAD total ion count measured in parallel from same samples in (E). (N=3).

702

703 Figure 6. Labeling of mitochondrial NAD tracks that of total NAD, but not of total NMN. For all panels, data

704 representing whole cells are depicted as solid bars, whereas data from isolated mitochondria are shown with a

705 stippled pattern. (A) Differentiated C2C12 parental and LentiCRISPR transgenic myotubes were analyzed for NAD

706 content. The cells are as follows: ctrl- parental line with no vector; R26- vector control; 1a and 1b- two separate

707 guide RNAs targeting NMNAT1; 2a and 2b- two separate guide RNAs targeting NMNAT2; 3a and 3b- two separate

708 guide RNAs targeting NMNAT3. (N=3). (B) Mitochondria isolated from differentiated C2C12 cells were held in state

709 2 (MirO5 respiration buffer containing 10mM Pyruvate, 5uM Malate) at 37°C with shaking for 30 min. They were

710 then collected and lysed in perchloric acid immediately, or transitioned into state 3 by adding ADP (12.5mM, final

711 concentration) with or without supplementation with NMN (0.5mM, final concentration) and maintained for 60

712 min at 37°C with shaking before collection. (N=2-4). (C-D) Total ion counts for NAD and NMN in extracts from 30

713 C2C12 LentiCRISPR whole cells (C) and isolated mitochondria (D) following a 4-hour incubation with isotopically-

714 labeled NR or NAR tracer. (Single measuements). (E-F) Fractional labeling of metabolites (NAD and NMN)

715 measured in C2C12 LentiCRISPR whole cells (E) and isolated mitochondria (F) after a 4-hour incubation with

716 isotopically-labeled NR or NAR tracer. (Sinle measurements). (G) Fractions of double-labeled NAD and NMN

717 measured in C2C12 LentiCRISPR whole cell and mitochondrial lysates following 4-hour incubation with isotope-

718 labeled NAR (means ± SEM). (N=3). (H) Immunoblot confirming NMNAT1 knockout in CRISPR C2C12 cell line. (*, P

719 < 0.05; **, P < 0.001; 2-tailed, unpaired Student’s t-test versus R26)

720

721 Figure 7. Direct injection of methanolic extracts reveals preferential labeling and mitochondrial uptake of NAD

722 over NMN. (A) NMN concentration and labeling in differentiated C2C12 cells extracted with -80°C 80:20

723 methanol:water, analyzed either by hydrophilic interaction chromatography (no drying step), or dried under N2,

724 re-suspended in water and analyzed by reversed-phase ion-pairing chromatography (with drying step). Blue bars

725 show unlabeled NMN resulting from intracellular NMN + NAD degradation after the drying/resuspension step;

726 Red bars indicate labeled NMN from spiked-in standard (1 uM, dual labeled). (N=2). (B) NAD total ion count

727 measured in parallel from same samples in (A). (N=2). (C) NAD and NMN labeling (mass +2 fraction) in

728 differentiated C2C12 cells treated with dual labeled NAR for 4 hours (whole cell vs. isolated mitochondria, N=3).

729 Data are compiled from three biological replicates and are displayed as means ± SEM. (D) NAD labeling (mass +2

730 fraction) in the human cell lines HEK293 and HL-60 following a 4-hour incubation with dual labeled NAR (whole

731 cell vs. isolated mitochondria, N=3). Data are compiled from three biological replicates and are displayed as

732 means ± SEM.

733 734 735

31

736 Figure 5 – figure supplement 1. Characterization of doubly-labeled nicotinic acid riboside. (A) NMR spectra

737 confirming the NAR synthesis product. Red NMR trace represents unlabeled β–NAR. Blue NMR trace represents

738 double-labeled NAR (~15% ɑ-NAR based on integration). (B) β–NAR-deuterium labeled on ribose and C-13 labeled

739 on the nicotinic acid moiety.

740

741 Supplementary File 1. Table of gRNA sequences cloned into LentiCRISPR v2 vector backbone. Two independent

742 guides were used to target each NMNAT isoform, and were compared to a control targeting the ROSA26 locus.

