Molecular Cell 23, 607–618, August 18, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.06.026
Substrate and Functional Diversity
of Lysine Acetylation Revealed
by a Proteomics Survey
Sung Chan Kim,1Robert Sprung,1Yue Chen,1
Yingda Xu,1Haydn Ball,1Jimin Pei,1Tzuling Cheng,3
Yoonjung Kho,1Hao Xiao,4Lin Xiao,5
Nick V. Grishin,1,2Michael White,3Xiang-Jiao Yang,5
and Yingming Zhao1,*
1Department of Biochemistry
2Howard Hughes Medical Institute and
Department of Biochemistry
3Department of Cell Biology
University of Texas Southwestern Medical Center
Dallas, Texas 75390
4ImmuneChem Pharmaceuticals Inc.
204-8678 Greenall Avenue
British Columbia V5J 3M6
5Molecular Oncology Group
Department of Medicine
McGill University Health Centre
Quebec H3A 1A1
posttranslational modification that is known to play
a key role in regulating transcription and other DNA-
dependent nuclear processes. However, the extent of
this modification in diverse cellular proteins remains
largely unknown, presenting a major bottleneck for
lysine-acetylation biology. Here we report the firstpro-
teomic survey of this modification, identifying 388
acetylation sites in 195 proteins among proteins de-
rived from HeLa cells and mouse liver mitochondria.
In addition to regulators of chromatin-based cellular
processes, nonnuclear localized proteins with diverse
functions were identified. Most strikingly, acetyllysine
including many longevity regulators and metabolism
enzymes. Our study reveals previously unappreciated
roles for lysine acetylation in the regulation of diverse
cellular pathways outside of the nucleus. The com-
bined data sets offer a rich source for further charac-
terization of the contribution of this modification to
cellular physiology and human diseases.
Lysine acetylation is a reversible and highly regulated
posttranslational modification. The posttranslational
modification was initially discovered on histones about
four decades ago (Vidali et al., 1968). However, it took
another 30 years before the first nonhistone protein,
p53, was identified to be lysine acetylated (Gu and
Roeder, 1997). The modification regulates diverse pro-
tein properties including DNA-protein interactions, sub-
cellular localization, transcriptional activity, and protein
stability. In addition to its important roles infundamental
biology, lysine acetylation and its regulatory enzymes
(histone acetyltransferases and histone deacetylases
[HDACs]) are intimately linked to aging and several
major diseases such as cancer, neurodegenerative dis-
orders, and cardiovascular diseases (Blander and Guar-
ente, 2004; Carrozza et al., 2003; McKinsey and Olson,
2004; Yang, 2004a). Despite intensive research over the
past decade, fewer than 90 lysine-acetylated proteins
have been identified (Kouzarides, 2000; Yang, 2004a).
The diverse cellular and physiological functions of ly-
sine acetylation cannot be exclusively explained by its
templated processes. This implies a wider influence for
the modification among proteins. In particular, lysine
acetylation has the potential to be connected to calorie
restriction and longevity, because the activity of Sirt1,
a mammalian ortholog of the yeast deacetylase Sir2,
can be modulated by calorie restriction (Bordone and
Guarente, 2005). Sirt1 appears to target diverse nonhis-
to regulate different cellular functions including stress
response, apoptosis, and energy metabolism (Brunet
et al., 2004; Cohen et al., 2004; Hubbert et al., 2002;
Vaziri et al., 2001). Nevertheless, the mechanism under
which Sirt1 regulates various cellular pathways and
physiological functions, including calorie restriction, re-
in epigenetic regulation and in the regulation of onco-
genic processes, HDAC inhibitors have been developed
and are currently under more than 30 clinical trials as
therapeutic agents for both hematologic malignancies
and solid tumors (Egger et al., 2004). Suberoylanilide
hydroxamic acid (SAHA), the most advanced clinical
compound of HDAC inhibitors, affects cell growth,
differentiation, and apoptosis preferentially on a broad
spectrum of transformed cells, but not normal cells
(Kelly et al., 2003). However, effects on histones cannot
explain such specificity, as histones are ubiquitously
expressed in both normal and transformed cells. Unfor-
tunately, the downstream targets of SAHA, as well as
the mechanism of its antitumor activity, are largely un-
known. Identification and functional characterization of
acetyllysine residues in cytosolic proteins and signaling
molecules (Cohen and Yao, 2004; Kaiser and James,
2004), and demonstration of HATs and HDACs in the
cytosol and mitochondria (Cohen and Yao, 2004;
Kovacs et al., 2005; McKinsey and Olson, 2004; Michi-
shita et al., 2005; Schwer et al., 2002) further support
the notion that the modification may be ubiquitous in
cells. Despite this evidence, the scope of lysine acetyla-
tion is largely unclear, creating one of the major black
boxes for its functional studies in DNA-independent
System-wide analysis of lysine acetylation is a poten-
tially powerful approach toward dissecting the complex
networks of such a highly versatile modification. Never-
theless, proteomics of lysine acetylation is no less
challenging than that of other protein modifications.
nonacetylated proteins with a wide range of expression
levels. Enrichment of lysine-acetylated proteins or pep-
tides is therefore necessary prior to proteomics analysis
in order to reduce the complexity and dynamic range of
the cellular proteome. However, no systematic analysis
has been reported for lysine acetylation.
