A role for the mitochondrial deacetylase Sirt3
in regulating energy homeostasis
Bong-Hyun Ahn*†, Hyun-Seok Kim†‡, Shiwei Song*, In Hye Lee*, Jie Liu*, Athanassios Vassilopoulos‡, Chu-Xia Deng‡§,
and Toren Finkel*
*Translational Medicine Branch, National Heart, Lung, and Blood Institute, and‡Genetics of Development and Disease Branch, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Salvador Moncada, University of London, London, United Kingdom, and approved August 1, 2008 (received for review April 18, 2008)
Here, we demonstrate a role for the mitochondrial NAD-dependent
deacetylase Sirt3 in the maintenance of basal ATP levels and as a
regulator of mitochondrial electron transport. We note that Sirt3?/?
mouse embryonic fibroblasts have a reduction in basal ATP levels.
the resting level of ATP correlates with organ-specific Sirt3 protein
expression. Remarkably, in mice lacking Sirt3, basal levels of ATP in
the heart, kidney, and liver were reduced >50%. We further dem-
onstrate that mitochondrial protein acetylation is markedly elevated
in Sirt3?/?tissues. In addition, in the absence of Sirt3, multiple
increased acetylation. Sirt3 can also physically interact with at least
Functional studies demonstrate that mitochondria from Sirt3?/?an-
incubation of exogenous Sirt3 with mitochondria can augment Com-
plex I activity. These results implicate protein acetylation as an
important regulator of Complex I activity and demonstrate that Sirt3
functions in vivo to regulate and maintain basal ATP levels.
acetylation ? sirtuins ? complex I ? electron transport
transcriptional silencing and acts as a regulator of life span (1, 2).
Remarkably, increased dosage of Sir2 extends the life span of yeast
as well as several other simple organisms (3, 4). Mammals have at
least seven different Sir2 homologs, with mammalian Sirt1 being
the closest structural relative of yeast Sir2. A number of Sirt1
protein deacetylase substrates have been previously identified
including p53 and the Foxo family of transcription factors (5).
Considerably less is known about the other sirtuin family members,
three of which, Sirt3,Sirt4, and Sirt5 appear to localize primarily to
the mitochondria (6–9). Evidence suggests that Sirt4 appears to
function predominantly as an ADP-ribosyltransferase. One target
(GDH) (10). Consistent with a complex interaction between mi-
tochondrial sirtuins, GDH also appears to be a deacetylase target
for Sirt3 (11). In addition, Sirt3-dependent deacetylation regulates
the activity of acetyl-CoA synthetase 2 (AceCS2) an important
mitochondrial enzyme involved in generating acetyl-CoA for the
tricarboxylic acid (TCA) cycle (12, 13).
A recent study demonstrated that mice deficient in Sirt3 are
exhibit normal body weight as well as normal body fat composition,
(11). Similarly, Sirt3?/?mice appeared outwardly healthy under
of starvation. In addition, although a previous report in a cell-
culture model had implicated Sirt3 in adaptive thermogenesis (9),
this physiological function was not apparently altered in Sirt3?/?
A proteomic survey of intracellular proteins that had internal
acetylation residues demonstrated that a disproportionate fraction
he sirtuins are a conserved family of proteins possessing NAD-
dependent deacetylase activity. In yeast, Sir2 is involved in
of identified proteins were in the mitochondria and/or associated
acetylation may represent an important mechanism to regulate
overall mitochondrial function. Indeed, given the mechanism of
action of the sirtuin family, levels of mitochondrial protein acety-
lation would seemingly be influenced and regulated by NAD?, a
key mitochondrial energetic intermediate. Nonetheless, the re-
ported unremarkable phenotype of Sirt3-deficient mice challenges
the physiological importance of NAD-dependent deacetylation in
regulating energy homeostasis. Here, using an independently gen-
erated model of Sirt3?/?mice, we provide evidence for Sirt3-
dependent regulation of global mitochondrial function. In partic-
ular, we demonstrate that the absence of Sirt3 results in a marked
that Sirt3 can reversibly bind to and regulate the acetylation and
activity of Complex I of the electron-transport chain.
