ARTICLE
DOI: 10.1038/s41467-017-01311-y

OPEN

Cysteinyl-tRNA synthetase governs cysteine
polysulﬁdation and mitochondrial bioenergetics

1234567890

Takaaki Akaike1, Tomoaki Ida1, Fan-Yan Wei2, Motohiro Nishida3,4, Yoshito Kumagai5, Md. Morshedul Alam6,
Hideshi Ihara7, Tomohiro Sawa8, Tetsuro Matsunaga1, Shingo Kasamatsu1, Akiyuki Nishimura3,
Masanobu Morita1, Kazuhito Tomizawa2, Akira Nishimura1, Satoshi Watanabe 9, Kenji Inaba9,
Hiroshi Shima10, Nobuhiro Tanuma10, Minkyung Jung1, Shigemoto Fujii1, Yasuo Watanabe11,
Masaki Ohmuraya12, Péter Nagy13, Martin Feelisch14, Jon M. Fukuto15 & Hozumi Motohashi 6

Cysteine hydropersulﬁde (CysSSH) occurs in abundant quantities in various organisms, yetlittle is known about its biosynthesis and physiological functions. Extensive persulﬁdeformation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells
and is believed to result from post-translational processes involving hydrogen sulﬁde-related
chemistry. Here we demonstrate effective CysSSH synthesis from the substrate L-cysteine, areaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs).
Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells
shows that CARSs have a crucial role in endogenous CysSSH production and suggests thatthese enzymes serve as the principal cysteine persulﬁde synthases in vivo. CARSs also
catalyze co-translational cysteine polysulﬁdation and are involved in the regulation of
mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulﬁdeproduction may thus clarify aberrant redox signaling in physiological and pathophysiologicalconditions, and suggest therapeutic targets based on oxidative stress and mitochondrial
dysfunction.

1 Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.
2 Department of Molecular Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan. 3 Division of
Cardiocirculatory Signaling, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, National Institutes of Natural Sciences,
Okazaki 444-8787, Japan. 4 Department of Translational Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka
812-8582, Japan. 5 Environmental Biology Section, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan. 6 Department of Gene Expression
Regulation, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan. 7 Department of Biological Science, Graduate School of
Science, Osaka Prefecture University, Osaka 599-8531, Japan. 8 Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University,
Kumamoto 860-8556, Japan. 9 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan. 10 Division ofCancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori 981-1293, Japan. 11 Laboratory of Pharmacology, Showa Pharmaceutical University,
Tokyo 194-8543, Japan. 12 Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan. 13 Department of Molecular
Immunology and Toxicology, National Institute of Oncology, Budapest 1122, Hungary. 14 Clinical and Experimental Sciences, Faculty of Medicine, University ofSouthampton, Southampton General Hospital and Institute for Life Sciences, Southampton SO16 6YD, UK. 15 Department of Chemistry, Sonoma State
University, Rohnert Park, CA 94928, USA. Tomoaki Ida, Fan-Yan Wei, Motohiro Nishida and Yoshito Kumagai contributed equally to this work.
Correspondence and requests for materials should be addressed to T.A. (email: takaike@med.tohoku.ac.jp)

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| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-yCysteine hydropersulﬁde (CysSSH) is found physiologicallyin prokaryotes, eukaryotic cells, and mammalian tissues1,2.
Previously, we unequivocally veriﬁed the presence ofremarkable amounts of CysSSH, glutathione persulﬁde (GSSH),
and longer chain sulfur compounds (polysulﬁdes, including CysS/
GS–(S)n–H) in cultured cells and tissues in vivo in mice andhumans3–6. The chemical properties and abundance of these
species suggest a pivotal role for reactive persulﬁdes (i.e., compounds containing an—SSH group) in cell-regulatory processes.
Researchers proposed that CysSSH and related species can behave
as potent antioxidants and cellular protectants, and may function
as redox signaling intermediates3–10. Persulﬁdes are also essential
structural components of several proteins and enzymes, e.g. serving as metal ligands in iron-sulfur clusters (or sulﬁde donors)
and in iron-cysteine and zinc-cysteine complexes11–15. In fact, theexistence of a cell reservoir for sulfane sulfur (sulfur-bonded
sulfur atoms with six electrons), including low-molecular-weight(LMW) and protein-bound cysteine polysulﬁdes, has long been
known1,3–7,15,16. Thus, although the prevalence of endogenous
polysulﬁdes is clearly established and their biological relevanceincreasingly being recognized, the chemical biology and physiological functions of these species are not known with any certainty. Current dogma holds that persulﬁde/polysulﬁde formationarises as a result of hydrogen sulﬁde (H2S) oxidation3,4,7–9 or
chemical reaction with nitric oxide3,17. Two H2S-generating
enzymes involved in sulfur-containing amino acid metabolism—
cystathionine γ-lyase (cystathionase, CSE) and cystathionine βsynthase (CBS)—can catalyze CysSSH biosynthesis using cystine(CysSSCys) as a substrate3,4,6–10,18–21. However, the observed Km
is high, and both cells and mice lacking CSE and/or CBS still
display appreciable levels of CysSSH20–24, which suggests thepossibility that alternative processes may be responsible for
endogenous persulﬁde production. Thus, it appears that otherbiosynthetic routes of CysSSH formation exist that have yet to be
identiﬁed.
This study reveals that cysteinyl-tRNA synthetases (CARSs), in
addition to their canonical role in protein translation, act as theprincipal cysteine persulﬁde synthases (CPERSs) in vivo. CARSs
play a novel and prominent role in endogenous production ofboth LMW polysulﬁdes and polysulﬁdated proteins that areabundantly detected in cells and in mice. Notably, CARS2, amitochondrial isoform of CARS, is involved in mitochondrial
biogenesis and bioenergetics via CysSSH production.
Results
Redox property of cysteine and protein polysulﬁdes. CysSSH
has unique redox-active properties that distinguishes it from thecysteine (CysSH) thiol. In evaluating the physiological rationale
for biological CysSSH production, our present study conﬁrmedthat cysteine persulﬁde/polysulﬁdes (CysSSH/CysS–(S)n–H) possess mixed sulfur reactivity—both nucleophilic and electrophilic(Supplementary Figs 1 and 2)—a property that is unique anddistinct from that of other simple biologically relevant thiols. The
dual electrophilic-nucleophilic character of hydropersulﬁdes iswell documented (the anionic RSS− species being nucleophilic
and the protonated RSSH species possessing electrophilic properties akin to disulﬁdes, RSSR)25–27. Moreover, dialkylpolysulﬁdescan also be nucleophilic and electrophile-mediated cleavage of S-S
bonds is established28. The unique properties and reactivity ofpolysulﬁdes allowed us to develop several analytical techniques
aimed at determining endogenous production of LMW andprotein-bound polysulﬁdes (Supplementary Fig. 3). We ﬁrst
developed a convenient method for selective detection of polysulﬁdated proteins: the biotin-polyethylene glycol (PEG)-conjugated maleimide (biotin-PEG-MAL) labeling gel shift assay
2(PMSA; Supplementary Fig. 3a)15. PMSA demonstrated extensive
protein-bound cysteine polysulﬁdation (Supplementary Fig. 4),
not only for recombinant proteins, prepared in an Escherichia coli
cell expression system (Supplementary Table 1) but also for
endogenous proteins expressed in mammalian cells.
We then used liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS) with β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) as a trapping agent to
identify and precisely quantify various hydropolysulﬁdes, andalso to verify the site speciﬁcity of polysulﬁdation as well as thenumber of sulfur atoms involved in proteins (Supplementary
Fig. 5, and Supplementary Table 2). We chose HPE-IAM for the
LC-ESI-MS/MS analyses, as described recently6 because of its
mild electrophilicity that ensures speciﬁc labeling of hydropolysulﬁdes to form stable adducts without appreciable artifactual
decay related to their dual nucleophilic and electrophilic character(Supplementary Fig. 2). In fact, we quantiﬁed CysS–(S)n–H
formed in alcohol dehydrogenase 5 (ADH5) and glyceraldehyde3-phosphate dehydrogenase (GAPDH) by LC-MS/MS analysis,
after pronase digestion of the HPE-IAM-labeled proteins, which
revealed that more than 70% of cysteine residues werepolysulﬁdated (Fig. 1a and Supplementary Fig. 6), a result
consistent with the PMSA proﬁle alluded to above (Supplementary Fig. 4). The treatment of ADH5 with N-ethylmaleimide(NEN) indeed completely abrogated the HPE-IAM labeling of
CysSH and CysSSH/SSSH as evidenced by LC-ESI-MS/MS
analysis shown in Supplementary Fig. 6b. This data indirectly
supports the electrophilic decomposition of protein-boundcysteine polysulﬁdes induced by a strong electrophile NEM.
Additional LC-quadrupole (Q)-time-of-ﬂight (TOF)-MS analyses
identiﬁed sites of polysulﬁde formation and the sulfur chain
length in each protein (Supplementary Fig. 7).
Protein polysulﬁdation induced by cysteinyl-tRNA synthetase.
Because such extensive protein polysulﬁdation is unlikely to occur
effectively by simple chemical means3,4,7–10, we hypothesized that
CysSSH and CysS–(S)n–H may be incorporated during proteintranslation. To evaluate this hypothesis, we analyzed the incorporation of CysSSH/CysS–(S)n–H into tRNA via cysteinyl-tRNA
synthetase (CARS) from E. coli (EcCARS) by using synthetic
CysS–(S)n–H and LC-MS/MS analyses (Supplementary Fig. 8).
We observed effective production of CysSSH-bound tRNA (CystRNACysSSH), which indeed suggests translational incorporationof CysSSH/CysS–(S)n–H into proteins. Unexpectedly, we identiﬁed extremely high levels (>80% of total cysteine residues) oftRNA-bound cysteine persulﬁde, trisulﬁde, and even tetrasulﬁde,when using simple (native) cysteine with EcCARS (Fig. 1b andSupplementary Fig. 9). As an important result, these cysteine
polysulﬁdes bound to tRNA were effectively incorporated into
nascent polypeptides, which is synthesized de novo in the ribosomes (Fig. 1c), as veriﬁed by a modiﬁcation of the puromycinassociated nascent chain proteomics (PUNCH-P) method29, heretermed PUNCH-PsP, PUNCH for Polysulﬁde Proteomics (Supplementary Fig. 10). This PUNCH-PsP analysis allowed us toobtain speciﬁc and selective identiﬁcation of the intact forms of
CysS–(S)n–H residues in the nascent peptides of GAPDH present
only within the ribosomes of E. coli, as Supplementary Fig. 10ashows. We clearly identiﬁed high degrees of polysulﬁdation
occurring at the 247Cys residue of the mature GAPDH protein
expressed and synthesized in E. coli. All native forms of CysSH,
CysSSH, and CysSSSH residues were efﬁciently recovered fromthe native whole GAPDH protein and the extension of polysulﬁdation reached more than 60% of the 247Cys residue ofmature protein (Supplementary Fig. 10e). All these rigorous LCQ-TOF analyses unambiguously revealed that extensive and

