JOURNAL OF P RO
J O U RN A L OF P R O TE O MI CS 74 ( 20 1 1 ) 4 3 1– 4 4 1
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α
N
-Acetylation
N -Acetylation of yeast ribosomal proteins
and its effectof
onyeast ribo
protein synthesis
protein synthesis
α
a
a
c
Kamita
, Yayoi
Masahiro Kamitaa , Yayoi Kimuraa , Yoko Inoa , Roza M. Masahiro
Kampb , Bogdan
Polevoda
, Kimura , Yoko
c
a,⁎
c
Fred Sherman , Hisashi Hirano
Fred Sherman , Hisashi Hiranoa,⁎
a
Graduate School of Nanobioscience, Yokohama City University, Suehiro 1-7-29, Tsurumi,
Yokohama 230-0045, Japan
a
Graduate School of Nanobioscience, Yokohama City Universit
University of Applied Science and Technology, 13347 Berlin, Germany
b
University of Applied Science and Technology, 13347 Berlin, G
c
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester,
NY 14642, USA
c
b
Department of Biochemistry and Biophysics, University of Roc
AR TIC LE I N FO
ABS TR ACT
Article history:
Nα-Acetyltransferases (NATs) cause the Nα-acetylation of the majority of eukaryotic
Received 21 November 2010
proteins during their translation, although the functions of this modification have been
Accepted 15 December 2010
largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA,
Available online 22 December 2010
NatB, NatC, and NatD. In this study, the Nα-acetylation status of ribosomal protein was
AR TIC LE IN FO
ABS TR A
Article history:
Nα-Acetyltransf
Received 21 November 2010
proteins during
Accepted 15 December 2010
largely unexplor
analyzed using NAT mutants combined with two-dimensional difference gel
Available online
December
2010
electrophoresis (2D-DIGE) and mass spectrometry
(MS). A22total
of 60 ribosomal
proteinsNatB, NatC, and
Keywords:
α
were identified, of which 17 were Nα-acetylated by NatA, and two by NatB. Theanalyzed usin
N -Acetylation
Nα-acetylation of two of these, S17 and L23, by NatA was not previously observed.electrophoresis
Ribosome
Furthermore, we tested the effect of ribosomal
protein Nα-acetylation on protein synthesiswere identified
Keywords:
α mutant. It was found that the protein synthesis α
using the purified ribosomes from each N
NAT
-Acetylation
Ribosomal protein
2D-DIGE
N -acetylation
activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%,
Ribosome
Furthermore, w
somal proteins were separated using 2-DE
CBB staining (Fig. 1C). A total of 59 protein
proteins, we analyzed the ribosomal proteins in t
strain and the NAT mutants using 2D-DIGE, and
Fig. 1 – Analysis of rRNAs and Nα-acetylated ribosomal proteins by 2D-DIGE. (A) Purified rRNA from yeast 80S ribosomes was
separated by agarose gel electrophoresis and stained with ethidium bromide. (B) Purified ribosomal proteins from yeast 80S
ribosomes were separated by SDS-PAGE and stained with CBB R-250. The details of MS data using an ESI-LIT-TOF MS was
shown in Supplementary Table 1. (C) Purified ribosomal proteins from yeast 80S ribosomes were separated by 2-DE using acidurea electrophoresis in the first dimension and SDS-PAGE in the second dimension and stained with CBB R-250. Protein spots
identified by an ESI-Q-TOF-MS are numbered and their details were indicated in Supplementary Table 2. (D–F) Identification of
the Nα-acetylated ribosomal proteins from the nat1, nat3, and mak3 mutants, respectively. Equal amounts of purified ribosomal
proteins from the normal strain and the NAT mutant were labeled with Cy3 and Cy5 respectively. The Cy-labeled ribosomal
proteins from the two strains were separated on the same 2-DE gel and the Cy3- and Cy5-images were compared. The
α
Fig. 1 – Analysis of rRNAs and Nα-acetylated ribosomal proteins by 2D-DIGE. (A) Purified rRNA from yeast 80S ribosomes was
separated by agarose gel electrophoresis and stained with ethidium bromide. (B) Purified ribosomal proteins from yeast 80S
ribosomes were separated by SDS-PAGE and stained with CBB R-250. The details of MS data using an ESI-LIT-TOF MS was
shownof
inrRNAs
Supplementary
1. (C) Purified
ribosomal
proteinsby
from
yeast 80S(A)
ribosomes
separated
by 2-DE
using
acid– Analysis
and NαTable
-acetylated
ribosomal
proteins
2D-DIGE.