743

744 Figure 6 – figure supplement 1. mRNA expression of NMNAT isoforms after CRISPR targeting. Total RNA was

745 extracted from differentiated cells in duplicate using Trizol (Invitrogen) according to the manufacturer’s

746 instructions. Subsequently, cDNA was synthesized using a high capacity cDNA reverse transcription kit (ABI). RT-

747 PCR was performed using Power SYBR Green PCR master mix (ABI) on the Quantstudio 7 Flex RT-PCR system (ABI).

748 (A-C) For all assays, the plots show gene expression values relative to the reference sample, R26 (control), and are

749 normalized to the control gene 36B4 (Rplp0). For each NMNAT isoform, two distinct gRNAs were generated (a and

750 b) near the 5’ end, which are separated by a short sequence (~40 bp) and the correspondingly named primers

751 amplify a region that is just downstream of the target site. The cells are named as follows: ctrl - parental line with

752 no vector; R26 - control; 1a and 1b - two separate guide RNAs targeting NMNAT1; 2a and 2b - two separate guide

753 RNAs targeting NMNAT2; 3a and 3b - two separate guide RNAs targeting NMNAT3.

754

755 Supplementary File 2. Primer sequences used to detect NMNAT transcripts. Each primer pair amplifies a region

756 just downstream of the guide RNA that bears the corresponding name (see Supplementary File 1).

757

758 Figure 6 – figure supplement 2. Partial digestion of mitochondrial preps with proteinase K impairs NAD synthesis,

759 but not respiration. Isolated murine skeletal muscle mitochondria were diluted in MiRO5 to equal concentrations

760 (1.1mg/mL) and were incubated on ice for 30 minutes with the following treatments: ctrl - diluted in MiRO5 only; 32

761 PI - incubated in MiRO5 only for 30 minutes followed by addition of protease inhibitor (1:100 dilution; Sigma); PI +

762 protK [0.5] - incubated with both protease inhibitor (1:100) and proteinase K (0.5 mg/mL; Roche); protK [0.25] -

763 incubated with proteinase K (0.25 mg/mL) followed by the addition of protease inhibitor; protK [0.5] - incubated

764 with proteinase K (0.5 mg/mL) followed by the addition of protease inhibitor. (A) Treatment with proteinase K

765 significantly impaired the ability of the mitochondria to generate NAD from NMN. (N=2). (B) Proteinase K-treated

766 does not impair mitochondrial OXPHOS activity. (N= 1).

767

768 Figure 6 – figure supplement 3. Overexpressing human NMNAT1 decreases NMN accumulation in NMNAT1

769 targeted cells and enhances the ability of mitochondrial preps to synthesize NAD from NMN. NMNAT1 protein

770 was restored by introduction of a viral vector containing the human form of the NMNAT1 gene in order to prevent

771 targeting by the mouse-specific NMNAT1 gRNA. The gateway compatible lentiviral vector, pLX304 (clone

772 ID:HsCD00434593, DNASU, Arizona), was co-transfected with the pMD2-G envelope and psPAX2 packaging

773 vectors into 293 cells using Fugene 6 transfection reagent (Promega). The consequent lentivirus was collected as

774 described and was used to infect the C2C12 CRISPR cells. (A) Expression of human NMNAT1 in C2C12 cells lacking

775 the murine isoform and the Rosa26 control line. (B) Human NMNAT1 reduced NMN accumulation in CRISPR-

776 targeted cells. (C) Human NMNAT1 cDNA enhanced the ability of mitochondrial preps to synthesize NAD from

777 NMN. Three biological replicates were used for the experiments reported in panels B and C.

778

33

Figure 1 A B

) 5

M ** ) 3 ** E

4 M S E + S g 3 + 2 m g / l m

o 2 /

l * m o

n 1

( 1 m n D (

A 0 D N ice st 3 st 3+ st 3+ st 3+ A 0 NAM NA NMN N t=0 t=30 t=60 NMN NMN +ADP st 2 hold C D st 2 30 min 30 min ) ) 5 2.5 M M E 4 addition of ADP E S S 2.0 + alone or ADP+NMN + g 3 g 1.5 m m / / l l o 2 o 1.0 m m n ( n 1 ( 0.5 D

state 2 D A A N 0 0.0 N 0 15 30 45 60 75 90 ADP ADP+ ADP+ ADP+ min 0.05mM 0.25mM 0.5mM st 2 30 min st 2 30 min st 3 30min NMN NMN NMN st 2 30 min st 3 + NMN 30min E F ***

) 5

M *

E 4 S +

g 3 m / l

o 2 m n

( 1 D

A 0 N ADP NMN NMN +ADP +ATP st 2 30 min 60 min Figure 2

** **

A ) 3 B ) 3 M ** ** M E E S S + + 2

g 2 g m m / / l l o o m m 1 1 n n ( ( D D A A N N 0 0 st 2 30 min ADP + + + + + st 2 30 min ADP + + + + NM N + NMN + NAM + + NAM + + PRPP + + PRPP +

C 2.5 *** ** ) M

E 2.0 S +

g 1.5 m / l

o 1.0 m n ( 0.5 D A

N 0.0 st 2 30 min st 3 60min + + + + NMN + + + Gallotannin + FK866 + Figure 3 A B ) g m ×

