Download Phosphorylation of the F1Fo ATP Synthase Я Subunit

Phosphorylation of the F1Fo ATP Synthase ␤ Subunit
Functional and Structural Consequences Assessed in a Model System
Lesley A. Kane, Matthew J. Youngman, Robert E. Jensen, Jennifer E. Van Eyk
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Rationale: We previously discovered several phosphorylations to the ␤ subunit of the mitochondrial F1Fo ATP
synthase complex in isolated rabbit myocytes on adenosine treatment, an agent that induces cardioprotection.
The role of these phosphorylations is unknown.
Objective: The present study focuses on the functional consequences of phosphorylation of the ATP synthase
complex ␤ subunit by generating nonphosphorylatable and phosphomimetic analogs in a model system,
Saccharomyces cerevisiae.
Methods and Results: The 4 amino acid residues with homology in yeast (T58, S213, T262, and T318) were studied
with respect to growth, complex and supercomplex formation, and enzymatic activity (ATPase rate). The most
striking mutant was the T262 site, for which the phosphomimetic (T262E) abolished activity, whereas the
nonphosphorylatable strain (T262A) had an ATPase rate equivalent to wild type. Although T262E, like all of the
␤ subunit mutants, was able to form the intact complex (F1Fo), this strain lacked a free F1 component found in
wild-type and had a corresponding increase of lower-molecular-weight forms of the protein, indicating an
assembly/stability defect. In addition, the ATPase activity was reduced but not abolished with the phosphomimetic mutation at T58, a site that altered the formation/maintenance of dimers of the F1Fo ATP synthase
complex.
Conclusions: Taken together, these data show that pseudophosphorylation of specific amino acid residues can have
separate and distinctive effects on the F1Fo ATP synthase complex, suggesting the possibility that several of the
phosphorylations observed in the rabbit heart can have structural and functional consequences to the F1Fo ATP
synthase complex. (Circ Res. 2010;106:504-513.)
Key Words: mitochondria 䡲 ATP synthase 䡲 phosphorylation 䡲 preconditioning
lated on subunits ␣,12–14 ␤,12,14,15 ␦,16 ␧,12 ␥,12,14,17 4,12
OSCP,12,14 c,14 and g,12 in a broad range of species. Phosphorylation has been correlated to dimerization of the ATP
synthase complex in yeast (subunit g)12 and in heart (␥
subunit),17 but there is currently no evidence that phosphorylation of ATP␤ regulates any aspect of the complex. Regulation of this complex could come at a variety of points:
transcription, translation, import, assembly, or direct functional regulation. The F1Fo ATP synthase is a well-conserved
enzyme complex that is known to exist in several different
assemblies, including both monomeric and dimeric
forms.17–19 Its subunits have a high degree of amino acid
sequence homology and similar assembly, structure, and
catalytic activity from Escherichia coli to mammals.18 Eukaryotic F1Fo ATP synthase is more sophisticated in both the
number of subunits and in the specific chaperones required
for assembly.20 It has been suggested that this increased
complexity could provide more regulated steps in the produc-
P
reconditioning (PC) is a phenomenon by which physiological and pharmacological interventions protect the
heart from damage during future ischemic episodes (for
review see1). Mitochondria have long been implicated in this
protective phenotype.2,3 In studying the link between PC and
the mitochondria, our group observed phosphorylation of the
F1Fo ATP synthase complex ␤ subunit (ATP␤) in response to
adenosine mediated PC.4 There have been several observations connecting modulation of the ATP synthase complex to
PC, including its specific downregulation to preserve ATP
pools during ischemia.5–7 The goal of the present work was to
gain insight into the functional aspects of phosphorylation of
ATP␤ by mutation of the amino acid residues in a model
system.
It is becoming increasingly clear that mitochondria participate in control by kinase cascades and protein phosphorylation (for reviews, see elsewhere8 –11). Several groups have
shown that the F1Fo ATP synthase complex can be phosphory-
Original received June 19, 2009; resubmission received December 2, 2009; revised resubmission received December 9, 2009; accepted December 10,
2009.
From Departments of Biological Chemistry (L.A.K., J.E.V.E.), Cell Biology (M.J.Y., R.E.J.), and Medicine (J.E.V.E.), Johns Hopkins University,
Baltimore, Md.
Correspondence to Jennifer E. Van Eyk, 602 Mason F Lord Bldg, Center Tower, Johns Hopkins University, 5200 Eastern Ave, Baltimore MD 21224.
E-mail [email protected]
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.109.214155
504
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tion of the enzyme,20 and phosphorylation adds yet another
level of regulation.
The present study focuses on defining the individual role of
each phosphorylated amino acid residue of ATP␤ discovered
in PC. To accomplish this, the genetically tractable model
system of Saccharomyces cerevisiae was chosen so that each
phosphorylation could be studied independently and in the
context of a complete knock out of the endogenous protein.