Here we describe a proteomics survey of the acetylly-
chondria by combining immunoaffinity purification of
tification by nano-HPLC/MS/MS analysis (Figure 1A).
This screening identified 388 lysine acetylation sites
from 195 proteins, dramatically extending the known in-
ventory of in vivo acetylation sites and substrates. Most
lysine-acetylated proteins identified in this study do not
have obvious roles in DNA-templated processes. More-
over, we identified 277 lysine acetylation sites in 133
mitochondrial proteins, thereby conclusively establish-
ing that lysine acetylation is an abundant posttransla-
tional modification in the mitochondrion. These results
clearly indicate that regulation of DNA-templated pro-
cess is just one of many roles that lysine acetylation
plays invivo. Integration ofthese data setsoffers a step-
ping stone toward understanding and characterizing
functional consequences of lysine acetylation in diverse
cellular regulatory schemes and in the pathophysiology
of human diseases.
Results and Discussion
Specific Detection of Lysine-Acetylated Proteins
To evaluate the specificity of anti-acetyllysine antibody,
we carried out immunofluorescent staining of HeLa cells
and revealed thattheantibody produced strong positive
signals in the nucleus and the midbody of the cells
two subcellular regions. Western blotting analysis with
nuclear and cytosolic protein extracts of HeLa cells
showed that multiple protein bands spanning a wide
mass range were detected in both extracts (Figure 2B).
Figure 1. Strategy for Proteomics Analysis of Lysine-Acetylated Proteins
(A) Cytosolic and nuclear protein fractions from HeLa cells were digested with trypsin. The resulting peptides were subjected to immunoaffinity
purification using an anti-acetyllysine antibody. The isolated peptides were analyzed by HPLC/MS/MS in an LTQ 2D ion-trap mass spectrometer
for peptide identification.
(B) An example MS spectrum with a peptide targeted for MS/MS analysis marked by an arrow.
(C) MS/MS analysis of the peptide marked in (B) led to identification of an acetylated peptide with sequence MCLVEIEK*APK (K* indicates
an acetylated lysine), unique to the NADH dehydrogenase75kDa subunit. The labels band ydesignatethe N-and C-terminal fragments,respec-
tively, of the peptide produced by breakage at the peptide bond in the mass spectrometer. The number represents the number of N- or
C-terminal residues present in the peptide fragment.
Signals from these bands could be competed away
efficiently by acetylated BSA (Figure 2C), but not non-
acetylated BSA (Figure 2B). Similarly, the antibody
detected numerous proteins in mitochondrial fractions
from either fed or fasted mice (Figures 2D and 2E).
These results indicate that the antibody is specific and
that many lysine-acetylated proteins remain to be
Immunoaffinity Purification and Identification
of Lysine-Acetylated Peptides
To identify lysine-acetylated peptides and map acetyla-
tion sites, agarose beads bearing immobilized anti-
acetyllysine antibody were used to affinity purify pep-
tides from a tryptic digest of a protein fraction of interest
(Figure 1A). The enriched peptides were analyzed by
nano-HPLC/MS/MS in an LTQ mass spectrometer (Fig-
ures 1B and 1C). The MASCOT search algorithm used
the resulting MS/MS spectra to identify peptide candi-
dates from a human protein database. To ensure the
quality of analysis, positive identifications were verified
by manual inspection of the MS/MS spectra (for the
procedure, see the Supplemental Data available with
this article online). The raw spectrum of each acetylated
peptide is available on our website (http://www4.
or upon request.
The analysis identified 388 lysine acetylation sites in
195 proteins, including 67 lysine acetylation sites in 37
sites in 38 proteins from the nuclear fraction, and 277
lysine acetylation sites in 133 proteins from mitochon-
dria (Figure 4, Tables S1 and S2). Only 13 of the 195
proteins were previously reported to be acetylated
(Table S1). Thus, this survey greatly expands the inven-
tory of lysine-acetylated proteins.
Verification of Lysine-Acetylated Peptides
To evaluate the quality of the proteomics data, three in-
dependent experimental approaches were taken. First,
a fraction of the newly identified lysine-acetylated pro-
teins was selected for confirmation by immunoprecipi-
tation and Western blotting analysis (Figure 3A). The
candidate proteins were selected based on commercial
availability of antibodies against them. Second, the
lysine-acetylated peptides were confirmed by MS/MS
spectra of synthetic peptides, a gold standard for verifi-
cation of peptide identification (Figure 3B, Figure S1).
Confirmation of lysine acetylation among all the candi-
date peptides, especially those with low MASCOT
scores, demonstrates that the peptide identification
algorithm and manual verification procedure were reli-
able. As the third approach, we analyzed how lysine
of the newly identified, lysine-acetylated proteins: p120
catenin, Rho GDI, and actin. The results indicate that the
acetylation is important for regulating the function of
each of these proteins (see below). Furthermore, identi-
fication of acetyl peptides corresponding to known
acetylation sites on core histones, p300, and CBP, as
well as to functionally important lysine residues on
aldolase, ING4, cyclophilin A, and TCP1, further support
the high quality of the data sets (Tables S1 and S2).