) direct Lyse

c 30000 3 e

M * s / E pellet l S o +

m 20000 2 supernatant g

p * ( m / l n o o i t 10000 m 1 p n ( m * u D s A n N

o 0 0

c Ice st 2 60min st 2 60min 2 ctrl NMN

O (no incub.) st 3 30min st 3 + NMN 30min

C D ) g m ×

) 10 c 15000 ctrl e M s E / 8 l * S NAD o + * m 10000 g ** 6 * p *** ( m / l n o o

4 i t m 5000 p n ( 2 m u D s A

* n N *

0 o 0 c

e 0 0 0 0 0 0 0 0 0 0 2 t=0 t=30 t=60 t=90 t=120 ic t= =3 =6 =9 12 t= =3 =6 =9 12 t t t t= t t t t= O ctrl NAD O O Figure 4

A N O H N NH2 O nicotinic acid (NA) O nicotinamide (NAM) + + N O H H 2N N O O HO O H O H HO NAPRT NAMPT + PRPP O H O H + PRPP nicotinic acid riboside nicotinamide riboside (NAR) NRK (NR) + ATP O O

+ + N O H N NH 2 O O

O H O H O O O O P O H Gallo P O H HO O H HO O H

nicotinic acid mononucleotide NMNAT nicotinamide mononucleotide (NAMN) + ATP (NMN)

O O

+ + N O H N NH 2 O O

O H O H O NH 2 O NH2 O O P O H N P O H N O H O H O N NADS O N – P O N – P O N O N +Gln O N O O O O O H O H

O H O H nicotinic acid adenine dinucleotide nicotinamide adenine dinucleotide (NAAD) (NAD)

B C ) ) % % NAD - 2 NMN-2 ( ( 100 100 n n NAD - 1 NMN-1 o o i i t t NAD - 0

c NMN-0 c 75 75 a a r r f f d d 50 50 e e l l e e b b a a 25 25 l l N D A 0 M 0 N No NR NR+ No NR NR+ N No NR NR+ No NR NR+ label 4hr Gallo label 4hr Gallo label 4hr Gallo label 4hr Gallo 4hr 4hr 4hr 4hr whole cell mitochondria whole cell mitochondria Figure 5 A B ) ) NAD - 2 NMN - 2 % % ( ( 100 NAD - 1 100 NMN - 1 n n o o i i NMN - 0 NAD - 0 t t c c 75 75 a a r r f f d d 50 50 e e l l e e b b 25 25 a a l l N D 0 0 A no NAR no NAR M no NAR no NAR N N label 4hr label 4hr label 4hr label 4hr whole cell mitochondria whole cell mitochondria

C D 1.0´ 108 7 NaAD t 1.2´ 10 n s 7 t NaMN u 8.0´ 10 n o u c

o 7 n 7 6.0´ 10 1.0´ 10 c o i n

o 7 R 6 3.0´ 10 i 4.0´ 10 l N a l t a 6 7 t 1.5´ 10 o 2.0´ 10 o T T 0 0 r r r r k ) M M M M M M d d e e e e n M n n n n n n le le c c c c la u 0 1 1 1 0 0 b e ra ra ra ra b 0 0 . 1 0 la b t t t t 0 . 0 1 n la R R R R 1 0 u n N N A A ( - u - - N N R C o- C o - - W it it C o N NAR (1uM) + increasing [NR] W m it m W m E F labeled fraction 30000 (from 1uM NMN) 8.0´ 107 t unlabeled fraction t n n 7 u (degraded from u 6.0´ 10 o 20000 o c 20uM NAD) c N D 4.0´ 107 A M N N l l 10000 a a 7 t t 2.0´ 10 o o T T 0 0 D N O A O 2 2 D N O A O A M S A M 2 S 2 N N H /B :H N H B H H N / : M d w M d w H u le O O u e O 0 e 2 e 0 l O e 2 b M 2 e 2 a H b H M l la M M u u 1 1 standards- NAD + NMN stds- standards- NAD + NMN stds- no incubation 30 min at 37°C no incubation 30 min at 37°C ) A ) 10 B 20 ice Figure 6 M M E E 8 st 2 30min S S 15 + + st 2 30min st 3 60min g g 6 * * * m m ** st 2 30min st 3 + / / l l 10 * * o o 4 NMN 60min m m n n ( ( 5 2 ** ** D D A A * * N N 0 0 ctrl R26 1a 1b 2a 2b 3a 3b ctrl R26 1a 1b 2a 2b 3a 3b NMNAT CRISPR KD NMNAT CRISPR KD CRISPR CRISPR whole cell lysates isolated mitochondria lysates C D 2.0´ 108 3.0´ 107 2.5´ 107 1.5´ 108 2.0´ 107 s s t t 1.0´ 108 1.5´ 107 n n 7 u u 1.0´ 10 o o 7 5.0´ 10 6 c c 5.0´ 10 n n 1.5´ 107 1.5´ 106 o o i i l l a 7 a 6 t 1.0´ 10 t 1.0´ 10 o o T T 5.0´ 106 5.0´ 105