The amino acid residues were mutated to nonphosphorylatable (alanine) or phosphomimetic (aspartic acid and glutamic
acid) residues. The acidic residue mutations incorporate a
charged residue to mimic phosphorylation. The alanine mutations act as a control mutation by ensuring backbone
spacing is retained, without the possibility of phosphorylation. The model system is an essential tool because 100% of
ATP␤ are modified at the same site and all ATP synthase
complexes contain modified subunit. In vivo mammalian
analysis would be complicated by the fact that the regulation
of the ATP synthase complex could involve either a single
modified subunit in each complex or multiple modifications
to different sites on each of the 3 ATP␤ in a given complex.
This study expands on our cardiac proteomic findings using a
model system to analyze phosphorylations independently of
the complications of controlling phosphorylation and dephosphorylation in mammalian systems. The mutant strains were
analyzed with respect to structure and function as compared
to wild-type (WT) and an ATP␤ deletion strain (atp2⌬). The
data show that phosphorylation of ATP␤ at unique amino
acid residues could act as important regulators of complex
function (ATPase rate) and structure (F1 and dimer formation), because several phosphomimetic mutants had dramatic
effects.
ATP Synthase ␤ Subunit Phosphorylation
505
Non-standard Abbreviations and Acronyms
ATP␤
BN-PAGE
DIG
EtBr
LM
MS
MS/MS
mtDNA
PC
WT
ATP synthase ␤ subunit
blue native polyacrylamide gel electrophoresis
digitonin
ethidium bromide
lauryl maltoside
mass spectrometry
tandem mass spectrometry
mitochondrial DNA
preconditioning
wild type
using sucrose gradient centrifugation (see the Online Data
Supplement).24
Blue Native PAGE and Two-Dimensional
BN/SDS-PAGE Gels
Blue native PAGE (BN-PAGE) was used to resolve the native, intact
mitochondrial protein complexes.25 Two-dimensional BN/SDS-PAGE
was also performed to analyze complex subunit composition.25 See the
Online Data Supplement for details.
One-Dimensional SDS-PAGE
One-dimensional electrophoresis, 4% to 12% NuPAGE Bis-Tris gels
(1 mm, Invitrogen), were run according to manufacturer’s protocols,
using LDS sample buffer and MES running buffer and stained with
silver26 or colloidal Coomassie.27
Western Blotting
Gels were transferred and blotted as described in the Online Data
Supplement.
Methods
An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org.
Media and Genetic Methods
Yeast media included YEPD (YEP with 2% dextrose), SD (synthetic
medium containing 2% dextrose), YEPD⫹EtBr (YEPD with 25
␮g/nL ethidium bromide), and SRaf (synthetic medium with 2%
raffinose); yeast genetic techniques are as previously described.21
Strains
The yeast strains used in this study are listed in Table 1. See the
Online Data Supplement.
ATPase Assays
In-solution ATPase assays were performed on sucrose gradient
isolated complex.28 In-gel ATPase analyses were performed following BN-PAGE.19 See the Online Data Supplement.
Tandem MS
Protein bands from 1D BN-PAGE and 2D BN/SDS-PAGE were cut
and prepared for digestion.29 See the Online Data Supplement for
data acquisition and protein identification details.
Three-Dimensional Structure
Mitochondria and ATP Synthase Isolation
Crude mitochondria were isolated from yeast homogenates22 and
purified using sucrose gradients.22,23 ATP synthase was isolated
The ␣3␤3 hexamer of the S cerevisiae ATP synthase from Protein
Data Bank (http://www.pdb.org) 2HLD structure30 was modeled
using DeepView/Swiss-PdbViewer v3.7.
Table 1. Phosphorylated Amino Acid Residues Identified in Rabbit Heart ATP␤ and the Relationship to the Yeast Protein and the
Phosphomimetic Mutants Used in This Study
Phosphorylated Amino Acid Residue
in Rabbit (Preprotein)
Corresponding Amino Acid
in Yeast (Mature Protein)
Percentage Identity (of the Tryptic
Peptide Surrounding the Residue)
Nonphosphorylatable
Mutant Strain
S106 and T107
T58
93%
T58A
T58E
T262/S263
S213
79%
S213A
S213D
T312
T262
95%
T262A
T262E
T368
T318
100%
T318A
T318E
Acidic amino acids (A and E/D) were substituted to mimic phosphorylations and alanine residues were substituted as control mutations.