Lysine Acetylation in Diverse Groups of Proteins
Lysine acetylation substrates with diverse cellular func-
tions were identified, including the four core histones,
regulators of transcription and chromatin structure,
splicing and translational proteins, chaperones, cyto-
skeletal proteins, signaling proteins, and metabolic
enzymes (Figure 4, Table 1, Tables S1–S4). Most nota-
bly, 133 mitochondrial proteins were found to be acety-
lated on lysine, conclusively demonstrating that lysine
Figure 2. Detection of Lysine-Acetylated Proteins by Using Anti-Acetyllysine Antibody
(A) Immunofluorescent image of HeLa cells using anti-acetyllysine antibody, showing strong lysine acetylation in nuclear and midbody localiza-
tion of acetylated proteins (indicated by an arrow).
(B and C) Western blotting analysis of two protein fractions (S-100, cytosolic; NE, nuclear) without (B) or with (C) competition with lysine-
(D and E) Western blot analysis of two mitochondrial fractions from livers of either fed or fasted mice, without (D) or with (E) competition with
lysine-acetylated BSA. BSA was included at 5% by weight with an additional 150 mg BSA in buffers for (B) and (D) or 150 mg acetylated BSA
for (C) and (E).
Global Analysis of Protein Lysine Acetylation
acetylation is an abundant posttranslational modifica-
tion in that organelle.
RNA Splicing and Translation Factors
ing or translation factors. We identified acetylated pep-
tides from hnRNP A1, an uncharacterized protein with
a potential role in splicing, and two translation factors
(EF1a and eIF-5A), suggesting that acetylation may play
a role in regulating RNA splicing and translation. Discov-
ery of acetyllysine residues in these splicing and trans-
lation factors suggests regulation of the two cellular
processes by previously unappreciated processes that
await further characterization.
Our analysis identified lysine acetylation in chaperone
proteins, including Hsp70, Hsp27, Hsp90, two subunits
Figure 3. Experimental Verification of Lysine-Acetylated Peptides by MS/MS of Synthetic Peptides and Reciprocal Immunoprecipitation
(A) Verification of lysine acetylation by Western blot analysis. The protein of interest was immunoprecipitated with an antibody and probed with
an anti-acetyllysine antibody by Western blotting analysis, with or without lysine-acetylated BSA (Ac-K-BSA).
(B) Tandem mass spectrum of a tryptic peptide from HeLa nuclear extracts, which resulted in identification of acetylated peptide
SAPAPK*K*GSK*K*AVTK*AQK (K* indicates an acetylated lysine) from histone 2B.
(C) Tandem mass spectrum of the synthetic peptide corresponding to the sequence identified in (B), showing similar ion intensity.
of the TCP1 ring complex TriC, cyclophilin A, and FK506
binding protein 4. Of these, only Hsp90 was previously
known to be lysine acetylated, but its acetylation sites
have not been mapped. Acetylation of Hsp90 influences
et al., 2005). Identification of the acetylation site should
activities of the receptor. Because the acetylation site
of Hsp70 is located at its substrate binding region, the
modification may affect substrate recognition.
Cyclophilin, a peptidyl-prolyl isomerase, binds the
immunosuppressive drug cyclosporin A and modulates
T cell activation by inhibiting the phosphatase calci-
neurin. Cyclophilin is acetylated at K125, a residue
located in the loop surrounding the drug pocket and a
potential site of ionic interaction with calcineurin. Muta-
tion of K125 to either an anionic or neutral residue
(K125E or K125Q) has minimal effects on isomerase
activity and cyclosporin A binding but renders the
mutant protein unable to associate with calcineurin or
inhibit its phosphatase activity (Etzkorn et al., 1994).
Acetylation of K125 could affect the interaction of cyclo-
philin with calcineurin.
Remarkably, a broad cadre of proteins participating in
microfilament formation and dynamics was identified.
Actin itself is acetylated at lysine 61, a residue immedi-
ately adjacent to a critical arginine required for actin
observed within cofilin, thymosin b10, profilin, moesin,
and tropomyosin. To begin to examine the conse-
quences of lysine acetylation on actin dynamics, epithe-
lial cells were incubated briefly in the presence of phar-
macological HDAC antagonists or agonists. As shown
in Figure 5, exposure to the class I/II HDAC inhibitor,
tricostatin A, resulted in a dramatic accumulation of fila-
mentous actin, suggesting stabilized actin stress fibers.