Purifiedwere
rRNA
from yeast
80S
ribosomes
wa
urea electrophoresis in the first dimension and SDS-PAGE in the second dimension and stained with CBB R-250. Protein spots
ated by agarose gel electrophoresis and stained with ethidium bromide. (B) Purified ribosomal proteins from yeast 80S
identified by an ESI-Q-TOF-MS are numbered and their details were indicated in Supplementary Table 2. (D–F) Identification of
omes were
separated by SDS-PAGE and stained with CBB R-250. The details of MS data using an ESI-LIT-TOF MS was
the Nα-acetylated ribosomal proteins from the nat1, nat3, and mak3 mutants, respectively. Equal amounts of purified ribosomal
n in Supplementary
1.strain
(C) Purified
from
yeast
ribosomes
were
separated
2-DE using acid
proteins from theTable
normal
and theribosomal
NAT mutantproteins
were labeled
with
Cy3 80S
and Cy5
respectively.
The
Cy-labeledby
ribosomal
proteins from
the two
were separated
on the same
2-DEsecond
gel and dimension
the Cy3- and and
Cy5-images
were
compared.
The Protein spo
electrophoresis
in the
firststrains
dimension
and SDS-PAGE
in the
stained
with
CBB R-250.
α
-acetylated
ribosomal
proteins
are
shown
in
Table
1.
N
fied by an ESI-Q-TOF-MS are numbered and their details were indicated in Supplementary Table 2. (D–F) Identification
α
-acetylated ribosomal proteins from the nat1, nat3, and mak3 mutants, respectively. Equal amounts of purified ribosom
ns from the normal strain and the NAT mutant were labeled with Cy3 and Cy5 respectively. The Cy-labeled ribosoma
Table 1 – Nα-Acetylation of ribosomal proteins.
Protein
MW (kDa) a
pI a
2-DE b
TOF-MS c
DIGE
NAT
60S
P0
P1 A/B
P2 A/B
L1 A/B d
L2 A/B d
L3
L4 A/B
L5
L6 A/B
L7 A/B
L8 A/B
L9 A/B
L10
L11 A/B
L12 A/B d
L13 A/B
L14 A/B
L15 A/B d
L16 A/B
L17 A/B
L18 A/B d
L19 A/B d
L20 A/B
L21 A/B
L22 A/B
L23 A/B d
L24 A/B
L25
L26 A/B
L27 A/B
L28
L29
L30
L31 A/B
L32
L33 A/B
L34 A/B
L35 A/B d
L36 A/B
L37 A/B
L38
L39
L40 A/B d
L41 A/B d
L42 A/B d
L43 A/B d
33.7
10.9/10.6
10.7/11.1
24.5/24.5
27.4/27.4
43.7
39.0/39.0
33.7
19.9/20.0
27.6/27.7
28.1/28.1
21.6/21.7
25.4
19.7/19.7
17.8/17.8
22.5/22.5
15.2/15.2
24.4/24.4
22.2/22.2
20.5/20.5
20.6/20.6
21.7/21.7
20.4/20.4
18.2/18.3
13.7/13.8
14.5/14.5
17.6/17.5
15.7
14.2/14.2
15.5/15.5
16.7
6.7
11.4
12.9/13.0
14.8
12.1/12.2