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-yacPolysulfidation of nascent GAPDH (C247) in ribosomes
247
247
SSH
SH

CysS–(S)n–H in ADH5300(m/z, 737.32)

CysSSSH(CysSSS-HPE-AM)

250(m/z, 753.32)

247 CysSH

200(m/z, 769.32)

Nascent
GAPDH
in ribosomes
CysSSSH
26.2%

247 CysSSSH

247 CysSSH

CysSH
29.3%

150
CysSSH(CysSS-HPE-AM)100

5.0 × 105counts

5.0 × 105counts

5.0 × 105counts

50
0

3.5
4.0
4.5
5.0 3.5
4.0
4.5
5.0 3.5
4.0
4.5
5.0
Retention time (min)Retention time (min)Retention time (min)CysSH CysS-(S)n-H

b

CysSSH
44.5%

d

CysS–(S)n–tRNA synthesis by EcCARS
HPE-IAM +O heat, pH 8.0
EcCARS
CysS–(S)n –CysSHtRNA O
HPE-AM+ tRNA
H2N (S)n H tRNAHO

CysSSSH(CysSSS-HPE-AM)

40

20
CysSSH(CysSS-HPE-AM)
CysSH CysS–(S)n–H

50 μg/mlO
EcCARS HO

HPE-IAM

H2N (S)n-H
CysS–(S)n–H

H2N SH
CysSH

CysS–(S)n–HPE-AM

100 μg/ml
200 μg/ml

CysSSH

4

60

0

O

81%
CysSSSSH(CysSSSS-HPE-AM)

EcCARS

EcCARS forming CysS–(S)n–H

CysSS-HPE-AM (μM)

80
CysS–(S)n–HPE-AM (nM)

247
SSSH

66.0%

3
2
1
0

CysSH
50 μM
100 μM
50 μM
100 μM
50 μM
100 μM

CysSSSH

0.6
CysSSS-HPE-AM (μM)

CysS–(S)n–HPE-AM (nM)

350

0.5
0.4
0.3
0.2
0.1
0

0

15
30
45
Reaction time (min)

60

0

15
30
45
Reaction time (min)60

Fig. 1 Formation of cysteine persulﬁde (CysSSH) and CysS–(S)n–H in proteins and their biosynthesis by EcCARS. a Quantitative identiﬁcation by LC-MS/
MS analysis of CysS–(S)n–H formed in recombinant ADH5 after pronase digestion of the HPE-IAM-labeled protein. b Formation of cysteine (CysSH) and
CysS–(S)n–H on tRNA (Cys-tRNACysS–(S)n–H) as identiﬁed by HPE-IAM labeling LC-MS/MS analysis, which determined the amounts of CysSH and CysS–(S)n–H released from Cys-tRNACys and Cys-tRNACysS–(S)n–H synthesized in the EcCARS enzymatic reaction after their heat or alkaline treatment. The
method employed is illustrated in the upper panel. c GAPDH cysteine polysulﬁdes are formed and incorporated into nascent polypeptides synthesized
de novo in ribosomes, as identiﬁed by PUNCH-PsP (Supplementary Fig. 10; cf. Supplementary Fig. 6). d CysS–(S)n–H formation from cysteine,
catalyzed by EcCARS, as dependent on enzyme and substrate (cysteine) concentrations and reaction time (lower panel). Schematic representation of
the EcCARS-catalyzed reaction (upper panel). HPE-AM, β-(4-hydroxyphenyl)ethyl acetamide; HPE-IAM, β-(4-hydroxyphenyl)ethyl iodoacetamide.
Data a, b are means ± s.d. (n = 3)

prevalent cysteine polysulﬁdation is introduced co-translationallyand sustained in the mature protein physiologically present evenin the post-translational processes of the cells.
Consistent with these ﬁndings, EcCARS itself appeared to havestrong catalytic activity for generating CysS–(S)n–H (CysSSH and
CysSSSH) from the natural substrate cysteine (Fig. 1d). The
persulﬁde synthase activity of EcCARS depended partly on added
pyridoxal phosphate (PLP) (Fig. 2a) but not on ATP and tRNA:the latter two being required for Cys-tRNACys biosynthesis by
EcCARS. Persulﬁde generation by EcCARS was enantioselective,
because only L-cysteine but not D-cysteine demonstrated activity,
which ruled out nonspeciﬁc post-translational persulﬁdation.
Furthermore, we performed a stable isotope (34S) tracer
experiment combined with LC-MS/MS-based HPE-IAM assay
to clarify the catalytic mechanism of cysteine polysulﬁdation by
EcCARS (Supplementary Fig. 11). Speciﬁcally, by means of LCMS/MS analysis for the enzymatic reaction with stable isotope(34S)-labeled cysteine as a substrate, we found that EcCARS
catalyzed the cleavage of a sulfur atom from one cysteine and its
transfer to another cysteine to form CysSSH, as Supplementary
Fig. 11a illustrates.
NATURE COMMUNICATIONS | 8: 1177

Identiﬁcation of CARSs as CPERSs. Kinetic analyses conﬁrmed
that, because of a very low Michaelis constant Km and high catalytic rate constant kcat, EcCARS is very efﬁcient in producing
CysSSH, i.e., functioning as a CPERS, with a high afﬁnity forcysteine (Supplementary Fig. 12 and Supplementary Table 3), in
particular when compared with the kinetic parameters of otherenzymes such as CSE (Supplementary Table 3)7,21. Although the
kcat/Km value is almost equal to values of EcCARS, CSE, and CBS
utilize only cystine (but not cysteine) as a substrate, which is quitedistinct from CARSs that use cysteine (but not cystine) for
CysSSH production3. In addition, because the intracellular cystinecontent range is physiologically at low micromolar or submicromolar concentrations, which are far lower than the Km
value of CSE (more than 200 μM), CSE cannot directly utilize
cysteine for persulﬁde production. Also, the cystine/CSE reaction
may not compete successfully with the reactions with otherenzymes metabolizing cystine and substance such as glutathione,
which exists abundantly in cells and thus readily interacts withcysteine under physiological conditions. The intracellular cysteine
concentration is reportedly 100–1000 μM in cells and majororgans3, which is much higher than the Km of CARS. These| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

e

100

60
30
Reaction time (min)C28

CysSS-HPE-AM (μM)

43

ALDH1A1
ADH5
GAPDH

32
29

ETHE1

K73/76 (KIIK)

C209

C28/209D

C209D

C28D

C28/209S

ALDH1A1
ADH5
GAPDH

43
32
29

ETHE1

K266/269 (KMSK)
Anticodon binding

Zn2+

PLP

C209S

72
53

Catalytic
Zn2+

C28S

WT

K266/269A

K73/76A

(kDa)

53

50

0

C28D
C209D
C28/209D

(kDa)
72

150

0.5

WT
C28S

*

200

1.0

EcCARS (–)

*

250

1.5

f
K266/269A

d
K269A
K73/76A

PLP (–)PLP (+)

K269A

WT

30
60
Reaction time (min)****** ***

K76A

***

K266A

0.5

EcCARS(–)

CysSSS-HPE-AM (nM)

***
***

0

300

0

1.0

K73A

*

CysSSH from EcCARS(WT/mutants)

2.0

1.5

WT
K73A

*

CysSSSHb

PLP (–)PLP (+)

2.0
CysSS-HPE-AM (μM)

CysSS-HPE-AM (μM)

CysSSH
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

CysSSH from EcCARS(WT/mutants)

C209S
C28/209Sc
EcCARS

K76A
K266A

a

28

PLP
73

76

KIIK

209

266 269

KMSK

Fig. 2 CysS–(S)n–H biosynthesis catalyzed by EcCARS and its various mutant EcCARSs. a CysS–(S)n–H (CysSSH and CysSSSH) biosynthesis from cysteine
catalyzed by EcCARS as a function of reaction time and the presence or absence of PLP. CysS–(S)n–H production was analyzed by using the HPE-IAM
labeling with LC-MS/MS analysis for the reaction of recombinant EcCARS (200 μg/ml) with 100 μM cysteine in the presence or absence of 50 μM PLP.
The data are means ± s.d. (n = 3). *P < 0.05. b General structure (upper panel) and conserved amino acid alignments (lower panel) of bacterial, human,and rodent CARSs. c, e Enzyme activities of EcCARS lysine (K) mutants c and cysteine (C) mutants e to form CysSSH. WT and EcCARS K and C mutants,
200 μg/ml each, reacted with 25 μM cysteine at 37 °C for 30 min. Data represent means ± s.d. (n = 3). ***P < 0.001. The enzyme activity of EcCARS Lys(d) and Cys (f) mutants was assessed by the PUREfrex assay with the cell-free translational reactions for ALDH1A1 (55 kDa), ADH5 (40 kDa), GAPDH(36 kDa), and ETHE1 (28 kDa), with protein syntheses being identiﬁed by western blotting

biochemical reports, therefore, strongly suggest that CARS canfunction as a major source of CysS–(S)n–H generation underphysiological conditions.
Investigation of EcCARS PLP-binding sites with LC-Q-TOFMS analysis and Mascot data searches indeed revealed that lysine(K) residues, including 73KIIK76 and 266KMSK269 motifs, boundto PLP (Supplementary Fig. 13). The sequence data showed thatseveral Lys residues, especially at the KIIK and KMSK motifs, areconserved in EcCARS and other homologues from different
organisms, including mammals (Fig. 2b and Supplementary
Fig. 14). Also, conserved two cysteine residues bound to the active
center Zn2+ (Fig. 2b and Supplementary Fig. 14). To clarify thefunction of PLP bound to EcCARS, we constructed a series of Lys
mutants of this enzyme (Supplementary Table 4) and measured
enzyme activities in terms of persulﬁde, i.e., CysS–(S)n–H,
formation and protein synthesis or translation. We observed,
via the HPE-IAM labeling LC-MS/MS analysis, a marked
decrease in CysSSH and CysSSSH synthesis, compared with thewild type (WT), for various Lys to Ala mutants at K73A, K76A,
K266A, K269A, and double mutants K73/76A and K266/269A of
EcCARS (Fig. 2c), all of which had intact protein synthesis
4potential as assessed by the PUREfrex cell-free protein synthesis
assay (Fig. 2d). We also quantiﬁed the amounts of PLP bound to
EcCARS by LC-ESI-MS/MS using 2,4-dinitrophenylhydrazine(DNPH). The DNPH-labeling LC-MS/MS analysis indicated thatthe amounts of PLP bound to WT EcCARS and four different Lys
mutants correlated well with their CPERS (persulﬁde producing)
activities (Supplementary Fig. 13b). In contrast, cysteine toaspartate mutants such as C28D (also C28S) and the double C28/
209D mutant still maintained high persulﬁde production, similar
to that of the WT cells (Fig. 2e), albeit their protein synthesis andtranslational activity were strongly attenuated (Fig. 2f).
Our computational modeling of the three-dimensional structure of EcCARS supported PLP binding to the particular Lys
residues at the 73KIIK76 and 266KMSK269 motifs of EcCARS(Fig. 3a). The present computational simulation predicts two
potential PLP-binding sites at K73 and K269 of KIIK and KMSK
motifs. Also, this modeling revealed that PLP-bound motifs havea vicinal location within 10–20 Å distance but apparently distinct
from both the ATP-binding HIGH motif and the Zn2+-binding
active site of the EcCARS for Cys-tRNACys biosynthesis. A
commensurate change in the binding capacity and/or stability of