In contrast, the agonist of class III HDACs, resveratrol,
potently inhibited stress fiber formation and/or stability
The Rho family GTPases are central regulators of
proteins described above, RhoGDI and p120 catenin
(p120), two proteins known to modulate RhoGTPase
function, were identified. RhoGDI selectively inhibits
Rac, Rho, or CDC42 activation and membrane localiza-
tion depending upon the phosphorylation of key regula-
tory residues in RhoGDI. To examine the potential con-
sequences of RhoGDI acetylation, the modified lysine
(K141) was replaced with glutamine or arginine to block
modification. Expression of RhoGDI in epithelial cells in-
and the K141R substitution did not grossly alter this
behavior. In contrast, the replacement of the acetylly-
sine with glutamine resulted in the accumulation of both
thickened stress fibers and numerous filopodia (Fig-
ure 5B). This observation suggests that the neutraliza-
tion of charge mediated by the acetylation of K141
may inhibit RhoGDI function and/or alter its G protein
specificity. Furthermore, p120 catenin (p120), a protein
Figure 4. Summary of Lysine-Acetylated Peptides and Proteins
Functional classification of lysine-acetylated proteins from HeLa cytosolic (S-100) fraction (A), HeLa nuclear fraction (B), and mitochondrial
fractions from either fed (C) or fasted (D) mouse liver, with the number of acetylated proteins in each functional group. Venn diagrams show
the number of lysine-acetylated peptides unique to and common between S100 and nuclear fractions (E) or fed mouse and fasted mouse liver
Global Analysis of Protein Lysine Acetylation
associated with E-cadherin in cell-cell adhesions, is
acetylated at three lysine residues, two of which are
known to be important for inhibiting Rho GTPase activa-
tion, thus promoting development of a dendritic mor-
phology in fibroblasts (Anastasiadis et al., 2000). The
three acetylable lysine residues are located in a seven-
residue lysine-rich stretch that is disrupted by a six-res-
idue insert in a rare alternative-spliced isoform (Fig-
ure 5C). Swapping the acetylated lysines with arginine
slightly decreased p120-induced ‘‘dendrite’’ formation
(Figure 5Dand datanotshown). Glutamine substitutions
completely abrogated this phenotype. These results
indicate that the acetylatable lysine residues are impor-
tant for p120’s functions in cells and suggest that acety-
lation may represent a new mechanism for regulation of
In addition to actin and microfilament regulators, two
intermediate filament proteins (Lamins A and C) were
also found acetylated. Our observations that the HDAC
inhibitor and activator regulate cytoskeleton structure
and that lysine acetylation is present among all the three
classes of cytoskeletal proteins further suggest that this
modification could represent a common mechanism for
modulation of the cytoskeleton structures.
Cytosolic Metabolic Enzymes
No acetylation has been previously identified among
metabolic enzymes in mammalian eukaryotes. In this
study, five cytosolic metabolic enzymes were found to
belysineacetylated: aldolase, enolase,triosephosphate
isomerase 1, phosphoglycerate mutase 1, and transke-
tolase. Lysine acetylation is likely to affect activities of
some of these enzymes. One good candidate is the gly-
colytic enzyme aldolase (or fructose 1,6-bisphosphate
aldolase), which is acetylated at K12, K41, and K146.
of the conserved K146 among orthologs dramatically
decreased enzymatic activity, by a factor of more than
500 with K146R and a factor of more than 1 3 105with
Table 1. List of Acetylated Longevity-Related Proteins and Acetylated Mitochondrial Dehydrogenases
Protein Namegi Functional Group
Fumarate hydratase 1*
Malate dehydrogenase 2
Isocitrate dehydrogenase 2*
NADH dehydrogenase (ubiquinone) flavoprotein 1*
NADH dehydrogenase (ubiquinone) Fe-S protein 1*
NADH dehydrogenase (ubiquinone) Fe-S protein 6*
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2*
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4*
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5*
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9*
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10*
NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3*
NADH-ubiquinone oxidoreductase subunit B17.2 , complex I*
Ubiquinol-cytochrome c reductase complex 11 kDa protein*
Succinate dehydrogenase Fp subunit
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g*
ATP synthase, H+ transporting, mitochondrial F1 complex, gamma 1*
Acyl-coenzyme A dehydrogenase, short/branched chain
Very-long-chain acyl-CoA dehydrogenase
Electron transferring flavoprotein, dehydrogenase
L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain
Hydroxyacyl-coenzyme A dehydrogenase
Hydroxyacyl-coenzyme A dehydrogenase beta subunit
17beta-hydroxysteroid dehydrogenase IV
Isovaleryl coenzyme A dehydrogenase
Aldehyde dehydrogenase family 6, subfamily A1
Aldehyde dehydrogenase family 4, member A1
Glutamate dehydrogenase 1
Phosphoglycerate mutase 1*
Triosephosphate isomerase 1*
Aldehyde dehydrogenase 2
Hydroxysteroid dehydrogenase-5, delta<5>-3-beta
PHD finger protein 15*
Fatty acid metabolism
Fatty acid metabolism
Fatty acid metabolism
Fatty acid metabolism
Fatty acid metabolism
Fatty acid metabolism
Fatty acid metabolism
Amino acid metabolism
Amino acid metabolism
Amino acid metabolism
Amino acid metabolism
Amino acid metabolism
Amino acid metabolism
Ketone body metabolism
Steroid hormone metabolism
Longevity-related proteins are marked with an asterisk (Browner et al., 2004; Hamilton et al., 2005). Protein name, gene index number, functional
classification, and number of lysine acetylation sites are specified.
K146Q (Morris and Tolan, 1994). Acetylation of K146 is
likely to have an effect similar to that of the K146Q muta-
tion. In addition to serving as an enzyme, aldolase has
other cellular activities, including actin binding. The
actin binding residues are located around the catalytic
pocket of the enzyme (Wang et al., 1996). Thus, in addi-
tion to modulating enzymatic activity, lysine acetylation
actin filaments. Similar to cytosolic metabolic enzymes,
many metabolic enzymes from the mitochondrion may
also be subject to dynamic regulation by lysine acetyla-
tion (see below).
Our analysis revealed lysine acetylation in three sig-
naling molecules: Rho GDI, annexin V, and phospholi-
paseCb1.Phospholipase Cb1catalyzesakey reaction
in diverse intracellular signal transduction pathways,
generating two second messengers, inositol 1,4,5-
trisphosphate, and diacylglycerol, from phosphatidy-
linositol 4,5-bisphosphate. The protein is activated by
diverse signals through G protein-coupled receptors
and receptor tyrosine kinases. Annexin V is a calcium-
dependent phospholipid binding protein that can inhibit
phospholipase A2 and protein kinase C. Identification
Figure 5. Acetylatable Lysine Residues of Rho GDI and p120 Catenin Impact Actin/Cytoskeleton Dynamics
(A) HeLa cells were treated for 2 hr with the indicated compounds. Filamentous actin was visualized with rhodamine-conjugated phalloidin (NT,
no treatment; TSA, tricostatin A; res, resveratrol).