The Sirt3 locus was inactivated by homologous recombination in
ES cells leading to the deletion of exons 2–4 [supporting
information (SI) Fig. S1]. Analysis of the tissues of homozy-
gously deleted mice revealed undetectable levels of Sirt3 protein
expression (Fig. S1). Consistent with a recent report (11), our
own extensive survey of multiple organs by both gross and
microscopic pathology revealed that Sirt3?/?mice appeared
indistinguishable from wild-type littermates. In addition, we
observed no increased or decreased mortality of these mice
during the first year of life (H.-S.K. and C.-X.D., unpublished
In an effort to more fully understand the role of Sirt3 in
mitochondrial biology, Sirt3?/?mice were bred to generate multi-
fibroblasts (MEFs). Given the central role of mitochondria in
generating ATP via the electron-transport chain (ETC), we first
asked whether deletion of Sirt3 altered basal ATP levels. We
measured ATP levels in five independent lines of wild-type MEFs
and a similar number of independent Sirt3?/?MEFs (Fig. 1A).
These analysis demonstrated that, on average, Sirt3?/?MEFs had
whether transient reconstitution of Sirt3 could restore this ATP
deficit. Sirt3?/?MEFs were transiently transfected with an empty
vector, wild-type Sirt3, or a deacetylase-inactive form of Sirt3,
Sirt3(HY). All transfections were performed with a cotransfected
GFP expression vector to allow for subsequent purification of
Author contributions: C.-X.D. and T.F. designed research; B.-H.A., H.-S.K., S.S., I.H.L., J.L.,
and A.V. performed research; C.-X.D. and T.F. analyzed data; and T.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
†B.-H.A. and H.-S.K. contributed equally to this work.
§To whom correspondence should be addressed at: National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9N105,
Bethesda, MD 20892. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
September 23, 2008 ?
vol. 105 ?
no. 38 ?
transfected cells using FACS sorting. As noted in Fig. 1B, expres-
sion of wild-type Sirt3 in the background of Sirt3?/?MEFs mark-
edly increased basal ATP, whereas the deacetylase inactive mutant
was ineffective in raising ATP levels. Similar results were obtained
in HeLa cells that contain endogenous Sirt3 (Fig. 1C).
Based on these observations, we asked whether Sirt3 might also
regulate ATP in vivo. We first took advantage of the known
variations in resting ATP levels in various tissue and organ beds. In
particular, we asked whether endogenous Sirt3 expression corre-
levels in pancreas, spleen, and skeletal muscle and higher levels in
heart, liver, and kidney. These resting levels of ATP appeared to
roughly, albeit not perfectly, correlate with the degree of Sirt3
expression in these organs. We then asked whether the absence of
Sirt3 could regulate organ-specific ATP levels. As noted in Fig. 1E,
analysis of matched pairs of wild-type and Sirt3?/?mice demon-
strated that in organs that normally express high amounts of Sirt3,
the absence of this mitochondrial deacetylase results in a ?50%
reduction in resting ATP. These differences in observed ATP
between wild-type and Sirt3?/?tissues could not be ascribed to
obvious differences in the level of expression for various compo-
nents of the mitochondrial electron transport (Fig. S2). In contrast
to what we observed in tissues expressing high levels of Sirt3, in
organs such as the pancreas, where we noted that normally there is
little to no Sirt3 expression, levels of ATP were not appreciably
different between wild-type and Sirt3?/?mice (Fig. 1E). We
therefore conclude that, consistent with the overall appearance of
the Sirt3?/?mice, the absence of Sirt3 does not produce a gener-
set and maintain tissue-specific levels of ATP.
We next asked whether these differences in ATP might result, at
least in part, to an alteration in ETC function when Sirt3 is absent.