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-yabc
Mouse CARS1

C209
C28

Zn
Cysteine

2.0
1.6
1.2
0.8
0.4
0

*

PLP (–)PLP (+)*

Human CARS2
HPE-IAM
adducts (μM)

HPE-IAM
adducts (μM)

EcCARS

CysSSH CysSSSH

PLPCysSSH
from human CARS2K188(KMSK)K73(KIIK)

HIGH
motif

K76
(KIIK)K269(KMSK)

e

1.6
1.2
0.8
0.4

+ 100 μM PLP+ 50 μM PLP+ 10 μM PLP
25 μM CysSH alone00 30 60 90 120
Reaction time (min)PLP-DNPH
adduct (μM)

PLP

K266
(KMSK)dK185(KMSK)CysSS-HPE-AM(μM)

Adenosine

2.0
1.6
1.2
0.8
0.4
0

*

PLP (–)PLP (+)*CysSSH CysSSSH
PLP bound tohuman CARS2

18
16
14
12
10
8
6
4
2
0

0 10 50 100
PLP treatment (μM)

Fig. 3 Computational modeling of EcCARS structure, and CysS–(S)n–H biosynthesis by CARS1/2. a A molecular docking model of PLP-bound EcCARS
generated by SwissDock using the crystal structure of EcCARS (PDB ID: 1LI5). Cysteinyl-tRNA is placed by superimposing the crystal structure of the
EcCARS-Cysteinyl-tRNA binary complex (PDB ID: 1U0B) to the docking model. b, c PLP-dependent CysSSH and CysSSSH biosynthesis by mouse
CARS1 and human CARS2. CysSSH and CysSSSH production was quantiﬁed by means of HPE-IAM labeling LC-MS/MS analysis in the reaction ofrecombinant mouse CARS1 and human CARS2 (200 μg/ml each) with 25 μM L-cysteine in the presence or absence of 100 μM PLP (37 °C, 2 h). The data
are means ± s.d. (n = 3). *P < 0.01. d Concentration-dependent effects of PLP on CysSSH and CysSSSH production by recombinant human CARS2. HumanCARS2 (200 μg/ml) reacted with 25 μM cysteine in the presence of 0, 10, 50, or 100 μM PLP at 37 °C for 30–120 min. No appreciable cysteine persulﬁdeproduction was detected in the reaction mixture of cysteine and PLP alone as long as no >100 μM PLP was used. e Precisely quantitative analysis for PLP
bound to human CRAS2. Human CARS2 treated with various concentrations of PLP (d) at 37 °C for 1 h was reacted with DNPH to form PLP-DNPH adduct,
followed by quantiﬁcation by LC-ESI-MS/MS analysis

PLP seems to exist, caused by the mutation of any one of four Lys
residue among four Lys residues because each single Lys mutation
at the KIIK and KMSK motifs greatly affected all CysS–(S)n–H
synthesis activity of EcCARS (Fig. 2c). One possible explanation
for the commensurate effect is that PLP may need multiple Lys
residues, rather than a single Lys binding, to exhibit stablebinding and full catalytic activity of CARS to function as CPERS
during CysS–(S)n–H formation. That is, for their stable binding
and catalytic activity, PLP-dependent catalytic activity may needstabilization by a multiple Lys binding, because CysSSH produced
by CARS, due to its highly nucleophilic nature, may readilyinterfere with the electrophilic aldehyde group of PLP to form an
imine (Schiff base) linkage on the Lys residues, which would
cause instability of the catalytic activity of PLP bound to theseparticular Lys residues of CARS. This interpretation receives
support from by the aforementioned computational structural
analysis showing the close localization (in 20 Å) of these Lys
residues at KIIK and KMSK motifs (Fig. 3a). Together these datasuggest that EcCARS is indeed an efﬁcient CPERS enzyme withindependent catalytic functions in aminoacyl-tRNA biosynthesis.
CARS2 functions as a CPERS conserved in mammals. Two
different CARSs exist in mammals: CARS1 (cytosolic) andCARS2 (mitochondrial)30–32. Both CARSs (mouse CARS1 andhuman CARS2, which we tested herein) had strong CysS–(S)n–H
producing activities, which depended on the presence of PLP(Fig. 3b–d). Also, a very nice correlation was found between the
CPERS activity and PLP content of CARS2 containing varied
amounts of PLP incorporated after treatment with different
concentrations of PLP (Fig. 3e). To clarify how much cellular
CysS–(S)n–H originated from CARS1 and CARS2 in human cells,
we attempted to disrupt CARS1 and CARS2 genes in
HEK293T cells via the CRISPR/Cas9 system in HEK293T cells.
We could not obtain CARS1-knockout (KO) cells, but we successfully established CARS2 KO cells. We selected one of theclones, carrying a 30-bp deletion plus an 8-bp insertion justdownstream of the translation-initiating codon in the CARS2 ﬁrst
NATURE COMMUNICATIONS | 8: 1177

exon, was selected for LC-MS/MS analysis (Supplementary
Fig. 15). CysS–(S)n–H and GSSH levels decreased signiﬁcantly in
CARS2 KO cells (Fig. 4a, b), which suggests that CARS2 is amajor producer of persulﬁde. Because we still detected a low levelof CARS2 in CARS2 KO cells (Fig. 4c), we also treated the cellswith siRNA against CARS2, which resulted in the 67 and 42%
decreases in CysSSH and GSSH levels, respectively (Fig. 4a, b).
When we knocked down CARS1 in CARS2 KO cells, CysSSH
decreased only marginally, which suggests a predominant role of
CARS2 in the production of CysSSH. Immunoblot analysis andimmunostaining veriﬁed the reduced CARS2 and CARS1 protein
levels in CARS2 KO cells and in cells with CARS1 or CARS2
siRNA (Fig. 4c and Supplementary Figs 16 and 17).
Markedly reduced persulﬁde formation in CARS2 KO cells wasrecovered by adding back WT CARS2. CARS2 C78/257D mutant
rescued the persulﬁde production of CARS2 KO cells, but K124/
127A, and K317/320A mutants (mutants of KIIK and KMSK
motifs, respectively), did not (Fig. 4d, e). The CARS2 KO cells had
a markedly decreased Cys-tRNA synthetase activity, and again
adding back the C78/257D mutant resulted in lost Cys-tRNA
synthetase activity, as assessed by the expression of mitochondrial
cytochrome c oxidase subunit 1 (MTCO1 encoded by mitochondrial DNA), but still retained full CPERS activity; conversely,
K124/127A and K317/320A mutants had impaired CPERS
functions but retained Cys-tRNA synthetase activity (Fig. 4f, g).
These results clearly verify that CARS2 truly functions as a
CPERS in mammals and that this function is separate fromcysteinyl-tRNA synthetase activity.
We also evaluated the potential contribution of CSE and CBS
to the endogenous persulﬁde production in HEK293T cells.
Silencing of CSE and CBS suppressed the persulﬁde production,
but notably, intracellular cysteine (CARS substrate) levels weresigniﬁcantly decreased (Supplementary Fig. 18). In CARS2 KO
cells, knockdown of CSE and CBS also reduced cysteine levels but
not persulﬁde production (Supplementary Fig. 18). Therefore,cysteine production is dependent on both CSE and CBS, and thus
cysteine is provided via the metabolic pathways mediated by CSE/
CBS in each cell line irrespective of CARS2 expression. In| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

5

CARS2

Mock

GAPDH

1.0

Mock

***

***

0.5
0

K317/320A

CARS2 KO

K317/320A

WT
C78/257D
K124/127A

WT
C78/257D
K124/127A

WTSDHA
MTCO1/SDHA

Relative intensity(arbitrary unit)

K317/320A

K124/127A

32
C78/257D

CARS1

Relative intensity(arbitrary unit)

Mock
WT

K317/320A
CARS2

72
53

43

5
0

MTCO1

32

C78/257D

53

10

(kDa)

WT

15

CARS2 KO

K124/127ACARS2 KO

K317/320A

K124/127A

C78/257D

WT

Mock

0

WT

5

**

WT

10

WT

g

(kDa)
72

20

Mock

15

**

WT

GSS-HPE-AM(pmol/mg protein)

**

***
***
***
*

1.0
0.5
0

CARS2 KO

N.S.
N.S.

25

CARS2/β-actin

siRNA

f
30

**

20

β-Actin

GSSH in cells

CysSSH in cells
N.S.
N.S.

25

CARS2

siRNA
CARS2 KOCARS2 KO

e
30

CARS1

WT

siRNA
CARS2 KO

WT

Mock

siRNA
CARS2 KO

***

CARS2

10

WT

CARS1 CARS2
1 2 1 2

CARS1/β-actin
1.0
0.5
0

Mock

CARS2: 42%

N.S.

15

0

d

CysSS-HPE-AM(pmol/mg protein)

20

CARS2

WT

0

CARS1

5

(kDa)
124
90
72
53
72
53
43
72
53
43
32

N.S.

CARS2

10

*

CARS1

N.S.

siRNA

Mock

15

*

25

WT

N.S.