(B) HeLa cells transiently expressing the indicated RhoGDI variants were stained with rhodamine-phalloidin to visualize F actin as in (A). Myc
epitope-tagged RhoGDI expression was verified by immunostaining (data not shown).
(C) Schematic representation of p120 isoforms and point mutants. Rectangles represent Armadillo-repeat domains. The sequence of residues
621–627, a lysine-rich stretch located between repeats 6 and 7, is shown with the three acetylatable residues shown in red. The p120 gene con-
tains three exons (A, B, and C) for alternative splicing. p120A, a major isoform, contains a peptide encoded by exon A, whereas p120AC
possesses this as well as a hexapeptide, encoded by the rare C exon (green) (Anastasiadis et al., 2000) inserted after K624. The KR mutant
(KR mut) contains arginine substitution of the three acetylatable lysines, and in the KQ mutant, these residues are replaced with glutamine.
cells were subjected to indirect fluorescence microscopy by immunostaining with anti-HA antibody and Cy3-labeled secondary antibody. The
images for p120 subcellular localization (a-HA staining) and nuclei (Hoechst staining) are shown, as well as their merged images. Unlike p120
Global Analysis of Protein Lysine Acetylation
of lysine-acetylated signaling proteins implies that ly-
sine acetylation might be involved in crosstalk between
signal transduction pathways.
Chromatin and Transcription Regulators
Chromatin binding proteins, such as high-mobility
group (HMG) chromosomal proteins, are known to be
acetylated. Our analysis identified K30 of HMG-1 as
a new acetylation site. Because K30 is located within
the DNA binding domain, acetylation at this site is likely
to affect the DNA binding affinity or DNA-bending ability
of HMG-1. Among chromatin-modifying enzymes, his-
tone acetyltransferases are known to be autoacetylated
(Yang, 2004b). Consistent with this, we identified acety-
lated peptides from p300, CBP, and MOZ (Table S1).
Autoacetylation of p300 is important for stimulating its
acetyltransferase activity (Thompson et al., 2004). For
p300, the four sites identified here correspond to the
previously mapped autoacetylation sites (Thompson
et al., 2004). These four lysine residues are conserved
in CBP and were also found to be acetylated, providing
further evidence of the high quality of the data sets.
ing enzymes. First, RbAp46, a core subunit of the Sin3a
and NuRD deacetylase complexes, is acetylated at
a lysine residue close to several WD40 repeats. This
WD40-repeat protein is also a key subunit of two other
complexes: chromatin assembly factor and HAT1 com-
plex. Second, ING4 is acetylated at three lysine residues
of ING4 is subject to frequent mutations in cancer cell
ing the acetyltransferase HBO1 and PHD finger protein
15 (also known as Jade2) (Doyon et al., 2006). Like
are acetylated. The histone methyltransferase MLL3 is
acetylated at K1869, while the DNA methyltransferase
DNMT1 is acetylated at three lysines on a GK repeat
linker. These repeats link the methyltransferase domain
resemble the N-terminal flexible tails of core histones.
Finally, lysine acetylation is present in proteins involved
in assembling, compacting, and remodeling chromatin.
protein) and SMARCA2 (an SNF2 homolog). Together,
these results support the notion that components of
chromatin, as well as chromatin-utilizing proteins, are
subjects of dynamic lysine acetylation.
Our proteomics survey also identified 22 acetylation
sites in core histones, including seven new and 15
known sites. The new sites are K11, K16, and K23 in
H2B; K36 in H3; and K4, K7, and K11 in an H2A isoform
(these three lysines are not conserved in the canonical
H2A sequence). Although functional roles of acetylation
at these newly identified sites remain to be established,
acetylation of K36 on H3 suggests a competition with
methylation on the same residue. Methylation of K36 is
a key modification event during transcriptional elonga-
tion (Margueron et al., 2005; Martin and Zhang, 2005).
This potential interplay is evocative of competitive acet-
ylation and methylation of K9 on H3.
A significant fraction of known lysine-acetylated pro-
teins, especially transcription factors, were not identi-
fied in this experiment. This could be caused by low ex-
pression levels of these proteins in HeLa cells (e.g., p53)
or by inhibitory effects of abundant, lysine-acetylated
proteins such as histones. Alternatively, those proteins
tightly associated with chromatin might not have been
extracted using our protocol for preparing nuclear ex-
tracts. Prior fractionation of the cell lysate with high res-
olution is needed to address the problem in the future.
Lysine Acetylation Is Abundant in the Mitochondrion
Our study shows that lysine acetylation is a common
posttranslational modification in the mitochondrion.
Proteomics analysis identified 277 unique acetylation
sites in 133 proteins from two fractions of mouse liver
mitochondria, one from fed mice and the other from
fasted mice (Figure 4). To our knowledge, in none of
these peptides and proteins has lysine acetylation been
previously observed. Among the acetylated proteins,
62% were identified in both fractions, 14% were specific
to fed mice, and 24% were specific to fasted mice. Most
lysine-acetylated proteins from mitochondrial fractions
were metabolic enzymes (91 proteins). ATP synthase
Fosubunit 8, one of 15 proteins encoded by mitochon-
drial DNA, was found to be acetylated, implying that
the acetylation reaction can occur in mitochondria.