0 0

26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 R R R R R R R R NR tracer NAR tracer NR tracer NAR tracer NR tracer NAR tracer NR tracer NAR tracer NAD total ion count NMN total ion count NAD total ion count NMN total ion count E whole cell lysates F isolated mitochondria lysates ) ) % % 100 100 ( ( n n

o +2 label o +2 label i i t t c c 75 75

a +1 label a +1 label r r f f d d e e l l 50 50 e e b b a a l l e e t t i i 25 25 l l o o b b a a t t e e 0 0 m m 6 1 3 6 6 6 26 1 2 3 26 1 2 3 26 1 2 3 26 1 2 3 2 2 2 1 2 3 2 1 2 3 2 1 2 3 R R R R R R R R NR tracer NAR tracer NR tracer NAR tracer NR tracer NAR tracer NR tracer NAR tracer NAD total ion count NMN total ion count NAD total ion count NMN total ion count ) %

( whole cell lysates isolated mitochondria lysates

e 25 t i G l H o b

a 20 t

e CRISPR (LC) m 15

d LC-NMNAT KD e l

e Ctrl LC-R26 LC-1a LC-1b LC-3a LC-3b LC-2a LC-2b b 10 * ** a l - e

l NMNAT1

b 5 u o

d b-actin f 0 o R26 1a 1b 3a 3b % NAD - whole cell NAD - mito NMN - whole cell NMN - mito A B Figure 7 Labeled fraction 600000 100000 (from 1uM labeled NMN) t t n n 80000 u Unlabeled fraction (cellular NMN + u o 400000 o c NMN degraded from cellular NAD) c 60000 N D A M N N 40000 l l 200000 a a t t o o 20000 T T 0 0

ct td ct td ct td ct td D D ra s ra s ra s ra s A A t N t N t N t N N N ex M ex M ex M ex M M M N N N N u u t+ t+ t+ t+ 5 10 ac ac ac ac tr tr tr tr ex ex ex ex

No drying step With drying step No drying With drying NAD C D step step Standards e t i l o D

b 20 30 A a t N e d m 15 e l d e 20 e b l a e l

10 - b e a l l - b 10 e u l 5 o b d u f o o d 0 0 f % o NAD NMN NAD NMN WC mito WC mito % Whole cell mitochondrial HEK293 HL-60 lysates lysates Figure 5 - figure supplement 1

A

O B N+ HO – O D C13

HO O H O

Figure 6 - figure supplement 2

2.0 A primer: NMNAT 1a primer: NMNAT 1b 1.5

Q 1.0 R

0.5

0.0 ctrl R26 1a 1b 2a 2b 3a 3b

B 3 primer: NMNAT2a primer:NMNAT2b

2 Q R

1

0 ctrl R26 1a 1b 2a 2b 3a 3b

6 C primer: NMNAT3a primer: NMNAT3b

4 Q R

2

0 ctrl R26 1a 1b 2a 2b 3a 3b Figure 6 - figure supplement 4 A state 2 30 min, state 3 8 state 2 30, st 3 60 min

) state 2 30, st 3 + NMN 60 min M 6 E Net synthesis S + ** g

m ** /

l 4 o m n (

D 2 A N

0 rl I ] ] ] t P .5 5 .5 c 0 .2 0 [ 0 [ K [ K t K t ro t ro p ro p + p I B P )

M 3000 E

S state 2 ± g state 3 m × c oligomycin e 2000 s / l FCCP o m

p Antimycin A ( n o

i 1000 t p m u s n o c 0 2

O rl I ] ] ] t P .5 5 .5 c 0 .2 0 [ 0 [ K [ K t K t ro t ro p ro p + p I P A Figure 6 - figure supplement 5

NMNAT1

b-actin

NMNAT1 cDNA rescue (O/E) B

6.0´ 106 ** * 3.0´ 105

N NAD ) M M N

E NMN ( S i + o

6 5 n g 4.0´ 10 2.0´ 10 c m / o s u t n n t u s o / m c

6 5 g

n 2.0´ 10 1.0´ 10 o + i ( S E D M A ) N 0 0 ROSA26 NMNAT1 ROSA26 NMNAT1 + O/E + O/E

C *** 8 * * Ice ** ) st 2 30 min, st 3 immed lyse M 6 E st 2 30 min, st 3 + NMN 60 min S + g m /

l 4 o m n (

D 2 A N

0 ROSA26 NMNAT1 ROSA26 NMNAT1 + O/E + O/E