GSS-HPE-AM(pmol/mg protein)

20

CARS2 KO*30

CARS2: 67%

**

25

**

Mock

CysSS-HPE-AM(pmol/mg protein)

30

**

CARS1

c
GSSH in cells

Relative intensity(arbitrary unit)b
CysSSH in cells

Mock

a

CARS2 KO

Fig. 4 Endogenous formation of persulﬁdes in HEK293T cells. Intracellular levels of CysSSH (a) and GSSH (b) in WT and CARS2 KO cells with CARS1 orCARS2 knocked down. Data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01; N.S., not signiﬁcant. c CARS1 and CARS2 Western blotting for cells used ina and b. Lane 1 and 2, duplicate determinations with each siRNA. The right panel shows the densitometric analysis for the western blot shown in the rightpanel. The data are means ± s.d. (n = 3). ***P < 0.001. Production of CysSSH (d) and GSSH (e) in CARS2 KO cells with WT or CARS2 C and K mutants
added back. The data are means ± s.d. (n = 3). **P < 0.01; N.S., not signiﬁcant vs. CARS2 KO mock. f CARS2 western blotting for WT and CARS2 KO cellswith WT or CARS2 C and K mutants added back. g Western blotting for the cells in d and e with different mitochondrial proteins: MTCO1, mitochondrial
cytochrome c oxidase subunit 1 (encoded by mitochondrial DNA) and SDHA, succinate dehydrogenase complex ﬂavoprotein subunit A (encoded bygenomic DNA). Supplementary Fig. 16 provides full blot images. The lower panel shows the densitometric analysis for the western blot. The data aremeans ± s.d. (n = 3). ***P < 0.001

addition, almost two thirds of CysSSH seems to be supplied by
CARS2 in HEK293T cells based on the decrease by almost two
thirds in the CysSSH levels. The rest of CysSSH in the CARS2 KO
cells were not derived from CSE/CBS expressed in
HEK293T cells, since no further reduction of CysSSH was
obtained even by CSE/CBS knockdown in CARS2 KO cells. These
results suggest that CSE and CBS do not contribute directly topersulﬁde production but rather may promote the biosynthesis ofcysteine and its supply to CARS, at least in this cultured cell
model under physiological conditions.
To further clarify CPERS functions of CARS2 in vivo, we
generated the Cars2-deﬁcient mice by using CRISPR/Cas9
technology. As Fig. 5 illustrates, a guide RNA (gRNA) was
designed against exon 1 of Cars2. We established a mutant mouseline with a mutant Cars2 allele (line 1) that had a 200-bp deletioncontaining a translation-initiating codon in exon 1 (Fig. 5a, b).
Mating of F1 Cars2 heterozygous KO (Cars2+/−) mice produced
WT and Cars2+/– mice, but not homozygous mice (viable
offsprings included 20 WT mice and 19 Cars2+/– mice), whichsuggests that Cars2–/– mice are embryonic lethal. Cars2+/– mice
were normally born without any apparent abnormalities inmacroscopic appearance or growth proﬁles during the observation period of at least 6 months after birth, but they demonstrated
reduced mitochondrial expression of CARS2 protein by half andmarked attenuation of CysSSH production; in contrast, we
6observed no appreciable change in mitochondrial DNA-encoded
MTCO1, which indicated intact Cys-tRNA synthetase activity in
Cars2+/– mice (Fig. 5c–e and Supplementary Fig. 19a). Therefore,
we quantiﬁed the sulﬁde metabolites in the liver of Cars2+/– miceand their WT littermates via LC-MS/MS analysis with HPE-IAM
as described earlier. As we expected, CARS2+/– mice showed astriking difference in persulﬁde production compared with
the WT littermates (Fig. 6a, b). Endogenous levels of CysSSH
and all other derivatives (e.g., GSSH, HS-, thiosulfate, andhydropolysulﬁdes) decreased by 50% or more in the liver andlung of Cars2+/– mouse compared with WT mice.
To exclude the possibility of off-target effects by the gRNA
used to produce line 1 Cars2+/− mice, we developed anotherstrain of Cars2+/– mice (line 2) with an alternative gRNAtargeting Cars2 exon 3. Line 2 Cars2+/– mice had phenotypes
almost identical to those of line 1 (Supplementary Figs. 20
and 21).
That heterozygous Cars2 mutant mice manifested a CysSSH
reduction by ~50% should be noted; it suggests that Cars2
contributes almost entirely to the CysSSH production in mouse
tissues under physiological conditions. As an important ﬁnding,Cars2 disruption did not alter expression levels of other sulﬁdemetabolizing enzymes, including CSE, CBS, and 3mercaptopyruvate sulfur transferase (3-MST) (Fig. 5e, Supplementary Figs. 19b, c and 21), which emphasized the sole

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-yab

500 bp

WT

Cars2

Cars2+/– M

WT
alleleKOalleleWT
Cars2 KO200-bp deletion

d

e

1.5

CARS2/
MTCO1/
SDHA 1.5 SDHA

1.0

1.0

0

WT

WT

0

*

150
100

1

*

200

*

Cars2+/–
WT
2 3 1 2 3
CARS1
CARS2
3-MST
CBS

50
0

0.5
Cars2+/–***

0.5

Cars2+/–

Relative intensity(arbitrary unit)

SDHA

*

WT
Cars2+/–

250

Mouse liver

CSE
β-Actin1
2
5
10
Mitochondria (mg/ml)

Relative intensity(arbitrary unit)

CARS2
MTCO1

300
CysSS-HPE-AM (nM)

WT
Cars2+/–
1 2 3 1 2 3CysSSH

Relative intensity(arbitrary unit)

Isolated mitochondria

Mouse
hepatocytesc1.5

CARS1/
β-actin

1.0
0.5
0

1.5
1.0

WT Cars2+/–CARS2/
β-actin

***

0.5
0

WT Cars2+/–

Fig. 5 Generation of Cars2-deﬁcient mice via the CRISPR/CAS9 system. a Schematic illustration of the mouse Cars2 gene structure and sequences of WT
and mutant alleles around the target locus. Green and black letters indicate the ﬁrst exon and intron of Cars2, respectively. The targeted locus of gRNA andprotospacer-adjacent motif (PAM) sequence were indicated in the WT sequence are indicated by underlined and bold letters, respectively. A modiﬁed
allele sequence obtained from the Cars2-edited mouse (line 1) is shown below. b Detection of mutations introduced by gRNA-Cas9 targeting Cars2 via PCR
with genomic DNA from WT and Cars2+/− mice. Cars2+/−, Cars2 heterozygous KO mice, M: DNA molecular weight marker. c Western blotting of CARS2
and mitochondrial proteins, e.g., MTCO1 and SDHA, from mitochondria isolated from the liver. The lower panel shows the densitometric analysis of thewestern blot. Data are means ± s.d. (n = 3). ***P < 0.001. d CysSSH production in mitochondria isolated from the liver of WT and Cars2+/− littermate mice.
Various concentrations of isolated mitochondria were reacted with HPE-IAM for 1 h, followed by LC-MS/MS analysis (see Supplementary Methods fordetails). Mitochondria were obtained from line 2 Cars2+/– mice (Supplementary Figs. 20 and 21). *P < 0.05, WT vs. Cars2+/– mice (two-way ANOVA).
e Western blotting of CARS1, CSE, CBS, and 3-MST with liver tissue obtained from WT and Cars2+/– mice. Supplementary Fig. 19 provides full blot images.
The right panels show the densitometric analysis of the CARS1 and CARS2 immunoblots. Data are means ± s.d. (n = 3). ***P < 0.001

contribution of CARS2 to endogenous persulﬁde biosynthesis
in vivo.
To explore the possibility that CARS2, a mitochondrial protein,can produce CysSSH and provide it to the whole cell, we isolated
mitochondria from mouse liver and measured the release of denovo-synthesized CysSSH from the mitochondria (Supplementary Fig. 22). A large fraction of CysSSH was indeed released frommitochondria, which supports the idea that CysSSH produced in
mitochondria is released into the cytoplasm and maintains
protein polysulﬁdation. As expected, CysSSH derived fromwhole-cell proteins was decreased in Cars2+/– mice, but cysteine(CysSH) did not (Fig. 7). Speciﬁcally, formation of 20–30% of
CysSSH in all cell proteins (polysulﬁdation) depended on CARS2
expression not only in the in vivo experiment using Cars2 KO
mice (Fig. 7a) but also in the in vitro cell culture study (Fig. 7b),
as identiﬁed by HPE-IAM labeling LC-MS/MS analysis with thewhole cell and tissues proteins isolated. These results suggest that
CysSSH derived from CARS2 signiﬁcantly contributes to thepolysulﬁdation of the whole-cell proteins. Because protein
polysulﬁdation appears to be mediated via post-translational aswell as co-translational processes, the former being controlled by
the thioredoxin (Trx)–Trx reductase (TrxR) system as recently
reported4, we expect that CysSSH generated in mitochondria isreleased into the cytoplasm and supplies sulfur to proteins forpolysulﬁdation (Fig. 7c). Our current evidence is the ﬁrst
demonstration that unequivocally veriﬁed in human cultured
cells and in vivo in mice that CARS2 is the major enzyme for
persulﬁde biosynthesis and thus functions as a CPERS in
mammals.
NATURE COMMUNICATIONS | 8: 1177

CARS-mediated polysulﬁdation and mitochondrial physiology.
Unexpectedly, CARS2 KO cells showed markedly altered mitochondrial morphology (i.e., shrunken or fragmented appearance),
which greatly improved when CARS2 was added back, as seen
with the MitoTracker Red ﬂuorescent mitochondrial stain (Fig. 8aand Supplementary Fig. 17c), transmission electron microscopy(Fig. 8b), and immunoﬂuorescence staining for translocase ofouter mitochondrial membrane 20 (TOMM20) and CARS2(Supplementary Fig. 17a, b). Not only WT CARS2 but also theC78/257D mutant induced a strikingly improved mitochondrial
morphology, but other Lys mutants tested did not (Fig. 8a, b andSupplementary Fig. 17c). In line with these ﬁndings, deletion of
CARS2 activated dynamin-related protein (Drp1), a major mediator of mitochondrial ﬁssion33, and Drp1 GTPase activity wassigniﬁcantly attenuated by adding back the WT CARS2 and C78/
257D mutant, thereby producing CysSSH without CARS activity,
but not by adding back the K317/320A mutant (Fig. 8c). Usually,
Drp1 in HEK293T cells was extensively polysulﬁdated (Fig. 8d),
as evidenced by our new biotin-PEG-MAL capture method(Supplementary Fig. 3b). However, Drp1 polysulﬁdation wasmarkedly suppressed by both CARS2 KO and additional CARS1/2
double-knockdown, respectively (Fig. 8d and Supplementary
Fig. 23). Because Drp1 is likely activated via chemical depolysulﬁdation or a post-translational process operated physiologically by the Trx–TrxR system, for example, we identiﬁed Drp1 asa major signal effector molecule reversibly regulated through aunique polysulﬁdation and depolysulﬁdation process (Fig. 8e).
We next examined CARS2 contribution to mitochondrial
biogenesis and function. Mitochondrial DNA normalized against| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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0