Phosphomimetic
Mutant Strain
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Figure 1. The amino acid residues of interest mapped onto the 3D structure of the ␣/␤ hexamer of S cerevisiae (Protein Data Bank
no. 2HLD).30 Amino acid residues are color-coded, with numbers given for the mature protein (with known mitochondrial targeting
sequence removed). Two of the residues (T58 [yellow] and S213 [red]) are located on the matrix-facing portion of the ␤ subunit,
whereas the other 2 (T262 [green] and T318 [blue]) are located within the center of the complex. Both buried (T262) and accessible
(T58) residues had observed assembly and functional differences.
Results
Phosphomimetic Mutations
Four of the 5 phosphorylated residues observed in the rabbit
heart protein are conserved in the yeast protein (see alignment, Online Figure I). The residues present were mutated to
phosphomimetic and nonphosphorylatable amino acids as
shown in Table 1. Two residues were located on the matrixfacing surface of the subunit (T58 and S213), and 2 were
located in the center of the complex (T262 and T318) (Figure
1). The 2 internal sites are located in close proximity, so
double mutations were made in an attempt to determine
whether simultaneous phosphorylation would result in a
different phenotype than the individual mutations.
Effect on Growth
The effects of the mutations were assessed by examining the
growth of all strains at 16°C, 30°C, and 37°C on either
SD-His or YEPD⫹EtBr media. These types of media were
chosen to examine the growth of the mutant strains in both a
selected, uninhibited manner (SD-His) and in the absence of
mitochondrial (mt)DNA (YEPD⫹EtBr). All strains grew
equivalent to WT at 30°C and 37°C, regardless of the media
(data not shown). However, at 16°C the T262E strain displayed reduced growth compared on SD-His (Online Figure
II, A) and did not form colonies on YEPD⫹EtBr media,
where there was no mtDNA (and thus no Fo), whereas the
T262A grew comparably to WT in both instances (Online
Figure II, B). This growth phenotype was mirrored by the
double mutants T262A/T318A (WT-like growth) and T262E/
T318E (T262E-like growth) (Online Figure II, C).
Phosphomimetic Mutants Exhibit a Differential
Arrangement of ATP Synthase Complex
by BN-PAGE
The ATP synthase assemblies present in each mutant strain,
as compared to both WT and the ATP␤ deletion strain
(atp2⌬), were analyzed by 1D BN-PAGE. Importantly, all of
the mutant strains had ATP␤ protein levels equivalent to WT,
as observed by SDS-PAGE of mitochondria (Figure 2A).
Because detergents are known to preserve complexes to
different extents,31 mitochondria from all strains were solubilized in either lauryl maltoside (LM) (Figure 2B and 2C) or
digitonin (DIG) (Figure 2D and 2E). LM disrupts protein
interactions to a greater degree than DIG, which is capable of
preserving more structures. Figure 2B and 2D shows equal
loading of gels by total protein stain (Coomassie) and Figure
2C and 2E shows Western blots probed with anti-ATP␤
antibody (␣ subunit blots gave the same pattern; data not
shown). Both LM- and DIG-solubilized mitochondria contain
an ATP␤ band at ⬇700 kDa. This is identified as the intact
F1Fo complex, based on apparent mass, previous observations32–35 and mass spectrometric (MS) data (Figure 2C and
2E; Table 2). The F1Fo band is present at equal amounts
(based on densitometry, n⫽3 each, Online Table II) in all
mutant strains solubilized with LM or DIG. The only exception being the LM-solubilized T262E/T318E, which is re-
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Figure 2. BN-PAGE of isolated WT and
mutant mitochondria. A, One-dimensional
SDS-PAGE (4% to 20%), denaturing/reducing gel. The total protein stain shows
equal protein loading and ␤ subunit content of all lanes (except atp2⌬, which lacks
the ␤ subunit). Total protein stain of
BN-PAGE gels shows equal protein content in all lanes for lauryl maltoside (B) and
digitonin (D) solubilized mitochondria. C
and E display representative Western blots
of the ATP␤ (n⫽3) for lauryl maltoside and
digitonin solubilized mitochondria, respectively. Bands are labeled according to the
complex that is present; F1Fo dimers, F1Fo
monomers, the F1 portion of the complex,
and ␣/␤ lower-molecular-weight bands. (For
intensity data values, see Online Table II.)
duced by 10% to 30% (P⫽0.03). Regardless of the detergent
used, ATP␤ was also present in a complex at 480 kDa. This
corresponds to the F1 portion of the complex (Table 2), which
is known to assemble independently of the Fo portion.32–35
The quantity of the F1 band is markedly different between
WT and several phospho-mutants in both detergent conditions (based on densitometry, n⫽3 each, Online Table II). In
Table 2. Number of Unique Peptides Observed by MS/MS for
Each Subunit Identified in Bands From WT BN-PAGE Digests
With Both Trypsin and Chymotrypsin
F1 Band
F1Fo Band
Dimer
Band
Digitonin
Lauryl
Maltoside
Digitonin
a
8
30
23
40
17
␤
9
30
18
25
18
7
6
6
3
␥
3
13
7
15
5
d
2
7
2
OSCP
6
12
5
4
2
9
3
Subunit
⑀
f
2
j
2
Lauryl
Maltoside
Digitonin
particular, T58E has much less of the F1 band than either WT
or T58A and the strains T318A and T262A/T318A also have
much lower levels than WT. Three of the phosphomimetic
strains (T262E, T318E and T262E/T318E) display an absence of this F1 band in both detergent conditions.