Our results suggest that w20% of mitochondrial pro-
teins are lysine acetylated, if we assume our analysis is
as sensitive as previous proteomics studies that identi-
fied w600 mitochondrial proteins (Mootha et al., 2003;
Taylor et al., 2003). Identification of such a large number
of lysine-acetylated mitochondrial proteins raises ques-
tions for future study. For example, which lysine acetyl
transferases and deacetylases are responsible for con-
tions really occur within the mitochondrion?
Energy Metabolism Proteins
Of the lysine-acetylated mitochondrial proteins that are
annotated with possible functions, more than half are in-
volved in some aspect of energy metabolism (Table S2).
Lysine-acetylated proteins are present among all the
tricarboxylic acid (TCA) cycle proteins, 26 proteins in-
volved in oxidative phosphorylation, 27 b-oxidation or
lipid metabolism proteins, eight associated with amino
acid metabolism, ten with carbohydrate metabolism,
three with nucleotide metabolism, and two with the urea
cycle. Transporter and channel proteins also play an im-
portant role in regulating energy metabolism. In this
study, 15 transporters or channel proteins were found to
be lysine acetylated. Although it remains unclear how
lysine acetylation of metabolic enzymes regulates their
state of these proteins influences the mode or rate of
energy production or other mitochondrial functions.
Notable among the acetylated metabolic enzymes are
the dehydrogenases, a group of enzymes that oxidize a
substrate by transferring hydrogen to an acceptor,
usually NAD, NADP, or a flavin coenzyme. Forty-eight
dehydrogenases were identified in previous proteomics
studies of mitochondrial proteins (Mootha et al., 2003;
Taylor et al., 2003). Our proteomics screening found that
at least 44% of mitochondrial dehydrogenases (21 pro-
tein complexes) were lysine acetylated. Among them,
14 use NAD as the electron acceptor to catalyze the
reactions in oxidative, catabolic routes.
Enzymatic activities of some of these proteins (e.g.,
the PDH complex and isocitrate dehydrogenase) are
modulated by the cellular energy charge (e.g., NAD+/
NADH ratio or acetyl-CoA level). On the other hand,
acetyl-CoA is used as a substrate for HATs, while NAD
is a cofactor for class III HDACs. NAD concentration
was suggested previously to modulate enzymatic activ-
ities of class III HDACs (Blander and Guarente, 2004).
Thus, it is tempting to propose that lysine acetylation
serves as a feedback mechanism for regulation of de-
hydrogenase activities in mitochondria in response to
cellular energy charge.
Acetylated mitochondrial dehydrogenases include
dihydrolipoyl dehydrogenase, nine subunits of NADH
dehydrogenase (oxidative phosphorylation complex I),
four TCA cycle-related dehydrogenases (fumarate hy-
dratase, succinate dehydrogenase, isocitrate dehydro-
genase, and malate dehydrogenase), five proteins in-
volved in b-oxidation, and ten proteins involved in other
metabolic reactions. For most of these enzymes, aber-
rant changes of their expression or functions lead to
a potential role for lysine acetylation in these diseases.
Longevity-Related Mitochondrial Proteins
Are Lysine Acetylated
Proteomics analysis identified acetyllysine in 16 pro-
teins believed to influence longevity (Table 1), including
three oxidative phosphorylation proteins (ATP synthase
g subunit, NADH dehydrogenase, NADH-ubiquinone
oxidoreductase), two proteins in the TCA cycle (fuma-
rate hydratase, isocitrate dehydrogenase), and two anti-
oxidant enzymes (superoxide dismutase 1, superoxide
dismutase 2). In addition to the mitochondrial proteins,
four lysine-acetylated proteins from cytosolic or nuclear
fractions are also involved in the regulation of aging.
These proteinsinclude Lamin A/C(an alternately spliced
nuclear matrix protein), profilin 1 (an actin binding pro-
tein), phosphoglycerate mutase (a glycolytic protein),
and a eukaryotic initiation factor (elF-5A). The roles of
ics analysis or RNAi screening (Browner et al., 2004;
Hamilton et al., 2005).
Regulatory roles of Sir2, Sir2 activators (e.g., resvera-
trol), and HDAC inhibitors (e.g., phenylbutyrate) in aging
or longevity pathways imply a link between lysine acet-
ylation and aging. It remains unclear, however, how this
modification affects aging and longevity. The lysine-
acetylated proteins related to aging and longevity iden-
tified in this study provide important leads for further
characterization of the roles of lysine acetylation in
Mechanistic Impact of Lysine Acetylation
As discussed above, analysis of the identified acetyla-
tion sites indicates that this modification exerts its
impact via alteration of protein-protein interactions, nu-
clear localization, enzymatic activity, and interplay with
other lysine modifications (e.g., methylation and sumoy-
lation). One striking feature of the group of acetylated
proteins identified here is that many are metabolic en-
zymes, which implies that alteration of enzymatic activ-
ity may emerge as a major consequence of acetylation.