13.6/13.6
13.9/13.9
11.1/11.1
9.8/9.7
8.8
6.3
14.5/14.5
3.3/3.3
12.2/12.2
10.0/10.0
4.6
3.6/3.7
3.8/3.9
9.7/9.7
11.1/11.1
10.3
10.6/10.6
6.4
10.1/10.1
10.2/10.2
10.0/10.0
9.7/10.5
10.8
9.9/9.9
9.4/9.4
11.2/11.7
10.4/11.6
11.4/12.0
10.5/10.5
10.9/10.9
11.7/11.7
11.4/11.4
10.3/10.3
10.4/11.2
5.9/6.0
10.3/10.3
11.3/11.4
10.1
11.4/10.5
10.4/11.2
10.5
12
9.8
10.0/10.0
11.2
11.1/11.1
11.6/11.6
10.6/10.6
12.2/11.6
12.2/12.3
10.9
–
10.6/10.6
–
11.4/11.4
11.2/11.4
–
–
–
–
○/–
○
●/–
–
–
○/–
○/–
–
–
●
–
–
–
○/–
●/●
–
–
○/–
–
–
–
–
–
○
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
●/–
○/○
●/●
–
–
–
–
●/–
○/–
○/–
–
○
●/●
–
–
●/–
–
–/●
○/–
–
–
–
○/–
○/–
–
–
○
–/○
○/–
○
○
○
○/–
○
●/–
–
–
●/ ○
–
○
○
–
–
–/○
–
–
–
–
●/●
○/○
○
●/●
○
○/○
○/○
○/○
○/–
–
●/●
○/○
○/–
●/–
○/–
●/●
○/○
○/○
○/○
○/○
○/–
○/–
●/●
○/○
○
–/○
○/–
○
–
○
○/○
○
○/○
–
○/○
–/○
–
○
–
–
–
–
–
–
–
–
NatA
–
–
NatA
–
–
–
–
–
–
NatA
–
–
NatA
–
NatA
–
–
–
–
–
–
NatA
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
40S
S0 A/B
28.0/28.0
4.5/4.5
–
●/–
–
–
Subunit
40S
436
S0 A/B
S1 A/B
S2
S3
S4 A/B d
S5
S6 A/B d
S7 A/B
S8 A/B d
S9 A/B
S10 A/B
S11 A/B d
S12
S13
S14 A/B
S15
S16 A/B d
28.0/28.0
28.7/28.8
27.4
26.5
29.3/29.3
25
27.0/27.0
21.6/21.6
22.5/22.5
22.4/22.3
12.7/12.7
17.7/17.7
15.8
17
14.5/14.6
16 J O U R N
15.8/15.8
A L OF P
4.5/4.5
10.0/10.0
10.4
9.4
10.1/10.1
8.6
10.4/10.4
9.8/9.9
10.7/10.7
10.8/10.1
8.7/9.92
10.8/10.8
4.5
10.4
10.7/11.3
R10.7
O TE O MI
10.3/10.3
CS 7 4 (2
–
●/–
●
○
–
●
–
●/–
–
–
–
–
–
–
●/–
0 –1 1 )
●/–
4 3 1–4 4 1
●/–
●/–
●
○
○/–
●
–
●/●
○/–
–
○/○
●/–
–
○
●/–
●
●/–
40S
Total
Protein
MW (kDa) a
pI a
2-DE b
TOF-MS c
DIGE
NAT
S17 A/B
S18 A/B d
S19 A/B
S20
S21 A/B
S22 A/B
S23 A/B d
S24 A/B d
S25 A/B
S26 A/B
S27 A/B
S28 A/B
S29 A/B
S30 A/B
S31
78
15.8/15.8
17.0/17.0
15.9/15.9
13.9
9.7/9.5
14.6/14.6
16.0/16.0
15.3/15.3
12.0/12.0
13.5/13.4
8.9/8.9
7.6/7.6
6.7/6.7
7.1/7.1
17.2
10.5/11.3
10.3/10.3
9.6/10.5
9.5
5.8/5.8
9.9/9.9
11.5/11.5
10.5/10.5
10.3/11.1
10.8/11.6
9.4/9.5
10.8/11.4
11.1/10.8
12.2/12.2
10.7
–
–
–
–
–
–
–
●/–
–
–
–
–
–
–
–
18
–
●/●
–/○
●
●/●
○/–
–
●/–
–
–
○/○
●/●
○/○
○/○
–
50
●/–
●/●
○/–
●
●/●
○/○
–
●/●
○/–
○/–
○/–
●/–
–
–
–
60
NatA
NatA
–
NatA
NatB
–
–
NatA
–
–
–
NatB
–
–
–
●: Nα-Acetylated ribosomal protein. ○: identified ribosomal protein.