To begin to address this hypothesis, we purified mitochondria from
biochemical analysis of hepatic mitochondria revealed a marked
change in the protein acetylation of Sirt3?/?mice that involved
multiple proteins. Similar results have been recently reported (11).
To more specifically pursue alterations in the ETC, we took
advantage of previously described methods that allow for the direct
In mice, Complex I is composed of ?40 separate proteins that
together function as an NADH dehydrogenase. Equal amounts of
liver protein lysate obtained from either wild-type or Sirt3?/?mice
was then used to immunocapture intact Complex I. These proteins
were subsequently resolved on an SDS/PAGE and acetylation
detected by using an antibody that recognizes internal acetyl-lysine
residues. As seen in Fig. 2B, the absence of Sirt3 results in an
increase in the acetylation of numerous proteins associated with
Complex I. In contrast, similar analysis with immunocaptured
between mitochondria isolated from the livers of wild-type or
Sirt3?/?mice (Fig. 2C).
ETC, treatment of HeLa cells with the broad-spectrum sirtuin
inhibitor, nicotinamide, led to markedly enhanced Complex I
Complex I acetylation was higher in cultured human HeLa cells
compared with isolated mouse liver mitochondria. We next asked
whether Sirt3 could directly deacetylate Complex I. Nicotinamide-
treated HeLa cells were used as a source to immunocapture
or Sirt4. Compared with samples exposed to Sirt4 or maintained in
deacetylase buffer alone, Complex I incubated in the presence of
exogenous Sirt3 protein demonstrated marked deacetylation of
numerous proteins (Fig. 2D). This result is consistent with both a
direct effect of Sirt3 on Complex I and with a previous result
demonstrating that Sirt4?/?mitochondria do not have significant
alterations in mitochondrial protein acetylation (11). Similar to
what we observed in vitro, transient overexpression in HeLa cells of
wild-type Sirt3 resulted in decreased acetylation of multiple pro-
teins comprising Complex I (Fig. 2E). In contrast, expression of a
deacetylase inactive form of Sirt3 [Sirt3(HY)] resulted in a modest
basal ATP in five independent isolates of primary wild-
type MEFs and a similar number of independent
Sirt3?/?MEF cell isolates. (B) Sirt3?/?MEFs were trans-
fected with an expression vector encoding GFP along
with an empty vector, epitope-tagged wild type, or a
deacetylase inactive (HY) Sirt3. Thirty-six hours after
transfection, GFP-positive cells were sorted by FACS
and ATP determined. ATP levels are expressed relative
to vector-transfected Sirt3?/?cells. Shown is the aver-
age of three independent experiments each per-
formed in triplicate. (C) HeLa cells were transfected
with an empty vector, epitope-tagged wild type, or
Sirt3(HY) and levels of ATP determined 48 h after
dent experiments. (D) Absolute level of ATP in various
tissues and organs of wild-type mice (n ? 3; mean ?
SD). Shown is the corresponding level of expressed
Sirt3 within each tissue as well as the 70-kDa complex
mitochondrial number and GAPDH for protein load-
ing. (E) Normalized levels of ATP in wild-type (black
bars) and Sirt3?/?mice (white bars) in various organs
with known high Sirt3 expression (heart, liver, and
kidney) as well as an organ (pancreas) with low or
group).*, P ? 0.01.
Sirt3 regulates basal ATP levels. (A) Levels of
www.pnas.org?cgi?doi?10.1073?pnas.0803790105Ahn et al.
increase in acetylation. This is consistent with other reports dem-
onstrating a dominant-negative effect of such inactivating muta-
tions (9, 17, 18).