Interestingly, the strains lacking the F1 band display
lower-molecular-weight bands of ATP␤ (Figure 2C and 2E)
and ␣ subunit (data not shown). DIG solubilization allowed
for the observation of a higher molecular weight form
(⬇1000kDa) of the ATP synthase complex, which likely
represent F1Fo dimers (Figure 2D). Some of the mutant
strains have differences in the amount of dimer as compared
to WT (22⫾4.9) (Online Table II). Most interestingly, T58A
had a greater quantity of dimer (56⫾10, P⬍0.02), whereas
the T58E had a WT level of dimer (27⫾5.1). Taken together,
the data in Figure 2 imply that, although all the mutant strains
have WT levels of the ATP␤ and of the intact F1Fo complex,
the strains differ greatly in the smaller and larger observed
assemblies.
Using 2D analysis (2D BN/SDS-PAGE) in which the
complexes are resolved first by BN-PAGE and then separated
into subunits by reducing and denaturing SDS-PAGE, we
were able to resolve the individual protein components of the
1D BN-PAGE bands (Figure 3). Figure 3A (LM solubilization) shows that the F1Fo (700-kDa) band of the WT, T262A,
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Figure 3. Two-dimensional BN/SDS-PAGE of isolated WT and mutant mitochondria. LM-solubilized mitochondria from WT, T262A, and
T262E yeast strains (A) and DIG-solubilized mitochondria from WT, T58A, T58E, T262A, and T262E yeast strains (B) were subjected
to BN-PAGE in 1D and SDS-PAGE in 2D (4% to 12% gels). This technique allows for the separation of complexes observed on 1D
BN-PAGE into individual subunits. Indicated protein gel spots were identified by MS/MS. These 2D BN/SDS-PAGE gels confirm the
presence of the Fo and/or F1 subunits at the correct location in the BN-PAGE. (For MS identification data, see Online Table III.)
and T262E strains is made up of at least ␣, ␤, ␥, and OSCP
subunits from both of the F1 and Fo components (based on
MS; Online Figure III and Online Table III). The suspected
F1 band (480 kDa) in the WT and T262A strains is missing
the Fo component OSCP subunit (Figure 3A). For the T262E
strain, in which there is no detectable F1 band by BN-PAGE,
no ␣, ␤, or ␥ subunit was resolved in this region. However,
the T262E strain gel contains additional ␣ and ␤ subunit spots
in the lower portion of the BN-PAGE. Two-dimensional
BN/SDS-PAGE of DIG-solubilized mitochondria confirmed
the subunit pattern observed in the F1Fo and F1 bands of
LM-solubilized mitochondria (Figure 3B). DIG 2D gels also
confirmed the presence of F1 subunits ␣, ␤, and ␥ and the Fo
subunit OSCP in the dimer area (based on MS; Online Figure
III and Online Table III).
To increase the coverage of the complex subunits, MS
analysis was performed directly on each 1D BN-PAGE band
of interest from WT mitochondria using trypsin and chymotrypsin independently. Several subunits of both the F1 and the
Fo portions of the complex were observed (Table 2). F1
subunits were observed in all bands, but Fo subunits were
only observed in the bands suspected to contain the full F1Fo
monomer and dimers (Table 2). For tandem MS (MS/MS)
peptide identification data, see Online Tables IV and V for
LM and DIG, respectively.
Several Phosphomimetic Mutants Have Decreased
ATPase Function
Functional assessment of the phosphomimetic and nonphosphorylatable mutations was performed on complexes isolated
from mitochondria by sucrose gradient centrifugation.24 Mitochondria isolated from the atp2⌬ strain were used as a
negative control. The in-solution ATPase rate for the complex
with the mutants at residue S213 (3.44⫾0.52 [S213A] and
3.14⫾0.51 ␮mol Pi/mg per minute [S213E]) did not differ
from WT (3.28⫾0.56 ␮mol Pi/mg per minute), but all other
mutations caused changes with respect to WT (Figure 4B).