The calculated molecular weight and pI of ribosomal proteins were obtained from SWISS-PROT database.
b
Nα-Acetylated ribosomal proteins were identified by 2-DE with an amino acid sequencer (Takakura et al.).
c
Nα-Acetylated ribosomal proteins were identified by MALDI-TOF-MS (Arnold et al.).
d
These ribosomal proteins A/B are used for duplicated genes that code proteins with identical sequence.
a
–
NatA
NatA
–
–
NatA
–
NatA
–
–
–
NatA
–
–
NatA
NatA
NatA
(continued
(continuedon
onnext
next page)
page)
Table 1 (continued)
Subunit
–
○/○
●
○
○/○
●
○/○
●/●
○/○
–/○
○/–
●/●
–
○
●/–
●
●/●
J O U RN A L OF P R O TE O MI CS 74 ( 20 1 1 ) 4 3 1– 4 4 1
Fig. 2 – The effect of Nα-acetylation on cell growth and protein synthesis. (A) Growth curves of the normal strain and the NAT
mutants. All strains were cultured in YPD at 30 °C until stationary phase. The absorbance of each culture was measured at 600 nm
every 2 h. (B) Effect of three different temperatures (20, 30 and 37 °C) on the growth of the normal strain and the NAT mutants.
Freshly grown yeast colonies were suspended in water, and 1/10 dilutions containing the same number of cells were spotted onto
YPD plates. Spotted plates were incubated at 20, 30 and 37 °C for 3 to 4 days. (C) Effect of Nα-acetylation on polyU-dependent poly
(Phe) synthesis. Purified 80S ribosomes from the normal strain and the NAT mutants were added to assay mixtures containing
soluble factor S-100 from the normal strain and radioactive Phe residues, and incubated at 30 °C for 30 min. The radioactivity of the
insoluble fraction, a measure of the incorporation of radioactive amino acids, was determined by liquid scintillation counter. The
P R O TE O MI CS 74 ( 20 1 1 ) 4 3 1–4 4 1
437
Fig. 2 – The effect of Nα-acetylation on cell growth and protein synthesis. (A) Growth curves of the normal strain and the NAT
mutants. All strainsαwere cultured in YPD at 30 °C until stationary phase. The absorbance of each culture was measured at 600 nm
Fig. 2every
– The
effect of N -acetylation on cell growth and protein synthesis. (A) Growth curves of the normal strain and the NAT
2 h. (B) Effect of three different temperatures (20, 30 and 37 °C) on the growth of the normal strain and the NAT mutants.
mutants.
All
strains
were
cultured
in YPD
at 30 °C
stationary
phase. The
absorbance
of each
culture
waswere
measured
600 nm
Freshly grown
yeast
colonies
were
suspended
inuntil
water,
and 1/10 dilutions
containing
the same
number
of cells
spottedatonto
everyYPD
2 h.plates.
(B) Effect
of three
different
temperatures
(20,
3037and
37 3°C)
the growth
normal strain
and the NAT mutants.
on polyU-dependent
poly
Spotted
plates
were incubated
at 20, 30
and
°C for
to on
4 days.