Given that Sirt3 appears to preferentially regulate Complex I
acetylation, we asked whether there might be a physical association
between the deacetylase and the ETC. Using equal amounts of
HeLa cell protein lysate, we immunocaptured Complex I or Com-
plex II. These immunocaptured ETC complexes were then probed
for associated Sirt3. As noted in Fig. 3A, endogenous Sirt3 protein
noted that, in contrast to mouse Sirt3, human Sirt3 exists in two
forms, a short form (equivalent to mouse Sirt3) and a longer form
containing a 142-aa human-specific N-terminal extension (7). Our
data would suggest that in HeLa cells, the association between
Complex I and Sirt3 involved both the long and short form of
human Sirt3. To begin to understand the dynamics of this associ-
ation, we purified Complex I from HeLa cells exposed to various
I after either a 2- or 6-h period of nutrient withdrawal (no serum
or glucose). In contrast, brief treatment with hydrogen peroxide or
with the Complex I inhibitor rotenone led to the apparent disso-
ciation of the Sirt3–Complex I interaction. These observations
rotenone or hydrogen peroxide. Consistent with this notion, treat-
a significant fall in ATP levels in wild-type MEFs (Fig. 3C). In
contrast, as previously noted, although basal levels of ATP were
reduced ?30% in Sirt3?/?MEFs, the addition of rotenone had
relatively little effect on these Sirt3-deficient cells. A similar dif-
the mitochondrial poison cyanide, a cytochrome c oxidase (Com-
plex IV) inhibitor, produced a similar magnitude reduction in ATP
levels in wild-type and Sirt3?/?MEFs.
To further assess the association between Sirt3 and Complex I,
we noted that whereas the acetylation of multiple proteins in
Complex I were effected by the absence or overexpression of Sirt3,
in HeLa cells, a protein of ?39 kDa appeared to be affected in the
most dramatic fashion. To pursue the identity of this protein, we
relied on a recent general proteomic survey of acetylated proteins
that identified 10 of the ?40 subunits of Complex I as having one
or more acetylation sites (14). One previously identified acetylated
component of Complex I with an estimated molecular mass of 39
kDa is subunit 9 (NDUFA9), in which lysine 370 is acetylated (14).
by Sirt3, we immunoprecipitated all acetylated protein from either
MEFs or liver protein lysates and subsequently analyzed these
immunoprecipitated proteins for NDUFA9. As evident in Fig. 3 D
and E, both in vitro and in vivo, the absence of Sirt3 resulted in a
marked increase in NDUFA9 acetylation. Similarly, treatment of
whereas in HeLa cells, overexpression of wild type but not
Sirt3(HY) reduced NDUFA9 acetylation (Fig. S4). These data
support the notion that NDUFA9 is one of several components of
Complex I, whose acetylation is regulated by Sirt3. Although,
not suitable for immunoprecipitation, after transfection of an
between Sirt3 and endogenous NDUFA9 (Fig. 3F).
Because Sirt3 appeared to selectively regulate the acetylation
of Complex I with little or no effect on Complex II, we next
sought to functionally test whether the absence of Sirt3 could
lead to selective alterations in ETC function. Mitochondria were
ylation. (A) Western blot (WB) analysis for internal
acetyl-lysine residues using total liver mitochondrial
protein extracts from two age-matched wild-type
(?/?) or Sirt3?/?mice. The mitochondrial protein
VDAC1 is used as a loading control, and Sirt3 expres-
sion is also shown. (B) Levels of acetylation from
immunocaptured hepatic Complex I in a wild-type
versus Sirt3?/?mouse. Subunit 9 (NDUFA9) of Com-
plex I was used as a loading control for the immuno-
capture. (C) Similar acetylation analysis for immuno-
captured Complex II. The 70-kDa Fp subunit of
Complex II (C II-Fp) was used as a loading control. (D)
Sirt3 deacetylates Complex I in vitro. Complex I was
isolated from nicotinamide-treated HeLa cells and
incubated for 2 hours in vitro with deacteylase reac-
tion buffer only (?) or with reaction buffer contain-
ing purified Sirt3 or Sirt4. The level of the Complex I
protein NDUFA9 is shown as a loading control as is
the level of exogenously added Flag-tagged Sirt3
and Sirt4. (E) Level of in vivo Complex I acetylation in
HeLa cells transfected with empty vector (V), wild-
(HY). IP, immunoprecipitation; MW, molecular mass.