400

15
10
5

200

0.4
0

0

**

12
8
4
0

**

10
pmol/mg protein

100

HSS–
16
pmol/mg protein

200

0.8

0
HS–

WT
Cars2 +/–

4

15
10
5
0

WT
Cars2 +/–

2
0

120
100
80
60
40
20
0

**

%

10

2
0

1.0

**

0.8

3
2
1
0

GSSH/
GSH

**

0.6

WT
Cars2 +/–

0.4
0.2
0
HS2O3–HSS–
4

WT
Cars2 +/–

4

5

140
120
100
80
60
40
20
0

**

6

0

HS–

HS2O3–

8
6

20

8

GSSH

25

*

1.2

100

0

300

1.6

10

*

15

GSH

2.0

*

300
%

pmol/mg protein

pmol/mg protein

25
20

GSSH/
GSH

GSSH

GSH

20

%

4

0

WT
Cars2 +/–

25

300
250
200
150
100
50
0

**

10
pmol/mg protein

8*pmol/mg protein

20

12

30
25
20
15
10
5
0

CysSSH/CysSH

CysSSH

pmol/mg protein

40

*
%

60

16

CysSH

pmol/mg protein

pmol/mg protein

pmol/mg protein

80

Lung from WT and Cars2 +/– micepmol/mg protein

20

100

CysSSH/
CysSH
pmol/mg protein

CysSSH

CysSH

pmol/mg proteinbLiver from WT and Cars2 +/– micepmol/mg proteina

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

8
6
4

**
WT
Cars2 +/–

20

Fig. 6 In vivo formation of various sulﬁde species in WT and Cars2+/− mice. Endogenous production of CysSSH and other related polysulﬁde compounds
was identiﬁed by means of HPE-IAM labeling LC-MS/MS analysis in the liver a and lung b obtained from WT and Cars2+/− littermates (21-week-old
males). The data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01

nuclear DNA was reduced in CARS2 KO cells, which was
similarly restored by WT CARS2 and C78/257D but not by Lys
mutants (Supplementary Fig. 24a), which suggests that CARS2derived persulﬁde enhances mitochondrial biogenesis. Mitochondrial membrane potential was decreased in CARS2 KO cells, but it
recovered or even increased when the WT and C78/257D mutant
were added back or overexpressed but not when Lys mutants
were used (Fig. 8f and Supplementary Fig. 25a). We also used an
extracellular ﬂux analyzer to measure the oxygen consumption
rate (OCR) in HEK293T CARS2 KO cells. The OCR in CARS2
KO cells was ~50% of that in WT cells (Fig. 8g), consistent with
the incomplete elimination of CARS2 protein and thereby
attenuated expression of MTCO1 in CARS2 KO cells (Fig. 4g).
The decrease of OCR in CARS2 KO cells was recovered byintroduction of WT CARS2 and C78/257D mutant but not by Lys
mutants (Fig. 8g and Supplementary Fig. 25b). A novel concept
emerging from these observations is that CARS2-derived cysteine
persulﬁdes play an important role in the electron transport chain(ETC) in mitochondria, which sheds light on a completely new
and fundamental role of persulﬁdes in supporting mitochondrial
bioenergetic function.

CARS2 linked up to mitochondrial ETC. In our efforts to elucidate the mechanism of how CARS2-derived CysSSH contributes
to the mitochondrial bioenergetics function, we noticed a quite
different proﬁle of the products of human CARS2 in the cell-free
enzyme reaction compared with cellular CARS2 metabolism in
HEK293T cells in culture (Fig. 9a, b). Although CARS2 synthesized mostly CysSSH/SSSH in a cell-free solution (Fig. 3c, d),preferential formation of HS− (H2S) together with thiosulfate(S2O32−) over CysSSH was evident with HEK293T cells. We thus
hypothesized that the mitochondrial compartment is a uniquemetabolic environment in which de novo CysSSH synthesized by
8

CARS2 may be further metabolized, possibly being coupled withthe mitochondrial ETC.
To understand how the ETC function and CysSSH derived
from CARS2 are associated (Fig. 8g and Supplementary Fig. 25b),
we examined the effect of ETC suppression on the metabolic
proﬁle of CysSSH and its derivatives in HEK293T cells (Fig. 9c–h).
We then used two approaches to inhibit the ETC in the cells: one
method was to use a speciﬁc inhibitor of complex III, antimycin
A (Fig. 9c–e), and the other ETC disrupter used was ethidium
bromide to induce mitochondrial DNA deprivation (Fig. 9f–h
and Supplementary Fig. 24b; see Supplementary Methods fordetails). Both ETC suppressive treatments caused a signiﬁcant
increase in CysSSH and simultaneous reduction of HS−
production, as assessed by the HPE-IAM labeling LC-MS/MS
analysis (Fig. 9c–h). These inverse and stoichiometric relationships between CysSSH and hydrosulﬁde anion (HS−) formationstrongly suggested an ETC activity-dependent conversion of
CysSSH to HS− mediated via the ETC occurring in the cells(Fig. 9e, h). We interpret these results to mean that CysSSH
derived from CARS2 in mitochondria is effectively reduced byaccepting an electron from the ETC to release HS− (H2S), as
Fig. 9i illustrates.
These data thus provide robust support for the idea that the
CARS2-CysSSH pathway is involved in the mitochondrial
function because CARS2-dependent CysSSH production isfunctionally integrated into and tightly linked to the mitochondrial ETC, which is in turn involved in the energy metabolism, as
Fig. 10 illustrates. In fact, low (nM) concentrations of H2Sreportedly sustained the ETC function possibly mediated by
sulﬁde:quinone reductase and other potential enzymes thatoxidize sulﬁdes to thiosulfate (S2O32−)7,34–38. How H2S issupplied endogenously in mitochondria remained unclear,
however. Our earlier and current studies suggest that CSE, CBS,and 3-MST are not major sources of H2S in mitochondria in

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-yaLiver from WT and Cars2 +/– mice
CysSSH

4
3
2
1
0

b

10

400

**

8

300
200

c

6

WT
Cars2 +/–

4

100

2

0

0

Mitochondria

100
0Protein

CysSSH/CysSH
12
*
10
%

8
6
4

SH
Protein polysulfidation
S-(S)n-H

19%

*

300
200

CARS2

CysSSHCysSSH400
pmol/mg protein

4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0

CysSH

CysSSH

HEK293T cells
CysSH

nmol/mg protein

CysSSH/CysSH
12

**

%

5

pmol/mg protein

nmol/mg protein

500

30%

CysSH
6

SH
WT
CARS2 KO

Trx/TrxR system
HS–/HSn–20

Fig. 7 Endogenous protein polysulﬁdation in vivo and in HEK293T cells. The amounts of CysSSH formed in whole cell protein recovered from the mouse
livers of WT and Cars2+/− (line 1) 21-week-old male littermates (a) and from WT and CARS2 KO HEK293T cells (b) were quantiﬁed by using HPE-IAM
labeling LC-MS/MS analysis. Data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01. c Schematic drawing of the mechanism of the extramitochondrial releaseof CysSSH into the cytosol, which may regulate whole cell protein polysulﬁdation

various mammalian cell lines and in mice in vivo (Fig. 5e andSupplementary Fig. 18)7,20–24. In this context, our study is theﬁrst to verify that HS− (or H2S) is indirectly formed from CARS2
via CysSSH generation in the mitochondrial environment (Figs. 9iand 10). Moreover, our recent study determined that CysSSH
contributed to the endogenous formation of iron-sulfur clusters14. Because iron-sulfur clusters are known to be synthesized
and utilized in complexes I-III of the ETC in mitochondria39, andare actively transported extramitochondrially, the CysSSHdependent HS− metabolism may be coupled with the generationof iron-sulfur centers of the mitochondrial ETC and cytosolic
formation and maintenance of various iron-sulfur complex
machineries as well. Our reasonable conclusion is, therefore, thatCARS2 functions as a major CPERS, which in turn promotes
mitochondrial biogenesis and bioenergetics (Fig. 10).
Discussion
Until now, endogenous persulﬁdes were thought to be formed asa result of H2S/HS− oxidation via post-translational processes,
and serve as protein cysteine thiol-bound intermediates ofdetoxiﬁcation enzymes3,7,21, and as metal ligands for iron andzinc complexes11–15. While CSE and CBS can catalyze CysSSH
biosynthesis by using cystine as a substrate3,4,6–10,18–21, several
cells and tissues without CSE/CBS expression and CBS/CSE KO
mice reportedly synthesized appreciable amounts ofpersulﬁdes3,20,22–24, but the source of the persulﬁdes (polysulﬁdes) or the sulfane sulfur reservoir has remained elusive. We
here demonstrate that CARSs catalyze CysS–(S)n–H formationfrom cysteine and co-translational protein polysulﬁdation. Also,
CSE and CBS may still play a major role in the CysSSH production via the direct catalytic reaction using cystine as thesubstrate especially under pathophysiological conditions associated with oxidative and electrophilic stress, where intracellularcystine concentrations are considerably approaching the high Km
value of CSE3,7,21,40–42.
The second, even more crucial, ﬁnding is that the mitochondrion is a key cellular compartment for the formation and actionof CysSSH and CysS–(S)n–H. Notably, CysSSH is mostly
NATURE COMMUNICATIONS | 8: 1177

generated by CARS2 localized in the mitochondria and is released
extramitochondrially into the cytoplasm so that it can effectively
produce CysS–(S)n–H and protein polysulﬁdation in whole-cellcompartments. The current study established that CARS2-derived
CysSSH (CysS–(S)n–H) indeed sustains mitochondrial biogenesis
and the ETC function. While the implications of these ﬁndings
await further investigation, a recent clinical study by Coughlin
et al. documented an intriguing result: CARS2 mutations identiﬁed in a patient were associated with ETC impairment andmitochondrial dysfunctions31. Although the patient’s clinicalsymptoms resulted from loss of a canonical function of CARS2,
which the neurological disorders might be caused by impairmentof CPERS activity of CARS2 is plausible, and thus this impaired
activity may overlap with the observed impairment of Cys-tRNAaminoacylation.
The nature of sulfane sulfur or polysulﬁdes has continued to bea puzzle for a long time, because of a complicated polysulﬁdechemistry with dual electrophilic and nucleophilic characteristics.
Previous reports demonstrated the ability of a trisulﬁde species toreact with numerous electrophiles. For example, Fletcher andRobson reported that thiocystine (cystine trisulﬁde, CysSSSCys)readily reacted with electrophilic halogens (e.g., Br2), which
resulted in cleavage of the S–S bond25. A review by Parker andKharasch also discussed numerous examples of the electrophiliccleavage of the S–S bond in disulﬁdes by electrophilic reagents
such as protons, sulfenium ions, and halogens26. More recently(and directly relevant to our studies), Abdolrasulnia and Woodreported that CysSSSCys reacted readily with iodoacetic acid (awell-established thiol-modifying agent) to ultimately give carboxymethylthiocysteine (CysSS–CH2COOH)27, which is consistent with the idea that a nucleophilic sulfur atom of thepolysulﬁde reacted with the electrophilic iodoacetic acid species
and led to S–S bond cleavage. Previous examination of thereaction of electrophiles with disulﬁdes (the simplest of allpolysulﬁdes) is entirely consistent with this idea28. Thus, ample
precedence for the nucleophilic character of polysulﬁdes exists, bycapitalizing on such a unique property, we are now able toidentify the cysteine and protein polysulﬁdation occurring
endogenously by means of a conventional PMSA or capturing| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

a

c

d

K317/320A

K124/127A

(kDa)
91
70

2
1
0

CARS2 KO

Total Drp1
(input)f

Electrophiles andTrx/TrxR system etc.