(C) Effectof
ofthe
Nα-acetylation
Freshly
grown
yeastPurified
colonies
were
suspended
and
1/10and
dilutions
containing
the same
cells were
spotted onto
(Phe)
synthesis.
80S
ribosomes
from in
thewater,
normal
strain
the NAT
mutants were
addednumber
to assayof
mixtures
containing
-acetylation
on polyU-dependent
YPD soluble
plates. factor
Spotted
plates
incubated
atand
20, radioactive
30 and 37 °C
for
3 to 4 days.
(C) Effect of
Nα°C
S-100
fromwere
the normal
strain
Phe
residues,
and incubated
at 30
for 30 min. The
radioactivity of thepoly
fraction,
a measure
of the incorporation
of radioactive
amino
determined
by liquid
scintillation
counter.
The
(Phe)insoluble
synthesis.
Purified
80S ribosomes
from the normal
strain and
theacids,
NAT was
mutants
were added
to assay
mixtures
containing
value
shown
in the
figure
was calculated
by subtracting
the
value
of the activity
at 0 min.at
(D)30
The
profiles
of the normal
soluble
factor
S-100
from
the normal
strain and
radioactive
Phe
residues,
and incubated
°C polysome
for 30 min.
The radioactivity
of the
strain
and
the
NAT
mutants.
Cytoplasmic
extracts
from
the
normal
and
the
mutant
strains
were
loaded
onto
7–47%
sucrose
insoluble fraction, a measure of the incorporation of radioactive amino acids, was determined by liquid scintillation counter. The
centrifuged,
The
fractions were
collected
from
the topat
to 0the
bottom
withpolysome
continuous
A254 monitoring.
valuegradients,
shown in
the figure and
wasfractionated.
calculated by
subtracting
the value
of the
activity
min.
(D) The
profiles
of the normal
strain and the NAT mutants. Cytoplasmic extracts from the normal and the mutant strains were loaded onto 7–47% sucrose
gradients, centrifuged, and fractionated. The fractions were collected from the top to the bottom with continuous A254 monitoring.
due to a failure to form 80S ribosomes or due to disruption in
40S subunit assembly. Thus, it is possible that decreased
protein synthesis activity in the nat3 mutant is at least in part
d protein
synthesis.
(A) Growth
curves
the normalor
strain
the NAT
due
to a failure
to form
80S of
ribosomes
dueand
to disruption
in
due to a defect in ribosome assembly, whereas the altered
dilution spot assays using YPD plates containing various
antibiotics that bind to the ribosome and inhibit translation
(Fig. 3A). We found that neither the normal strain nor the nat1
dilution spot assays using YPD plates containing various
mutant was sensitive to puromycin, which is known to cause
438
J O U RN A L OF P R O TE O MI CS 7 4 (2 0 1 1 ) 4 3 1–4 4 1
Fig. 3 – The effect of the NatA deletion on sensitivity to translation inhibitors. (A) The effect of various antibiotics on the growth
of the normal strain and the nat1 mutant. Freshly grown yeast colonies were suspended in water, and 1/10 dilutions starting at
0.1 OD600 were spotted onto YPD plates containing the indicated antibiotics. Spotted plates were incubated at 30 °C for 4 days.
(B) The effect of paromomycin on growth of the normal strain and the nat1 mutant. Freshly grown yeast colonies were cultured
in YPD containing increasing concentrations of antibiotics until the culture without antibiotic reached an OD600 of 1–1.5, which
was taken as 1.0.
results suggest that Nα-acetylation of ribosomal proteins by
NatA has no specific effect on translocation or peptidyl
transferase activity.