Although the A and E mutants of the residue T58 both had
reduced ATPase rates, the T58A strain had significantly
better function (2.42⫾0.38 ␮mol Pi/mg per minute) than the
T58E phosphomimetic strain (1.43⫾0.25 ␮mol Pi/mg per
minute; P⬍0.01 between T58A and T58E). Both mutations at
T318 reduced ATPase rates compared to WT (T318A
0.17⫾0.15 ␮mol Pi/mg per minute; T318E no detectable
activity). The T262A strain trended toward an increase in
ATP hydrolysis (4.01⫾0.36 ␮mol Pi/mg per minute;
P⫽0.054) compared to WT, whereas the phosphomimetic
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Figure 4. In-solution ATPase assays. A, Mitochondria were solubilized in lauryl maltoside and the complexes were separated on a sucrose gradient
to isolate the ATP synthase complex. A representative gel of the combined ATP synthase fractions from each of the strains is shown. Arrows indicate the contaminating bands also present in the atp2⌬ lanes. ATPase assays were performed on this deletion strain as a negative control. B,
In-solution ATPase assays were performed on fractions from each of the yeast mutant strains and were compared to WT and the negative control
(atp2⌬). ND indicates no detectable signal. *P⬍0.05 from WT; §P⬍0.05 between the nonphosphorylatable and the phosphomimetic strains (both
based on Mann–Whitney test). n⫽6 all observed values fell within the linear range of the assay.
(T262E) had no detectable activity. It is of note that the
double mutant of T262E/T318E (0.14⫾0.27 ␮mol Pi/mg per
minute) is even more impaired in its ATPase rate than the
T318E mutant alone (no detectable activity).
Because different quantities of the free F1 and the dimer
forms of the complex were observed by BN-PAGE in some
mutant strains, in-gel ATPase assays were also performed to
ensure that the in-solution ATPase data were affected by
these different complexes. DIG-solubilized mitochondria
were used because they allowed for the observation of all
ATP synthase bands (Figure 5). Although equal quantities of
F1Fo ATP synthase monomer are present in all strains (except
the atp2⌬ control), the activity stain shows increased intrinsic
activity in the T262A monomer compared to WT and no
observable activity in the T262E monomer (Figure 5). This
pattern was also observed in the dimer bands of the WT,
T262A, and T262E strains. The in-gel activity of the F1, F1Fo
monomer, and F1Fo dimer bands in Figure 5 show the same
functional patterns as the in-solution assays, indicating that
the level of intrinsic activity is driven by the ATP␤ protein,
not its assembled state. This is also confirmed by the fact that
though T262E and T318E have similar BN-PAGE band
patterns, T318E retains activity in all observed bands,
whereas T262E has no activity.
Discussion
The present work explores the ATP␤ phosphorylations,
originally observed in a rabbit heart, in the model system S
cerevisiae. Using this model system, we have shown that
mutations mimicking phosphorylation of specific residues of
ATP␤ can have unique effects on the structure and function
of the complex. The phosphomimetic of the T262 residue
blocks the ATPase function of the complex, whereas mutations at the T58 residue primarily affect dimer formation.
Because the T262 residue is buried in the interior of the intact
complex, it may be inaccessible to dynamic regulation
(although it could be phosphorylated before or during complex formation). Residue T58, which is on the surface of the
complex, could be modulated in the intact ATP synthase
complex in a faster time scale.
Figure 5. In-gel ATPase assays. In-gel
ATPase assays were performed on
BN-PAGE of digitonin solubilized mitochondria from all strains. Left, Representative Coomassie-stained gel to indicate
protein load. The right gel illustrates the
ATPase activity of each of the bands as
white lead phosphate precipitate on the
gel. These assays mirror the results
observed in Figure 4 for in-solution
ATPase assays (n⫽3).
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Figure 6. Schema of ATP synthase
structures and the effects of the phosphomimetic mutations. The various
structures observed by BN-PAGE are
listed and the subunit interactions that
occur within them. Mutants that caused
defects at any stage are listed.
Growth of Phosphomimetic Mutants
Although each strain grew well at 30°C on all media, the
added stress of growth at 16°C revealed a cold-sensitive
phenotype for the T262E strain. At this temperature, T262E
did not grow well on SD-His, and this deficiency was even
more pronounced on YEPD⫹EtBr. As growth of yeast on
media containing EtBr results in the loss of mtDNA,36 the
phenotype of the T262E mutant on this medium implies a role
of mtDNA in maintaining the viability of the T262E strain.
Yeast mtDNA encodes 3 subunits of the Fo portion of the
complex (a, c, and 8).18 The F1 portion of the complex is
essential to viability in the absence of the Fo section,37
possibly because of hydrolysis of ATP into ADP by the F1
component, allowing the ADP/ATP translocase to maintain
mitochondrial membrane potential.18,37 The inability of the
T262E mutant to grow only in the absence of mtDNA at 16°C
indicates that this phosphorylation most likely interferes in
some aspect of the F1 component assembly or function that
can be stabilized by the Fo portion of the complex.