Lysine acetylation also occurs in known or putative NLS
sequences (Table S5); therefore, change of subcellular
localization may be another effect of this modification.
to other posttranslational modifications, such as meth-
ylation, ubiquitination, and sumoylation. On a single ly-
Acetylation sites in 11 proteins conform to the sumoyla-
tion consensus sequence (Table S4), suggesting that
acetylation competes with sumoylation. Consistent
with this hypothesis, acetylation of p300 inhibits its su-
moylation (Bouras et al., 2005). Unlike sumoylation, con-
sensus sequences for methylation and ubiquitination
have not been defined. Further studies of the acetylated
proteins identified here will certainly reveal interplay of
acetylation with these two modifications, as has been
nicely shown for histones and the p53 tumor suppressor
(Berger, 2002; Brooks and Gu, 2003; Strahl and Allis,
2000). Thus, the large data sets compiled here (Tables
S1 and S2) present a golden opportunity for further
study of interplay among different lysine modifications.
Analysis of Lysine Acetylation Sites
Identification of the large set of acetylation sites here
made possible, for the first time, a preliminary analysis
of motif preference (Figure 6). The residue preference
for acetylated peptides from nonhistone S100 proteins
is asparagine at the 21 position, while histidine at the
+1 position is favored for nonhistone proteins from
nuclear extract. For histone proteins, a propensity for ly-
served. Lysine-acetylated peptides from mitochondria
show different preferences in their flanking sequences,
namely histidine or tyrosine at the +1 position (Figure 6).
The preference was more striking when the density map
was normalized by the relative abundance of amino acid
residues in human or mouse proteins (Supplemental
The differences in preference of amino acid residues
flanking acetyllysineresidues suggestthatthemodifica-
tion might be catalyzed by a subset of acetyltrans-
ferases with substrate preferences unique to the sub-
cellular organelles. It is highly likely that linear lysine
acetylation motifs exist among lysine-acetylated pro-
teins similar to motifs observed for phosphorylation.
Local Structural Properties of Acetylated Lysines
Acetylated lysine shows a different preference for sec-
ondary structure than nonacetylated lysine. Acetylated
lysines are found in a helical conformation about 9%
more frequently than the average lysine and in a coil
about 8% less frequently (both comparisons are statis-
tically significant with p < 0.03; see Supplemental Data
for the analytical method used). Thus, acetylation ap-
pears to be favored in the context of specific secondary
structural characteristics. Acetylated lysines have an
average relative side chain solvent accessibility of
61.5, which is higher than that of all lysines (50.5), sug-
gesting that acetylated lysines prefer more exposed en-
vironments. Of all the lysines in these proteins, about
14% are predicted to be in disordered regions (at a false
positive rate threshold of 5%). In contrast, only 7.4% of
acetyllysines are predicted to be in disordered regions.
This result is consistent with the secondary structure
Global Analysis of Protein Lysine Acetylation
type analysis. Acetylation sites appear to have local
structural preferences that are quite different from those
of protein phosphorylation sites; phosphorylation sites
have a strong correlation with disordered regions
(Iakoucheva et al., 2004).
A major difficulty in the field of lysine acetylation biology
is the unavailability of lysine acetylation substrates.
Lysine-acetylated proteins cannot be as easily detected
as protein phosphorylation, because [14C]-acetyl-CoA
has low radioactivity and anti-acetyllysine antibody
has low affinity. Such a challenge is well represented
by the fact that few lysine-acetylated substrates have
been identified in organelles other than nuclei.
The proteomic survey presented here represents the
first systematic analysis of lysine-acetylated proteins,
identifying 388 acetylation sites in 195 proteins. Thus,
our study significantly expands the inventory of proteins
containing this modification. In addition, we demon-
strated that lysine acetylation is abundant in mitochon-
ylation and mitochondrial functions. Identification of
previously unknown lysine-acetylated proteins, with di-
verse cellular functions and from multiple cellular com-
partments, implies that lysine acetylation is involved
in the regulation of cellular pathways far beyond DNA-
The finding that more than 20% of mitochondrial pro-
teins are acetylated is totally unexpected. Acetyl-CoA
and NAD (or NAD-to-NADH ratio) are the key indicators
as a substrate for lysine acetylation by HATs, while NAD
Figure 6. Bioinformatics of the Lysine-Acetylated Sites
The relative abundance of each amino acid residue (including acetylated lysine) surrounding sites of lysine acetylation was calculated and sche-
matically represented by density maps. Prevalence of specific amino acids at positions surrounding lysine acetylation sites are shown for
(A) S100 (cytosolic) fraction (excluding histones), (B) nuclear extract (excluding histones), (C) histones, and (D) mitochondrial fractions from
both fed- and starved-mouse mitochondria. (E) Distribution of acetylated and nonacetylated lysine residues in protein secondary structures.
groups. Identification of acetyllysine residues in a large
number of metabolic enzymes and longevity proteins
tory pathway of energy metabolism. Lysine acetylation,
together with the concentrations of acetyl-CoA and
NAD, might serve as a liaison connecting cellular energy
level with HAT/HDAC activities and enzymatic activities
of acetylated substrates.