Sirt3 regulates mitochondrial protein acet-
Ahn et al.
September 23, 2008 ?
vol. 105 ?
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isolated from age-matched wild-type and Sirt3?/?mice, and
respiration was determined by using Complex I- and Complex
II-dependent substrates. As noted in Fig. 4 A and B, the use of
the Complex I-dependent substrates glutamate and malate and
the absence of Sirt3 results in a noticeable decrease in State
3-dependent respiration. Analysis of multiple wild-type and
Sirt3?/?littermates revealed an ?20% reduction in measured
Complex I activity, with no corresponding change in Complex II
activity (Fig. 4 C and D). Respiratory control ratios and uncou-
pled respirations were similar between wild-type and Sirt3?/?
mitochondria (Table S1). Next, in a fashion analogous to our
previous experiments where we had demonstrated efficient in
vitro deacetylation (see Fig. 2D), we sought to assess the direct
effects of Sirt3 on Complex I activity by incubating permeabil-
ized HeLa cell mitochondria with purified sirtuin proteins. In
this case, after mitochondria were exposed to purified sirtuin
proteins, we assessed the level of rotenone-sensitive NADH
oxidation, a direct measurement of Complex I activity. Although
Complex I activity was roughly similar in vector-treated samples
and Sirt4-treated mitochondria, incubation of HeLa cell mito-
cell lysate was used to immunocapture either Complex
I or Complex II. These ETC components were then
resolved on SDS/PAGE and probed for association with
Sirt3. Both the short and long form of human Sirt3
associates with Complex I. The purity of the immuno-
capture complexes are demonstrated by probing the
stripped blot for the Complex I component NDUFA9
association of endogenous Sirt3 with Complex I. Im-
munocaptured Complex I was probed for associated
Sirt3 under fed conditions (?), after 2 or 6 h of starva-
tion, after hydrogen peroxide (0.5 mM, 30 min) treat-
ment, or after rotenone (10 ?M, 30 min) treatment.
The arrows indicate the short and long form of human
Sirt3. Below, total levels of Sirt3 or the Complex I
component NDUFA9 were assessed for each condition
by using 30 ?g of mitochondrial protein lysate. (C) ATP
levels in wild-type (?/?) or Sirt3?/?MEFs under basal
conditions (black bars), or after a 30-min exposure to
bars), or hydrogen peroxide (0.5 mM; gray bars). Basal
levels of ATP are reduced in the Sirt3?/?cells and are
relatively resistant to rotenone or hydrogen peroxide
challenge but exhibit normal ATP sensitivity to cya-
(D) or liver protein lysates from wild type (?/?) or
Sirt3?/?mice (E). (F) HeLa cells were transfected with a
myc-tagged empty vector, wild-type Sirt3, or a deacetylase-inactive Sirt3(HY) and equal amounts of lysate immunoprecipitated with a myc-epitope antibody or
an irrelevant Flag-epitope antibody. Sirt3 immunoprecipitation reveals the presence of coprecipitated endogenous NDUFA9. IP, immunoprecipitation; WB,
Sirt3 associates with Complex I of the ETC. (A)
consumption using Complex I-dependent substrates
for liver mitochondria obtained from a wild-type (A)
rate for Complex I from intact liver mitochondria of
four wild-type and four Sirt3?/?mice (mean ? SD;*,
P ? 0.02). (D) Calculated State 3 respiration rate for
Complex II-dependent substrate (succinate ? rote-
none) in wild-type and knockout mice. NS, not signif-
icant. (E) Rates of NADH consumption for purified
HeLa cell mitochondria after in vitro incubation with
deacetylase buffer containing purified Flag-Sirt4
(green), Flag-Sirt3 (red), or Flag-vector (purple). Nor-
malized NADH absorbance was monitored at 340 nm.
The arrow indicates the time of addition of rotenone
(4 ?M final concentration). Shown is the mean rate
(?SD) of NADH consumption of triplicate determina-
tions from one of two similar experiments.