F1Fo ATP Synthase Complex Monomer Assembly
The F1 and Fo components of the ATP synthase complex
form independently in the matrix and the inner membrane.18
Different complex assemblies are observed on BN-PAGE
gels depending on the type of gel used and the protein/
detergent ratio.31,35,38,39 In this study, LM-solubilized WT
mitochondria had 2 prominent ATP synthase bands: the F1Fo
complex monomer (⬇700 kDa) and the F1 portion alone
(⬇480 kDa). Solubilization with DIG allowed for the additional observation of the F1Fo complex dimer. It is possible
that the independent F1 portion may be a product of the
detergent extraction31 and not relevant in vivo, but the stark
differences between the phosphomimetic strains and the
consistency between both LM- and DIG-solubilization give
insight into the subunit interactions affected by these phosphorylations (Figure 6).
The F1Fo monomer is present at equal quantities in all
strains, implying that intact complex monomer can assemble
and is stable as the holoenzyme (Figure 2C and 2E). However, there is a striking difference in the amount of free F1
complex between WT and some mutant strains. The phosphomimetic mutants T262E, T318E, and T262E/T318E have
reduced quantities of the F1 complex (Figure 2C and 2E). It
is probable that the F1 portion of the complex is unstable in
these phosphomimetic strains and is either labile under the
detergent extraction conditions or cannot form unless it is
assembled with the Fo component. In other words, the Fo
component of the complex is capable of stabilizing the F1
when the complex is fully assembled. This hypothesis is
consistent with the stunted growth phenotype of the T262E
mutants described above on EtBr containing media, where a
functional F1 is essential in the absence of the Fo.
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T262 is located on the interior of the F1 portion of ATP
synthase complex (Figure 1). There are no published data to
suggest that the T262 residue has either interior intrasubunit
interactions that would explain this apparent structural interference, suggesting unknown biophysical aspects of this
amino acid residue. All of the other sites that caused a
decrease in the quantity of the F1 component compared to WT
have known interactions. For example, T318 is in close
proximity to sites known to be involved in hydrogen bonding
between the ␣ and ␤ subunits in E coli.40 The T58 residue is
also located within a region involved in subunit interactions,
the ␤-barrel domain.41,42 Although T318 and T58 have less F1
by BN-PAGE and known subunit interactions, they did not
show defects in growth assays, indicating that any functional
effect of this F1 destabilization in the accessible T58 or buried
T318 mutants is incomplete.
The ATPase rate of T262A trended toward being higher
than WT, whereas the T262E strain had essentially no
activity, indicating that this residue could be an important
regulatory site. T262 is buried within the center of the F1
portion of the complex (Figure 1), and, as such, phosphorylation would have to occur before assembly or by some
autophosphorylation. This residue is located near 3 residues
known to interact with oxygen atoms of phosphate during
catalysis (␤Asp256, ␤Asn257, and ␤Arg260).30 Although
there is no indication in the literature that T262 is important
for catalysis, this study clearly indicates that modifications
here have significant implications for the function of the F1Fo
complex. An in vivo phosphorylation of the T262 equivalent
residue in rabbit hearts would likely result in decreased function
of this complex, which has been shown to occur in PC.
F1Fo ATP Synthase Complex Dimer Assembly
Although the S. cerevisiae provided an excellent model
system in which to study the phosphorylation of ATP␤, there
are a few important caveats to this study when considering it
in the context of the mammalian heart. Most importantly, the
phosphomimetics in this study are constitutively present,
mimicking universal phosphorylation at a single site in each
ATP␤ mutant strain. This is likely not representative of the in
vivo cardiac situation, because the phosphorylated forms
represent a small portion of the total ATP␤ in the rabbit heart.
Also, it is possible that the phosphorylations exert regulation
at only discrete steps in import, assembly or function of the
F1Fo complex. Because the phosphomimetic mutations are
constitutively present, it is impossible to clarify these steps
and some consequences of in vivo phosphorylation may be
missed. The other main difference between the model system
approach and in vivo phosphorylation is that all of the ATP␤
in the yeast mutants contain the same modified site. As such,
the ATP␤ composition in all complexes is homogenous rather
than a combination of unphosphorylated or heterogeneous
phosphorylation on each ATP␤ in the complex. This singlesite analysis is beneficial as it allows the direct assignment of
function to a particular phosphomimetic residue. However, in
vivo it is possible that regulation would involve different
stoichiometry at any site on each of the complex’s 3 ␤
subunits, and this differential residue phosphorylation could
act to produce a unique phenotype. Even so, the observations
of this study provide significant evidence that phosphorylation of ATP␤ can have important implications to the structure
and function of the F1Fo ATP synthase complex.