CARS2 KO

K317/320A

K124/127A

C78/257D

0

WT

0.2

100
50
0

K317/320A

0.4

150

K124/127A****

C78/257D

0.6

200

WT

**

***
***
***
***

250

WT

**

Mitochondrial OCR**
OCR (pmol/min)

**

0.8

Mock

CARS2 KO+ K317/320A

1.0

WT

CARS2 KO+ K124/127ACARS2 KO
+ C78/257D

JC-1 aggregate/monomer
intensity

CARS2 KO

InactiveDrp1

gJC-1 membrane potentialTEM
CARS2 KO+ WTActiveDrp1

CARS2 KOb
WTPolysulfidation

Depolysulfidation

91
70

Mock

C78/257D

WT

0

WT

20

**

C78/257D

40

**

3

CysSSHCARS2 KOsiRNA
WT MockCARS1/2
1 2 1 2 1 2Polysulfidated
Drp1 (eluted)

K317/320A

**

WT

**

**

Drp1 polysulfidation

**

4

Mock

60

**

WT

**

**

e

Drp1 activity
Drp1-GTPase/Drp1 input

80

Mock

Length of mitochondria(AU)

MitoTracker

CARS2 KO

Fig. 8 CARS2-dependent mitochondrial morphogenesis and bioenergetics. a Mitochondrial morphological analyses with MitoTracker Red ﬂuorescent
mitochondrial stain: morphometric analysis of mitochondrial length in HEK293T cells (WT and CARS2 KO; CARS2 WT and mutants added back). AU,
arbitrary unit. The data are means ± s.d. (n = 3). **P < 0.01. b Transmission electron microscope (TEM) images of the cells in a. Scale bars, 1 μm.
c Identiﬁcation of Drp1 activity in HEK293T cells (WT and CARS2 KO; CARS2 WT and mutants added back). The GTP-agarose pulldown assay was
performed. The data are means ± s.d. (n = 3). **P < 0.01. d Drp1 expressed in extensively polysulﬁdated (biotin-PEG-MAL capture method) HEK293T cells.
Drp1 was markedly suppressed and nulliﬁed by CARS2 KO and CARS1/2 knockdown. Lanes 1 and 2 show duplicate determinations with each siRNA.
Supplementary Fig. 23 provides full blot images. e A schematic drawing of Drp1 activity as regulated by protein polysulﬁdation and depolysulﬁdation, as
affected by polysulﬁdes vs. electrophiles and Trx/TrxR system. f Changes in membrane potential as assessed by using JC-1 staining of HEK293T cells (WTand CARS2 KO; CARS2 WT and mutants added back). The data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01. g Assessment of mitochondrial electron ﬂow
in HEK293T CARS2 KO cells with or without adding back WT and C78/257D, K124/127A, and K317/320A mutants, as analyzed by measuring OCR using
an extracellular ﬂux analyzer. Time dependence of oxygen consumption before/after inhibition of mitochondrial respiration at complexes I and III byrotenone/antimycin A, and its statistical summary; the data are means ± s.d. (n = 3). ***P < 0.001

assays and even by using HPE-IAM labeling LC-MS/MS analysis.
The present discovery of a novel polysulﬁde biosynthesis, therefore, can now explain substantial endogenous generation of sulfane sulfur, which we clariﬁed as composed of various polysulﬁdederivatives and which is biosynthesized by CPERSs and CARSs.
Our ﬁndings raise a number of important questions; however,for example, why are such protein-bound cysteines abundantly
polysulﬁdated, does polysulﬁdation affect protein folding? And,
what function does this modiﬁcation play in compartments otherthan mitochondria? Determining how CPERS activity is regulated
will also be important. Given the powerful effects of persulﬁdeson mitochondrial morphology and bioenergetics, the availabilityof persulﬁdes in cells must be subject to stringent regulation.
Although CPERSs play a critical role in generating CysSSH, theTrx–TrxR system may help maintain cellular persulﬁde concentrations within certain limits by controlling the rate of persulﬁde degradation4.
Some aminoacyl-tRNA synthetases reportedly possess functions in physiological processes besides their role in translation43.
The mitochondria-promoting functions of CARS2 suggest its
non-canonical roles and therefore may therefore represent
10“moonlighting” roles of CARS2. However, CARSs effectively
synthesize cysteine polysulﬁdes, and this process is closely related
to the initial translational process of de novo synthesis of nascent
polypeptides in ribosomes (cf. Fig. 1b and Supplementary
Fig. 10). The CPERS function of CARSs is apparently associatednot only with translation but also with the mitochondrial
respiration, which indicates that CARSs, rather than having a
moonlighting role, have a primary function of producing
persulﬁdes.
In conclusion, our discovery of reactive persulﬁde production
mediated by the CARS or CPERS pathway and the potent effects
on mitochondrial functions observed would seem to represent asigniﬁcant evolution of molecular and cell biology, thereby
inviting a paradigm shift in the current understanding of cellulartranslation, redox signaling, and energy metabolism (Fig. 10). Our
discovery of CARS and CPERS as a major sources of reactive
persulﬁdes in biology may usher in a new era of modern redox
biology and life science research that hold great potential toinvigorate translational studies in a variety of disease processes
known to be associated with aberrant redox regulation andmitochondrial dysfunction.

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

b

0
H

C
ys
SS

H
S

C
ys
SS

2O
3 –

H
S–

e

10
0
1.25

2.5

0

Antimycin A (μM)

f

g
*

h

0

(–)

(+)

mtDNA elimination**30
HS–: 28.0

10

Bis-S-HPE-AM(pmol/mg protein)

CysSSH: 19.2

20

8
4
1.25

20
10
0

(–)

(+)

mtDNA elimination

40

2.5

Antimycin A (μM)

Changes in
CysSSH/HS–

40

30

12

0

2.5

HS–

CysSSH40

1.25

Changes in CysSSH/HS–CysSSH
N.S.
20
HS–
N.S.
16

Antimycin A (μM)

HPE-IAM adducts(pmol/mg protein)0*
HS–: 15.1

20

*

80
70
60
50
40
30
20
10
0

HS–: 11.8

30

CysSSH: 14.1

CysSSH: 12.0*
Bis-S-HPE-AM(pmol/mg protein)

*

40

HS–
HPE-IAM adducts(pmol/mg protein)

SH

C
ys
SS

C
ys
SS

HS–

10

d
CysSSHCysSS-HPE-AM(pmol/mg protein)

20

CysSSSHc

CysSS-HPE-AM(pmol/mg protein)

30

2O
3 –

CysSSHCysSSHHS2O3–40

H
S

HPE-IAM adducts(pmol/mg protein)

1.8
1.5
1.2
0.9
0.6
0.3
0

CARS2 in cells

HS2O3–

HS–

SH
H
S–

CARS2 cell-free

H

HPE-IAM adducts(μM)

aCysSSH
HS–
N.S.

30

i
ETC
e–

O2

CysSSH

HS–

CARS2

CysSH

HS2O3–

20
100
mtDNA elimination

Fig. 9 Mitochondrial ETC-mediated reduction of CysSSH. a, b Sulﬁde metabolite proﬁling for the reaction of the recombinant human CARS2 in vitro (a) andof CARS2 expressed in HEK293T cells (b). c–h Changes in amounts of CysSSH (ΔCysSSH) and HS− (ΔHS−) induced by complex III inhibition by antimycin
A c–e or by mitochondrial DNA (mtDNA) elimination induced by ethidium bromide (f–h) in WT and CARS2 KO HEK 293 T cells. The values of CysSSH andHS− shown in b–h indicate the quantity of each compound produced in the cells in a manner dependent on CARS2 expression, which was determined bysubtracting each amount in CARS2 KO HEK293T cells from that in the WT cells, after quantiﬁcation of each metabolite via HPE-IAM labeling LC-MS/MS
analysis. e, h Stoichiometric alterations (conversion) between CysSSH and HS− in cells by the ETC inhibition. i Schematic diagram of ETC-mediatedCysSSH reduction to form HS− and possible further conversion to S2O32−. The data are means ± s.d. (n = 3). *P < 0.05; **P < 0.01; N.S., not signiﬁcantMethods
LC-ESI-MS/MS analyses for per/polysulﬁdes. LC-ESI-MS/MS analysis with
HPE-IAM (Supplementary Fig. 5 and Supplementary Table 2) was used todetermine CysSSH or CysS–(S)n–SH formed from EcCARS and CARSs. To identify
CysS–(S)n–H formed and incorporated into Cys-tRNA via the enzymatic reactionof EcCARS, 200 μg/ml recombinant EcCARS was reacted with 0.5 mg/ml tRNA
(Sigma-Aldrich) and CysS–(S)n–H or 10 μM cysteine as the substrate, in 50 mM
HEPES buffer (pH 7.5) containing 1 mM ATP, 25 mM KCl, and 15 mM MgCl2 at
37 °C, followed by alkylation with 1 mM HPE-IAM for 20 min at 37 °C. CysS–(S)n–H were formed from 10 μM cystine and 30 μM Na2S2 in 30 mM HEPES buffer
pH 7.5 at 37 °C for 5 min. The Cys-tRNACys–(S)n–H synthesized by EcCARS was
precipitated by adding 10% trichloroacetic acid to the reaction mixture, followed bytrapping by cotton wool ﬁlters (100 μl) placed in pipette tips. The precipitated totaltRNA containing Cys-tRNACys–(S)n–H was washed with 10% trichloroacetic acid(200 μl twice) and with 70% ethanol (200 μl twice) to completely remove the freecysteine and CysS–(S)n–H. CysS-HPE-IAM and CysS–(S)n-HPE-IAM adducts
were dissociated by alkaline heat hydrolysis of the ester bond of aminoacyl moietiesof the Cys-tRNACys and Cys-tRNACys–(S)n–H. The hydrolysis was performed in
20 mM Tris-HCl (pH 8.0), which contained known amounts of stable isotopelabeled internal standards, at 70 °C for 15 min. The eluted solutions were acidiﬁedwith formic acid and analyzed via LC-ESI-MS/MS. Also, Cys-tRNA-bound CysSSH
was identiﬁed by detecting a CysSSH-adenosine adduct formed in the Cys-tRNA
molecules synthesized by EcCARS from the substrate cysteine. The CysSSHadenosine adducts in the reaction of EcCARS with cysteine and Cys-tRNA were
measured by using LC-ESI-MS/MS analysis. In brief, CysSSH incorporated into
NATURE COMMUNICATIONS | 8: 1177

tRNA as catalyzed via EcCARS with cysteine was prepared in the same manner asthat described above, followed by alkylation with HPE-IAM and acetylation withacetic anhydride, as described earlier44. After precipitation and washing of samples
with ethanol, the acetylated and HPE-IAM-labeled Cys-tRNACysSSH was digested
to generate acetylated CysSS-HPE-AM-bound adenosine by treatment with RNase
ONE (Promega, Madison, WI) at 37 °C for 1 h, after which LC-ESI-MS/MS analysis was performed. To measure CysS–(S)n–H generated directly by EcCARS andCARSs, recombinant EcCARS, mouse CARS1, or human CARS2 was incubatedwith cysteine in 50 mM HEPES buffer (pH 7.5) containing 25 mM KCl and 15 mM
MgCl2 with or without 1 mM ATP at 37 °C. The mixtures were then reacted with
1 mM HPE-IAM in methanol at 37 °C for 20 min to form CysS–(S)n–HPE-IAM
adducts. After centrifugation, aliquots of the supernatants were diluted 10–100
times with 0.1% formic acid containing known amounts of isotope-labeled internal
standards and were subjected to LC-ESI-MS/MS. To clarify the molecularmechanism of CysSSH formation, 50 μM 34S-labeled L-cysteine was reacted with
200 μg/ml EcCARS as a substrate in 50 mM HEPES buffer (pH 7.5) containing
25 mM KCl and 15 mM MgCl2 at 37 °C for 15–60 min. The reaction products
treated with HPE-IAM were diluted with 0.1% formic acid containing known
amounts of isotope-labeled internal standards, which were then subjected to LCESI-MS/MS as described above. To determine kinetic parameters, WT EcCARS
and C28S EcCARS were incubated with different concentrations of L-cysteine in
50 mM HEPES buffer (pH 7.5) containing 25 mM KCl and 15 mM MgCl2 at 37 °C
for 30 s. The reaction mixtures were treated with 1 mM HPE-IAM, followed by LCESI-MS/MS as described above. The data were ﬁtted by nonlinear regression to theMichaelis–Menten equation by using GraphPad Prism software ver. 6.0 (GraphPad| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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a
HO
H 2N