The absence of Sirt3 selectively affects Com-
www.pnas.org?cgi?doi?10.1073?pnas.0803790105Ahn et al.
chondria with exogenous Sirt3 led to increased Complex I
activity (Fig. 4E and Fig. S5).
basal ATP and, hence, overall energy homeostasis. In tissues such
as the liver, heart, and kidney that normally express high levels of
Sirt3, the absence of the deacetylase leads to marked reduction in
ATP. Surprisingly, we observed no outwardly observable untoward
effects of this marked energy reduction. A recent study also
concluded that Sirt3?/?mice appear to have normal weight, body
fat, oxygen consumption, activity levels, and food consumption
(11). These normal general metabolic parameters stand in contrast
to the significant reduction in ATP we observed within certain
Sirt3?/?tissues. It should be noted, however, that in organs such as
the heart, concentrations of ATP are normally in the millimolar
range (19). As such, even with a 50% reduction in ATP concen-
all intracellular enzymes. It will, however, be interesting to test in
future experiments whether the function of Sirt3?/?mice are
impaired under certain stress conditions, especially those condi-
tions that might result in increased energetic demand or reduced
nutrient or energy supply.
Based on reconstitution with either wild type or the deacetylase-
function of Sirt3 is necessary for maintaining ATP levels. Given
previous observations demonstrating that Sirt3 deacetylates GDH
I, the effects of Sirt3 on ATP levels are undoubtedly complex and
presumably represent the coordinated regulation of both substrate
presented here (Fig. 2A) as well as elsewhere (11, 14) suggest that
the list of Sirt3-dependent mitochondrial targets undoubtedly will
expand. Nonetheless, our results are supportive of the notion that
Complex I activity can be regulated by acetylation and deacetyla-
role in ETC function.
Our results also suggest that the association of Sirt3 with Com-
plex I is reversible and that either the Complex I inhibitor rotenone
or hydrogen peroxide exposure can lead to the apparent release of
Sirt3 from the ETC. Interestingly, strengthening the connection
with rotenone or hydrogen peroxide significantly reduces ATP in
wild-type, but not Sirt3?/?, MEFs. In contrast, treatment with the
Complex IV inhibitor cyanide resulted in nearly equivalent reduc-
tion in ATP levels in wild-type and Sirt3?/?MEFs. These obser-
vations are consistent with our other data demonstrating that the
association of Sirt3 with Complex I participates in setting and
maintaining optimal ATP levels. These results also raise the
hypothesis that Sirt3 might protect the respiratory chain from
oxidative stress and that ETC function after hydrogen peroxide
might be different in wild-type and Sirt3?/?MEFs. Surprisingly,
to a similar degree in both wild-type and Sirt3?/?MEFs after
glutathione before and after hydrogen peroxide were similar in
wild-type and Sirt3?/?MEFs (Fig. S7). Thus, we conclude that,
under the conditions used, the absence of Sirt3 does not appear to
alter the inhibition of mitochondrial oxygen consumption that
occurs after hydrogen peroxide exposure (20–22). Additional ex-
periments with extended ranges of concentrations of hydrogen
role of Sirt3 in the cellular response to oxidative stress await
Finally, it is of interest to note that Complex I is an NADH
dehydrogenase and that Sirt3 is, in turn, an NAD-dependent and
-regulated deacetylase. The direct physical association of Sirt3 with
Complex I suggests that Sirt3 might act as a rheostat for the ETC,
overall energy homeostasis (Fig. S8). This is also supported by our
observation that the interaction of Sirt3 with Complex I is sensitive
function and, hence, regulate the synthesis of acetyl CoA, Sirt3 is
uniquely poised to sense and/or respond to two separate interme-
diates, NAD and acetyl CoA, that are, in turn, critical outputs of
mitochondrial metabolism. Given that the absence of Sirt3 leads to
a dramatic increase in the acetylation of numerous mitochondrial
proteins and that this is accompanied by significant alterations in
basal ATP levels, our results suggest that reversible protein acet-
ylation might represents an underappreciated but fundamental
mechanism to regulate overall mitochondrial activity.