Here, we have shown that, in a model system, mutations of
ATP␤ mimicking the phosphorylations observed in rabbit
heart can impact both the structure and function of the F1Fo
ATP synthase complex. Specifically, the T58E mutant affected dimer formation and decreased ATPase function, and
the T262E mutant ablated ATPase function. The matrixfacing, accessible nature of the T58 site makes it a likely
candidate for rapid regulation of the ATP synthase complex
into dimers or a mild phosphorylation-dependent decrease in
function. The T262 residue is buried in the center of the
complex, making it likely that it is phosphorylated before
assembly and may be involved in longer-term regulation of
the complex. This would imply that in the original PC model,
The other difference on the BN-PAGE gels is in dimer
formation between the T58A and T58E strains (Figure 2E).
Formation of the ATP synthase complex into dimers and
oligomers has been observed in mammalian cells and yeast
and can affect cristae formation,35 improve efficiency of the
enzyme,43 and even affect mitochondrial membrane potential44 (for review, see elsewhere45). Phosphorylation of ATP
synthase complex subunits, g subunit in yeast12 and ␥ subunit
in bovine,17 have been correlated to the formation of dimers.
The difference in dimer formation between the T58A and
T58E strains implies an additional role for phosphorylation of
ATP␤ in the regulation of dimer formation or maintenance.
F1Fo ATP Synthase Complex Function
The amino acid residues of ATP␤ involved in the binding of
nucleotide/phosphate and catalysis have been defined in
yeast.30 None of the residues in this study is known to be a
part of the catalytic site, yet it is clear that the substitution of
the phosphomimetic mutations can affect function (Figures 4
and 5), probably because of conformational or other allosteric
changes. Functional consequences of ATP␤ phosphorylation
were examined by 2 ATPase assay methods and both yielded
similar results. Two of the amino acid residues (S213 and
T318) had no significant differences in ATPase rate between
the phosphomimetic and the nonphosphorylatable mutations.
Both of the mutations of residue T318 displayed no ATPase
activity, implying its importance to overall function. The
double mutation of the internal sites mimicked the ablation of
function observed for T318 alone and thus could not provide
any insight into cooperation or inhibition of these 2
internally-located phosphorylations. Two of the phosphorylated amino acid residues exhibited a large difference between A and E forms, indicative of potential regulation by
phosphorylation in vivo. There was a 2-fold difference in
ATPase rate between the T58A and T58E strains. As discussed above, the T58 amino acid residue is located within a
␤barrel domain that has structural interactions with other F1
subunits.41,42 The interactions of this domain could be disrupted by the T58E mutation (or by phosphorylation), causing
an inefficient complex and producing the lower ATPase rate.
Implications of ATP␤ Phosphorylation
512
Circulation Research
February 19, 2010
some portion of the ATP␤ is being imported and assembled
in the 60-minute timeframe. Although there are no data on the
ATP␤ protein specifically, measurable amounts other cardiac
mitochondrial proteins can be imported, in vitro, in as quickly
as 3 minutes.46 The data from this model system analysis
suggest that an increase in phosphorylation of ATP␤ during
PC would result in functional changes to ATP synthase
complex. The kinase and phosphatase involved in the regulation of this phenomenon are still unknown and must be
uncovered to gain a full understanding of the consequences of
ATP␤ phosphorylation.
13.
14.
15.
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Acknowledgments
16.
We thank the Technical Implementation and Coordination Core of
The Johns Hopkins National Heart, Lung, and Blood Institute
Proteomics Center for the LTQ MS/MS analysis; Steven Elliott for
4800 MALDI TOF/TOF analysis; and Cory Dunn for creation of the
atp2⌬ strain. We also thank Shandev Rai for editorial comments
and suggestions.
17.
Sources of Funding
This work was supported by the National Heart, Lung, and Blood
Institute Proteomic Initiative (contract NO-HV-28120 to J.E.V.E.),
NIH grant P01HL081427 (to J.E.V.E.), American Heart Association
Predoctoral Fellowship 0715247U (to L.A.K.), and NIH Predoctoral
training grant 2T32-GM07445 (to M.J.Y.).
Disclosures
None.
18.
19.
20.
21.
22.
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Novelty and Significance
What Is Known?
●
●
Cardiac preconditioning stimuli can affect mitochondrial function and
specifically the F1Fo ATP synthase enzyme complex.
Phosphorylation can occur on several subunits of the mitochondrial
F1Fo ATP synthase, including 5 specific sites on the ␤ subunit
found in pharmacological preconditioning.
What New Information Does This Article Contribute?
●
●
In a model system, phosphomimetic mutations of T262 ablated
function, indicating that in vivo phosphorylation could result in
down-modulation in ATP activity.
Phosphomimetic mutations to the T58 site cause changes in the
function and the maintenance of complex dimers, which play a
large role in overall mitochondrial shape and function.