DNA

Cysteine persulfide
synthases
HO(CPERSs)OO

H2N (S)n H
CysS-(S)n-H

SH

L-Cysteine

Transcription

CARS
mRNA

H
(S)n (S)n (S)n (S)n (S)n
S S S
S
S
S

SH

Protein polysulfidation(co-translational)

SOx–

R

H

Trans-sulfidation(post-translational)

Translation

Cys-tRNA

Trx/TrxR system
SH

Protein

HS–/HSn–

Depolysulfidation

Polysulfidation

Redox signal regulationb
Antioxidant
effectsCysSH
CARS2/CPERS
HS2O3–

Glucose

Depolysulfidation

PLP
CysSSH

O2
ST
SD

ATP
TCA
Pyruvatee–

ActiveDrp1

Inactive

Polysulfidation
HS–
SQR

II

Electrophiles

e–

O2

H+/e–

H2O

Q
I

III
ETC

IV

H+

Fig. 10 CARS-mediated protein polysulﬁdation and mitochondrial functions. a The physiological relevance of co-translational protein polysulﬁdation that isreversibly regulated by various post-translational modiﬁcations, including depolysulﬁdation. b A CysS–(S)n–H regulation mechanism for mitochondrial
functions with regard to mitochondrial biogenesis and bioenergetics. CysSSH is reductively metabolized to CysSH and HS−, which may be oxidized by
sulﬁde:quinone reductase (SQR) and other enzymes, e.g., sulfur dioxygenase (SD) and sulfur transferase (ST), in a manner linked to ETC in mitochondria.
The CysS–(S)n–H-dependent HS− metabolism may be coupled with formation of the iron-sulfur clusters, as being controlled by the mitochondrial ETC. I, II,
III, and IV: complexes I, II, III, and IV; TCA tricarboxylic acid (Krebs) cycle

Software, San Diego, CA) to obtain the kinetic parameters. Each calculated enzyme
parameter was compared with that of recombinant CSEs (rat and human), which
we obtained from the enzymatic reaction with L-cystine as the substrate, according
to our previous report3. For analysis of intracellular persulﬁde levels in cultured
HEK293T cells, and livers and lungs obtained from WT and Cars2+/– littermate
mice, the cultured cells and mouse tissues were lysed or homogenized in a cold
methanol solution containing 1 mM HPE-IAM, after which cell lysates wereincubated at 37 °C for 20 min. After centrifugation, aliquots of the supernatants of
the lysates were diluted 20 times with 0.1% formic acid containing known amounts
of isotope-labeled internal standards, which were then analyzed via LC-ESI-MS/MS
for per/polysulﬁde determination. A triple quadrupole (Q) mass spectrometer
LCMS-8050 (Shimadzu) coupled to the Nexera UHPLC system (Shimadzu) wasused to perform LC-ESI-MS/MS. Per/polysulﬁde derivatives were separated bymeans of Nexera UHPLC with a YMC-Triart C18 column (50 × 2.0 mm innerdiameter) under the following elution conditions: mobile phases A (0.1% formicacid) with a linear gradient of mobile phases B (0.1% formic acid in methanol)from 5 to 90% for 15 min at a ﬂow rate of 0.2 ml/min at 40 °C. MS spectra wereobtained with each temperature of the ESI probe, desolvation line, and heat blockat 300, 250, and 400 °C, respectively; and the nebulizer, heating, and drying
nitrogen gas ﬂows were set to 3, 10, and 10 liters/min, respectively. Various per/polysulﬁde derivatives were identiﬁed and quantiﬁed by means of multiple reaction
monitoring (MRM). Supplementary Table 2 summarizes the MRM parameters foreach derivative.
Identiﬁcation of CysS–(S)n–SH formed in nascent peptides. CysS–(S)n–SH
species synthesized endogenously and formed in nascent polypeptides by EcCARS
in E. coli cells in culture were analyzed by means of puromycin-associated nascent
chain proteomics (PUNCH-P)29, which was speciﬁcally modiﬁed here for12

polysulﬁdated proteins (PUNCH for Polysulﬁde Proteomics, henceforth called
PUNCH-PsP). The E. coli JM109 cells transfected with an hGAPDH expression
vector (pGE-30) were cultured and hGAPDH expression was induced with IPTG
as described earlier, followed by collecting and sonication of the cells in cell lysis
buffer containing 0.3 mg/ml lysozyme and 2 mM IAM without any reducing
agents. The supernatant obtained by centrifugation was applied to the Ni-NTA
agarose column for puriﬁcation of the mature GAPDH protein. From the resultant
pellet of the E. coli cell lysate, the ribosomal fraction was isolated via sucrose
density gradient ultracentrifugation, as reported previously29. The ribosomal
fraction was suspended in polysome buffer (50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, and 25 mM KCl), containing an EDTA-free protease inhibitor cocktail (asindicated by the manufacturer), and was then reacted with 2 mM IAM at room
temperature for 30 min. After the ribosomal fraction was washed with the polysome buffer, the ribosomes were treated with 5′-biotin-dC-puromycin (Jena
Bioscience, Jena, Germany) in TTBS (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween
20, pH 7.6) at 37 °C for 15 min and were then reacted with avidin magnetic beads(Wako Pure Chemical Industries) to ﬁnally capture the newly synthesized polypeptides in ribosomes in the E. coli cells in culture. The puromycin-labeling conditions were optimized for the E. coli ribosomes used in the present study,
according to the original report29. The CysS–(S)n–H residues in GAPDH weredetected by means of LC-Q-TOF-MS as described earlier, with tryptic digests of themature GAPDH puriﬁed simultaneously and the same digest of the nascent
GAPDH polypeptides within the cultured E. coli ribosomes captured with andrecovered from the biotin-puromycin-bound avidin beads. CysS–(S)n–H in thenascent polypeptides can be selectively identiﬁed by using PUNCH-PsP, which we
successfully developed and describe here (Fig. 1c and Supplementary Fig. 10).
During this PUNCH-PsP analysis, the cysteine and CysS–(S)n–H residues locatedin the polysulﬁde exit tunnel in the ribosomes are not accessible to exogenously
added IAM and can thus be protected from alkylation by IAM because of the

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

unique physicochemical properties of the interior structure of the polypeptide exit
tunnel in the ribosome45–47, which allowed us to obtain speciﬁc and selective
identiﬁcation of the intact forms of CysS–(S)n–H residues in the nascent peptides
present only within the ribosomes, as Supplementary Fig. 10a shows. As soon as
the mature GAPDH isolated from E. coli. with the Ni-NTA agarose was treated byquick digestion with 10 μg/ml trypsin at 37 °C for 30 min, which was promptly
subjected to the LC-ESI-Q-TOF analysis, in a similar manner as shown for the
PUNCH-PsP method.
Preparation and puriﬁcation of recombinant CARS proteins. To generate
recombinant CARSs, open-reading frames of these genes were transferred into
AG1 (Agilent Technologies, Santa Clara, CA) competent cells. Recombinant
EcCARS, mouse CARS1, and human CARS2 proteins were puriﬁed by using thefollowing standard procedure. Brieﬂy, these proteins were produced in AG1, andthey were puriﬁed by using nickel nitrilotriacetic acid agarose; resultant puriﬁedproteins were extensively dialyzed against phosphate buffer and stored at −80 °C
until use. Protein concentration was determined by using the Protein Assay CBBSolution (Nacalai Tesque, Kyoto, Japan), and protein purity was conﬁrmed via
SDS-PAGE.
Generation of CARS2 KO cell lines. The genome editing CRISPR/Cas9 system
was used to generate human CARS2 KO cell lines. To obtain gRNA, which is highly
speciﬁc for the ﬁrst exon of the human CARS2 locus and has fewer off-target sites
within the human genome, we based an optimal gRNA design on the software
program CRISPRdirect48. To express Cas9 and gRNA in HEK293T cells, the pX459
V2.0-CARS2 gRNA vector was created by inserting annealed oligonucleotide pairs(5′-caccTGGGCCTTGGGCGGGCTGGG-3′ and 5′-aaacCCC AGCCCGCCCAAGGCCCA-3′) into the BpiI sites of pX459 V2.0. pX459 V2.0 vector, whichenables expression of a gRNA (directed to the CARS2 exon 1; Supplementary
Fig. 15), SpCas9, and a puromycin resistance gene from a single vector, wasobtained from the Zhang laboratory via Addgene plasmid 6298849. HEK293T cells
were plated in 6-well plates (1.0 × 105 cells per well) 24 h before transfection.
Cultured cells were transfected with 2 μg of pX459 V2.0-CARS2 gRNA by using
Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The medium was changed 24 hafter transfection. After another 24 h of incubation, the cells were replated on 10cm dishes and cultured for various time periods at 37 °C with a selection medium
containing 2.0 μg/ml puromycin (Invitrogen). Puromycin-resistant clones werearbitrarily selected and used for screening CARS2 KO cell lines to ﬁnally obtainstable CARS2 KO cell lines. Disruption of the CARS2 gene was veriﬁed by loss of
CARS2 protein expression as determined by western blotting.
Construction of mammalian hCARS2 expression vectors. To produce an
hCARS2 expression vector (pPyCAGIP-FLAG-hCARS2), the XhoI fragment of
pET-15b-hCARS2 was cloned into the XhoI site of pPyCAGIP-FLAG. The same
vectors containing various mutant hCARS2 genes were obtained via site-directedmutagenesis by using inverse PCR with pPyCAGIP-FLAG-hCARS2 as a template
and primer sets for generation of pPyCAGIP-FLAG-hCARS2 C78/257D, K124/
127A, and K317/320A.
Transfection of various CARS2 genes and knockdown of CARS1/2. WT andvarious mutant CARS2 genes were transfected into HEK293T WT and mutant cells asreported recently3 by using expression plasmids such as pPyCAGIP-FLAG-hCARS2
and CARS2 mutant vectors. Transfection of the expression plasmid was performed byusing Lipofectamine 2000 according to the manufacturer’s instructions. In brief, we
incubated WT and CARS2 KO HEK293T cells seeded in 24-well plates (6 × 105 cells
per well) and 8-well culture slides (2 × 105 cells per well) for 12 h at 37 °C. For
transfection, we mixed 1.5 μg per well of the expression plasmid with 50 μl of OptiMEM (Invitrogen) in a tube. Before plasmid DNA and transfection reagent solutions
were added to the cells, solutions were mixed together and incubated for 5 min atroom temperature and then added to the cells, after which incubation proceeded for
30 h or 3 days. Also, knockdown of CARS1 and CARS2 was performed as reported
recently3 by using the following small interfering RNAs (siRNAs): CARS1,CARSHSS101368 (Invitrogen), and CARS2, CARS2HSS128464 (Invitrogen). siRNAtransfection was performed by using Lipofectamine RNAiMAX (Invitrogen)according to the manufacturer’s instructions. The siRNA was introduced into WT
and CARS2 KO cells, as described above for CARS2 gene transfection.
Generation of Cars2-deﬁcient mice. All experimental procedures conformed to“Regulations for Animal Experiments And Related Activities at Tohoku University”, and were reviewed by the Institutional Laboratory Animal Care and Use
Committee of Tohoku University, and ﬁnally approved by the President of University. We generated two lines of Cars2-deﬁcient mice as follows. Cars2 gRNAs
vectors were constructed with use of a pT7-sgRNA and pT7-hCas9 plasmid (a giftfrom Dr. M. Ikawa, Osaka University)50. After digestion of pT7-hCas9 plasmidwith EcoRI, hCas9 mRNA was synthesized by using an in vitro RNA transcription
kit (mMESSAGE mMACHINE T7 Ultra kit; Ambion, Austin, TX), according to
the manufacturer’s instructions. A pair of oligonucleotides targeting Cars2 wasannealed and inserted into the BbsI site of the pT7-sgRNA vector. The sequences of
the gRNAs were designed as follows: 5′-GGACAGATCCAGCGAACAGG-3′ and
NATURE COMMUNICATIONS | 8: 1177