Materials and Methods
Generation of Sirt3 Knockout Animals. To construct the targeting vector, we
isolated recombinant phages containing overlapping regions of genomic DNA
fragment originating 5? to exon 2 of Sirt3 was subcloned into the NotI and XhoI
1 before formation of the final targeting construct designated ploxPneoSirt3.
TC1 ES cells were then transfected with NotI-linearized pLoxPneoSirt3 and se-
confirmed positive by Southern blot analysis. In brief, Southern blot analysis
involved digestion of genomic DNA with EcoRV and subsequent hybridization
with a 3? flanking probe. Wild-type allele generated an ?20-kb band, whereas
the recombined allele generated a 5-kb band.
germ-line transmission. Male mice bearing germ-line transmission were subse-
quently mated with female FVB EII-Cre mice (26) to generate complete deletion
of exons 2–4 of Sirt3. The animals were genotyped by either Southern blot
analysis or by PCR analysis using the following primers: F1; 5?-gagatccatcagcttct-
gtg, R1; 3?-ccctcaatcacaaatgtcgg, F2; 5?-gggagcactctcatactcta, R2; 3?-ttactgctgc-
ctaacgttcc. Primers F1 and R1 are located within intron 4 and amplify the wild-
type allele (450 bp). Primers F2 and R2 are located within intron 1, and the
combination of primers F2 and R1 amplify the deleted allele (486 bp).
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10%
DMEM containing 15% FCS. To transfect HeLa cells or MEFs, we used either
Fugene HD (Roche) or Effectene (Qiagen) according to the manufacture’s rec-
ommendations. For determining the effects of Sirt3 expression on HeLa cell ATP
levels, we transfected a single 10-cm plate with 10 ?g of the indicated plasmid
an ?70% transfection efficiency. For isolation of Complex I from transfected
HeLa cells, we, in general, transfected four 15-cm plates per indicated condition
and, when indicated, used 20 ?g of plasmid per plate. Forty-eight hours after
transfection, cells were lysed for ETC immunocapture as described below. For
were transfected with 1 ?g of GFP expression plasmid along with 5 ?g of an
empty vector, wild-type Sirt3, or the deacetylase-inactive Sirt3. Thirty-six hours
diately lysed for ATP analysis.
Constructs and Antibodies. The human wild-type Sirt3 expression and the cata-
lytically inactive HY mutant in which a single amino acid residue has been
modified (histidine-to-tyrosine at amino acid residue 248) were generated by
standard PCR amplification and subsequent cloning into a Myc-tagged expres-
in vitro deacetylase assay, we used Flag- rather than Myc-tagged Sirt3 or Sirt4
(Addgene). For immunodetection of human Sirt3, we used a commercial anti-
antibodies were used: acetylated-lysine polyclonal antibody, and tubulin (Cell
Signaling Technology), c-Myc monoclonal antibody (Santa Cruz Biotechnology),
the 39-kDa Complex I component NDUFA9 and COX IV antibody (Abcam), the
Complex II 70-kDa Fp subunit antibody, the Complex I 30-kDa subunit NDUFS3,
the Complex III 49-kDa Core1 subunit, and the Complex I and Complex II Immu-
Ahn et al.
September 23, 2008 ?
vol. 105 ?
no. 38 ?
nocapture kit (Mitosciences), VDAC1/porin (Invitrogen), GAPDH (Ambion), and
Flag antibodies (Sigma).
Mitochondrial Isolation, Acetylation Assays, and Immunoprecipitation. Mito-
chondria were isolated from cultured cells by incubating cells in Isolation Buffer
I [1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 50 mM Tris (pH 7.4), 10%
Glycerol] containing Complete EDTA-free protease inhibitor mixture (Roche).
procedures in accord with the manufacturer’s recommendation (Pierce). For
isolation of functional mitochondria from mouse tissues, we rapidly harvested,
The livers were then homogenized in this buffer with a glass–Teflon motorized
homogenizer. The mitochondrial fraction was isolated by differential centrifu-
at a concentration of 0.5 mg/ml before functional assessment.