At the mechanistic level, lysine acetylation not only
neutralizes a positive charge but also increases the hy-
drophobicity and size of lysine’s side chain. As ex-
pected, the modification is likely to induce significant
conformational changes of substrate proteins, possibly
leading to changes in function. Identification of lysine
acetylation sites among proteins with known or pre-
dicted biological function confirms previously estab-
lished mechanisms of functional regulations and sug-
gests new ones. The impact of lysine acetylation on
protein function is reminiscent of the effects of protein
In eukaryotic cells, each protein may be subject to
multiple modifications, which can interact with each
other to form a dynamic regulatory program (Pawson
tionsites, as illustrated for acetylation sitesin this study,
is one of the first steps toward understanding the funda-
mental question how such a program operates in vivo.
Given the critical roles of lysine acetylation in regulating
diverse cellular physiology, it is likely that proteins regu-
lating lysine acetylation (HDACs and HATs) and their
substrates constitute a rich source of candidate pro-
teins relevant to human disease and therapeutic targets
for drug design. In addition, the acetylation data sets
generated in this study could drive experimental efforts
toward functional characterization of lysine-acetylated
proteinsand theanatomy oflysine acetylation pathways
specific to human diseases.
Cell Culture, Transfection, and Immunocytochemistry
Suspension HeLa S3 cells were grown in DMEM supplemented with
5% newborn calf serum, nonessential amino acids, 100 units/ml
penicillin, and 100 mg/ml streptomycin in 2 liter round-bottom side-
arm spinner flasks. Cells were treated with 0.3 mM tricostatin A
(TSA; class I/II HDAC inhibitor) and 50 mM of sirtinol (class III
HDAC inhibitor) at a density of 5 3 105cells/ml for 24 hr before har-
vesting, for preparation of cytosolic and nuclear extracts.
HeLa adhesion cells and NIH 3T3 cells were grown in DMEM sup-
plemented with 10% fetal bovine serum and 10% newborn calf se-
rum, respectively. Transfections were carried out using Superfect
transfection reagent for NIH 3T3 cells and Lipofectamine 2000 for
For subcellular localization of acetyllysine proteins, HeLa cells
were fixed and stained with anti-acetyllysine polyclonal antibody
(0.65 mg/ml) using a 1:50 dilution with or without competition with
50 mg of acetylated BSA. To assess the effects of acetylation on
stress fiber formation, HeLa cells were treated with 1 mM TSA or
0.2 mM resveratrol (class III HDAC agonist) or were left untreated
for 2 hr. Rhodamine-conjugated phalloidin was used to visualize
Cytosolic Extracts, Nuclear Extracts, and Mitochondrial Lysates
Preparation of cytosolic (S-100) and nuclear extracts from HeLa S3
cells, and protein lysate from mouse liver mitochondria, is described
in detail in Supplemental Experimental Procedures.
In-Solution Tryptic Digestion
Ten milligrams of proteins of interest were precipitated with ace-
tone, followed by centrifugation at 22,000 3 g for 10 min. The result-
ing pellet was rinsed twice with cold acetone to remove residual
salts, resuspended in 50 mM NH4HCO3(pH 8.5), and digested with
trypsin at an enzyme-to-substrate ratio of 1:50 for 16 hr at 37?C.
The tryptic peptides were reduced with 5 mM DTT at 50?C for
30 min and then alkylated using 15 mM iodoacetamide at RT for
30minindarkness. The reaction wasquenchedwith 15mMcysteine
at RT for 30 min. To ensure complete digestion, additional trypsin
at an enzyme-to-substrate ratio of 1:100 was added to the peptide
mixture and incubated for an additional 3 hr. The peptides were
dried in a SpeedVac.
Affinity Purification of Lysine-Acetylated Peptides
The affinity-purified anti-acetyllysine antibody (ImmunoChem Phar-
maceuticals Inc., Burnaby, British Columbia, Canada) was immobi-
lized onto protein A-conjugated agarose beads at 3–4 mg/ml by in-
cubation at 4?C for 4 hr. The supernatant was removed and the
beads were washed three times with NETN buffer (50 mM Tris$HCl
[pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP40).
The tryptic peptides obtained above were resolubilized in NETN
buffer. Insoluble particles were removed by centrifugation at
100,000 3 g for 10 min. Affinity purification was carried out by incu-
immobilized agarose beads at 4?C for 6 hr with gentle shaking. The
beads were washed three times with 1 ml of NETN buffer and twice
with ETN (50 mM Tris$HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA). The
bound peptides were eluted from the beads by washing three times
with 50 ml of 0.1% TFA. The eluates were combined and dried in
a SpeedVac. The resulting peptides were cleaned in mC18 ZipTips
instructions, prior to nano-HPLC/mass spectrometric analysis.
Protein Identification by Nano-HPLC-MS/MS and Manual
Verification of Identified Peptides
The procedures are detailed in Supplemental Experimental Proce-
perimental Procedures, Supplemental Results, and Supplemental
References and can be found with this article online at http://
This work was supported by The Robert A. Welch Foundation
(I-1550 to Y.Z. and I-1414 to M.W.), NCI/NIH’s ‘‘Innovative Technol-
ogies for the Molecular Analysis of Cancer’’ program (CA107943 to
Y.Z.), the National Cancer Institute of Canada (to X.-J.Y.), and the
Canadian Institutes for Health Research (to X.-J.Y.). We thank Steve
McKnight and Shanhai Xie for preparation of liver mitochondria and
Qin, and Helen Yin for their insightful suggestions. We also thank
John Cottrell from MatrixScience for his assistance.
Received: February 6, 2006
Revised: May 3, 2006
Accepted: June 27, 2006
Published: August 17, 2006
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