5′-AATAATCAAGAGAGCTAACG-3′, located at exons 1 and 3 of Cars2 gene, togenerate CARS2-deﬁcient lines 1 and 2 mice, respectively. After digestion of pT7sgRNA with XbaI, gRNAs were synthesized by using the MEGAshortscript kit(Ambion). We used C57BL/6N female mice (purchased from Crea-Japan Inc.,Tokyo, Japan) to obtain C57BL/6N eggs, and we performed in vitro fertilization
with these eggs. In brief, Cas9 mRNA and gRNA were introduced into fertilized
eggs by injecting using a Leica Micromanipulator System, according to the protocols reported previously50, after which we transferred the eggs to the oviducts ofpseudo-pregnant females on the day of the vaginal plug. A founder mouse harboring the Cars2 mutant alleles was crossed with WT mice to obtain Cars2 heterozygous mice. After segregating the Cars2 mutant alleles, heterozygous mice witha 200-bp deletion in exon 1 (line 1) and with a 1-bp insertion in exon 3 wereselected for additional analyses (Figs. 5–7; Supplementary Figs. 20 and 21).
MitoTracker Red staining for mitochondrial morphology. To analyze mitochondrial morphogenesis under several experimental conditions in cells, mitochondria were imaged by using the ﬂuorescent probe MitoTracker Red CMH2Xros (Invitrogen). In brief, culture slides were coated with 0.5% polyethylene
imine for more than 1 h and washed twice with PBS. CARS2 KO cells weretransfected with expression plasmids for WT and individual mutants of humanCARS2 via Lipofectamine 2000. At 3 days after transfection, cultured cells were
washed with Hank’s buffer, incubated with 1 μM MitoTracker Red CM-H2Xros at
37 °C for 30 min, rinsed twice with Hank’s buffer, and examined with a Nikon EZC1 confocal laser microscope (Tokyo, Japan). We used ImageJ and Prism software
for image processing and quantiﬁcation of mitochondrial dimensions including
their length.
Mitochondrial bioenergetic functions. To determine the membrane potential(ΔΨm) of mitochondria under several experimental conditions, tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining was performed according
to the manufacturer’s protocol. Accumulation of the cell-permeable JC-1 probe(Abcam) in mitochondria depends on the membrane potential, associated with aﬂuorescence emission shift from green to red. Brieﬂy, WT and CARS2 KOHEK293T cells, cultured in 8-well multichamber Millicell slides coated with PEI,
were treated with various CARS2 vectors or were untreated, as described above. For
JC-1 staining, cultured cells were washed with HKRB buffer (20 mM HEPES,
103 mM NaCl, 4.77 mM KCl, 0.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM KH2PO4,
25 mM NaHCO3 and 15 mM glucose, pH 7.3), incubated with 20 μM JC-1 at 37 °C
for 30 min, rinsed twice with HKRB buffer, and examined with a Nikon EZ-C1
confocal laser microscope. ImageJ software was used for image processing andquantiﬁcation of the JC-1 ﬂuorescent responses.
Mitochondrial bioenergetic functions. Mitochondrial function was investigated,according to a previous report with a slight modiﬁcation51, by measuring the basal
OCR of the mitochondria under various experimental conditions in WT andCARS2 KO cells, using the XF96 Extracellular Flux Analyzer (Seahorse Bioscience,
Agilent). At the end of the experiment, rotenone and antimycin A (2.4 μM each)were added to inhibit complexes I and III of the mitochondrial electron transport
chain, respectively, to determine the remaining mitochondria-independent OCR.
Net OCR was normalized to the cell number determined at the end of theexperiments by means of sulforhodamine B staining (Sigma-Aldrich, St. Louis,
MO). To obtain the mitochondria-speciﬁc OCR, only the rotenone/antimycinsensitive part of cell respiration was used.
Effect of suppression of ETC on metabolic proﬁles of CysSSH. The mitochondrial ETC in HEK293T cells was inhibited either by a complex III inhibitor,
antimycin A, or by elimination of mitochondrial DNA (mtDNA) induced byethidium bromide. For the direct but partial ETC (complex III) inhibition, WT andCARS2 KO cells were treated with various concentrations of antimycin A for 1 h,followed by methanol extraction for measurement of CysSSH and its related sulﬁdederivatives by HPE-IAM labeling LC-ESI-MS/MS analysis as described earlier. To
indirectly suppress all ETC components (complexes), mtDNA from WT andCARS2 KO HEK293T cells was eliminated speciﬁcally by treatment with ethidium
bromide (50 ng/ml, 127 nM) for 12 days under standard cell culture conditions(37 °C, humidiﬁed, 5% CO2/95% air) with DMEM containing 10% FBS, 1%
penicillin-streptomycin, sodium pyruvate (1 mM), nonessential amino acids (1%),
and uridine (50 μg/ml), according to a previous method with a slight modiﬁcation52. The cells without mtDNA were then subjected to HPE-IAM labeling LCMS/MS analysis for persulﬁde metabolic proﬁling, similar to antimycin-treatedcells. The efﬁcacy of the present mtDNA elimination and the resultant ETC suppression were assessed by measuring mtDNA as described below (Supplementary
Fig. 24b), and these results were conﬁrmed by substantial suppression of mitochondrial cytochrome c oxidase subunit 1 (MTCO1: encoded by mtDNA), as
identiﬁed by western blotting. In contrast, MitoTracker Red staining showed noappreciable altered morphology of mitochondria in HEK293T cells with or without
ethidium bromide treatment, at least under the present experimental conditions.
The quantity of each sulﬁde produced from CARS2 in the cells was determined bysubtracting the amount of each sulﬁde in CARS2 KO HEK293T cells from that in
the WT cells, after quantiﬁcation of each metabolite via HPE-IAM labeling LC-MS/| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

13

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

MS analysis. Changes in the amounts of CysSSH (ΔCysSSH) and HS− (ΔHS−)
induced by complex III inhibition by antimycin A or by mtDNA elimination in
WT and CARS2 KO HEK293T cells were then calculated.
Statistical analysis. Results are presented as means ± s.d. of at least three independent experiments unless otherwise speciﬁed. For statistical comparisons, we
utilized two-tailed Student’s t test or two-way analysis of variance followed by theStudent–Newman–Keuls test, with signiﬁcance set at P < 0.05.
Data availability. The data that support the ﬁndings of this study are available
from the corresponding author upon request.

Received: 23 September 2016 Accepted: 29 August 2017

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Acknowledgements
We thank J.B. Gandy for her editing of the manuscript and evaluating concepts andterminology of the paper with regard to the understanding by non-specialist readers.
Thanks are also due to S. Akashi, H. Abdul Hamid, Y. Kakihana, and M. Matsubara for
their technical assistance in our study. P.N. is a János Bolyai Research Scholar of the
Hungarian Academy of Sciences. M.F. is supported by the National Institute for HealthResearch through the NIHR Southampton Biomedical Research Centre. This work was
supported in part by Grants-in-Aid for Scientiﬁc Research ((S), (A), (B), Challenging
Exploratory Research, and Innovative Areas “Oxygen Biology: a new criterion for integrated understanding of life”) from the Ministry of Education, Sciences, Sports, andNATURE COMMUNICATIONS | 8: 1177

| DOI: 10.1038/s41467-017-01311-y | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01311-y

Technology (MEXT), Japan, to T.A. (26111008, 26111001, 15K21759, 25253020,
16K15208), Y.K. (25220103), H.M. (15H04692), K.I. (26116005), T.S. (15H03115), and
M.N. (15K14959); a grant from the JST PRESTO program to T.S. (10104025) and M.N.
(13417243); grants from The Hungarian National Science Foundation (K 109843) andfrom the National Institutes of Health (R21AG055022-01) to P.N.; and grants from the
National Institutes of Health (HL106598) and the National Science Foundation (CHE1148641) to J.M.F.

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T.A. and H.M., experiment design, biochemistry and cell biology, data analysis, animalstudies, and writing the paper; T.I., M.M.A., H.I., T.S., M.J. and T.M., MS analysis,biochemistry and molecular biology, cell biology, and cell imaging; S.W., K.I., protein
structural analysis; F.-Y.W., S.K., Akir.N., H.S., N.T. and K.T., cell biology and mitochondria study; M.M. and M.O., CRISPR/Cas9 technology, cell biology, and animalstudies; M.N., Akiy.N., S.F., K.Y. and Y.W., cell signaling, cell biology, and data analysis;
P.N., M.F., J.M.F., chemical analysis and editing the paper.

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Supplementary Information accompanies this paper at doi: 10.1038/s41467-017-01311-y.
Competing interests: The authors declare no competing ﬁnancial interests.

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