The isolation of either Complex I or Complex II was achieved by using
isolated mitochondria with the relevant immunocapture beads (Mitoscience)
2 mg of mouse hepatic mitochondria or 1 mg of isolated HeLa cell mitochon-
dria for ETC complex isolation. For measurement of basal tissue expression of
organs and probed with the rabbit polyclonal antibody described above. For
detection of NDUFA9 acetylation, we immunoprecipitated overnight either
derived from either wild-type or Sirt3?/?mice with a polyclonal acetyl-lysine
antibody. All immunoprecipitated acetyl-lysine proteins were washed five
times in Isolation Buffer I and then resolved by SDS/PAGE and processed for
Western blotting to assess for specific levels of NDUFA9 acetylation.
To assess the in vitro deacetylation of Complex I, we used immunocaptured
Complex I purified from nicotinamide-treated HeLa cells (10 mM nicotinamide
for 16 h before harvest). Each reaction contained equal amounts of purified
NAD) for 2 h at 37°C along with the addition of purified Sirt3 or Sirt4. These
purified sirtuins were prepared by transfecting HeLa cells with expression con-
structs encoding either Flag vector, Flag-Sirt3, or Flag-Sirt4 (Addgene). We used
transfection, HeLa cells were harvested in Isolation Buffer I, and 2 mg of trans-
ity gel (Sigma). Purified proteins were subsequently eluted competitively with
five 1-column volumes of a solution containing 100 ?g/ml FLAG peptide (Sigma)
in TBS [50 mM Tris?HCl, 150 mM NaCl (pH 7.4)]. Isolated Complex I was then
samples purified as above but initially derived from HeLa cells transfected with
Flag-vector only. The reaction was stopped after 2 h by the addition of SDS-
loading buffer. The eluted Complex I was analyzed for acetylation by Western
blotting using the acetyl-lysine polyclonal antibody.
To assess the in vitro effects of Sirt3 on Complex I activity, we isolated mito-
chondria from HeLa cells in Isolation Buffer I as described above. Permeabilized
mitochondria (200 ?g of protein) were then incubated with an equal volume of
2? deacetylase reaction buffer in a total reaction buffer of 250 ?l. Where
or a similar volume obtained from Flag elution of Flag vector-transfected cells.
The mitochondria were incubated with Flag-eluted proteins for 45 min at 37°C
volume of 500 ?l. Absorbance of NADH was measured at 340 nm for 12 min,
of NADH metabolism that was rotenone sensitive.
ATP Measurements and Mitochondrial Respiration. ATP was measured by using
the ATP determination kit (Molecular Probes). For in vivo measurements, mice
samples then underwent two 10-s rounds of sonication. Lysates were than cen-
trifuged at 13,000 ? g for 30 min and the supernatant measured for protein
We always measured ATP from freshly isolated, not frozen, tissues. A similar
Assay kit II (Calbiochem) according to the manufacturer’s recommendation.
sucrose, 10 mM KCl, 10 mM Tris?HCl, and 5 mM KH2PO4at pH 7.2. Glutamate
(5 mM), malate (5 mM), and ADP (1 mM) were used to assay respiration
through complex I. Succinate (5 mM), rotenone (1 ?M), and ADP (1 mM) were
were determined after inhibition of mitochondrial ATPase with oligomycin.
ACKNOWLEDGMENTS. We are grateful to I. Rovira for help with manuscript
preparation. We thank B. Schwer, M. Hirschey, and E. Verdin (University of
California at San Francisco, CA) for the gift of Sirt3 antibody. This work was
supported by National Institutes of Health Intramural funds (to C.D. and T.F.)
and a grant from the Ellison Medical Foundation (to T.F.).
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