On the other hand, both paromomycin and hygromycin
caused a specific decrease in the growth of the nat1 mutant as
compared to the normal strain. Therefore, we examined the
effect of various paromomycin concentrations on growth of
translational termination. In addition, our reporter construct
is useful for the analysis of translational fidelity in yeast.
fect on translocation or peptidyl
is useful for the analysis of translational fidelity in yeast.
oth paromomycin and hygromycin
in the growth of the nat1 mutant as
strain. Therefore, we examined the
mycin concentrations on growth of
B) and found that sensitivity to
ng at higher antibiotic concentratranslational error-inducing antibioding center on the ribosome's 40S
conformational changes affecting
nticodon helix between mRNA and
it appears that the Nα-acetylation of
atA may be required to maintain a
ty.
mal protein Nα-acetylation in
mutant's translational fidelity using
ne consisting of genes encoding a
and a FLAG peptide (1 kDa) (Fig. 4A).
n is accurate, the 14 kDa peptide is
n readthrough occurs, the 15 kDa
the FLAG tag) is produced. In the
d the affect of paromomycin on the
oduct in a concentration-dependent
und that the readthrough product
g concentrations of paromomycin.
e compared translational fidelity
n and the nat1 mutant (Fig. 4C). The
readthrough product in the nat1
gher than in the normal strain.
the readthrough product in the nat1
uenced by the addition of paromoon by NatA is required for optimal
Fig. 4 – The role of ribosomal protein Nα-acetylation in
translational readthrough activity. (A) The structure of the
stop codon readthrough construct used in this study. The
14 kDa protein A fragment is a predominant translation
product in the normal strain. The 14 kDa protein A fragment
combined with the FLAG tag protein (1 kDa) which resulted in
15 kDa peptide is the mistranslated protein product.
(B) Protein production in the normal strain with high
concentration of paromomycin induced stop codon
readthrough. Cells were grown in YPD containing the
indicated concentration of paromomycin. Protein samples
were loaded onto 15% SDS-PAGE and detected using Western
blot with anti-peroxidase antibody. (C) Comparison of stop
codon readthrough activity between the normal strain and
the nat1 mutant.
J O U RN A L OF P R O TE O MI CS 74 ( 20 1 1 ) 4 3 1–4 4 1
439
on
ng has revealed that yeast contains 137
enes, encoding 78 unique ribosomal proteins
duplicate genes [27]. Takakura et al. detected
mal proteins in the yeast 80S ribosome by 2gel electrophoresis in the first dimension
the second dimension, and identified 14
which were Nα-acetylated by NatA using
n [12]. Arnold et al. found that 30 of the
mal proteins, including the isoforms, were
lated by NatA, NatB, or NatC using shotgun
TOF MS [13]. In the present study, we applied
MS to identify the Nα-acetylated ribosomal
ast 80S ribosome. By these techniques, we
ated proteins and their non-acetylated counof differently colored spots with slightly
points. The numbers of ribosomal proteins
ribosomal proteins, not including isoforms,
pectively, showing that we detected 77% of
roteins encoded and 83% of the known 69
teins (pI > 7). We did not detect the remaining
ins by 2D-DIGE, because of low molecular
Da) or isoelectric point (pI < 5).
his is the first report to describe the use of
MS techniques in the differential display
tylated and non-Nα-acetylated ribosomal
etected more ribosomal proteins and Nαmal proteins than in the previous studies.
ated the effect of the lack ribosomal protein
the ribosome function using the NAT
tion of MAK3 (component of NatC) did not
otein Nα-acetylation or protein synthesis. In
utant, two ribosomal proteins lost the Nαthe protein synthesis activity of purified
ecreased (Fig. 2). Although the abnormal
Fig. 5 – Location of NatA Nα-acetylated ribosomal proteins on
the 80S ribosome structure. The 25S rRNA is in light blue, the
18S rRNA is in green, the non-Nα-acetylated ribosomal
proteins are in dark blue, and the Nα-acetylated ribosomal
proteins are in red. The graphic visualization was done with
the program PyMol (PDB ID: 1S1H and 1S1I).