This study is an important step forward in our understanding of
the posttranslational regulation of the mitochondrial F1Fo ATP
synthase complex. The study moved a novel proteomic discovery that the ␤ subunit was phosphorylated to a system that
allowed the functional effect of each modification to be assessed. It was shown that 2 of the phosphorylation sites
(mimicked by pseudophosphorylation mutants) affect structure
and function of ATP synthase. This study is the first to connect
site-specific phosphorylation of an F1Fo ATP synthase subunit
with modulation of the holoenzyme. This opens several new
questions regarding the kinases involved in phosphorylation and
the dynamic nature of these phosphorylations in vivo. Understanding the mechanisms of these phosphorylations provides a new
context for mitochondrial involvement in PC.
Phosphorylation of the F1Fo ATP Synthase β Subunit: Functional and Structural
Consequences Assessed in a Model System
Lesley A. Kane, Matthew J. Youngman, Robert E. Jensen and Jennifer E. Van Eyk
Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Circ Res. 2010;106:504-513; originally published online December 24, 2009;
doi: 10.1161/CIRCRESAHA.109.214155
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2009 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/106/3/504
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2009/12/23/CIRCRESAHA.109.214155.DC1
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SUPPLEMENTAL MATERIAL
MATERIALS AND METHODS
Strains. The ATP2 gene (β subunit) was cloned from genomic DNA using the primers 5’ATAAGAATGCGGCCGCTAAACTATCATATATATGTATTTCCTTTC-3’ and 5’CCGCTCGAGCGGGAACGGTAATTTGGAATACAG-3’ with restriction sites for NotI and
XhoI, respectively (underlined). The resulting PCR product was digested with NotI and XhoI
ligated into vector pRS313. Site-directed mutations were introduced into the ATP2 gene using a
QuikChange Kit (Stratagene) with the oligonucleotides corresponding to either an alanine
mutation or glutamic or aspartic acid for each phospho-residue (The oligonucleotides are listed in
the Online Table I). DNA sequencing was used to confirm that appropriate mutations were
present. Yeast with no expression of the ATP2 gene (MATa met15Δ 0 his3Δ 200 leu2Δ 0 trp1Δ
63 ura3Δ 0 atp2Δ::LEU2) were transformed with a vector containing the WT ATP2 gene, one of
the mutant ATP2 genes or an empty vector control.
ATP synthase isolation. The F1Fo ATP synthase complex was enriched using a discontinuous
sucrose gradient separation, essentially as described1. Briefly, 1mg of intact mitochondria was
gently solubilized at 4°C in PBS (3.2 mmol/L Na2HPO4, 0.5 mmol/L KH2PO4, 1.3 mmol/L KCl,
135 mmol/L NaCl, pH 7.4) with 1% n-dodecyl-β-D-maltoside and centrifuged at 18,000 x g for
1h at 4°C. The supernatant was separated using a discontinuous sucrose gradient (15-35%) and
spun at 72,000 x g for 16.5h at 4°C. 400μL fractions were collected from the bottom of the tube.
To identify the fractions containing the F1FoATP synthase complex, a 10μg aliquot of each
fraction was separated on a denaturing 1D gel (as described below) and silver stained2. The F1Fo
ATP synthase complex spread across 2–3 fractions of a sucrose gradient (Online Figure IV) and
these fractions were pooled and used as the isolated complex (Figure 4A) protein quantification
(BCA assay, Pierce) performed. Pooled fractions were also run on a denaturing 1D gel and
stained with colloidal Coomassie,3 equal protein content was confirmed by densitometric analysis
(10μg per lane) (Progenesis, Nonlinear Dynamics). Aliquots of the pooled F1Fo ATP synthase
fractions (100μL of 0.1μg/μL) were flash frozen and stored at -80°C for further analysis.
Separation was performed in triplicate for each yeast strain.
Blue Native PAGE gels. Blue Native PAGE (BN-PAGE) resolved the native, intact mitochondrial
protein complexes.4 Briefly, samples of intact mitochondria were gently solubilized in BNPAGE sample buffer (50mmol/L Tris-HCl, 50mmol/L NaCl, 10% glycerol w/v, 0.001% Ponceau
S, pH 7.2) with 1.3% n-dodecyl-β-D-maltoside w/v and incubated at 4°C for 30min. Each sample
was centrifuged for 20min at 18,000 x g at 4°C. The supernatant was collected and 40μg loaded
into BN-PAGE sample buffer with 0.4% w/v Coomassie G250, then loaded on a 3-12%
NativePAGE Novex gel (1mm, Invitrogen) and run according to manufacturer’s protocols. Gel
were utilized in one of four ways: fixed with 50% v/v methanol, 10% v/v acetic acid and bands of
interest excised for LC MS/MS; transferred to PVDF membrane (Millipore, 45μm) for western
blotting; separated by a second dimension SDS-PAGE (as described below); or used for in-gel
ATPase assays (as described below).
Two-dimensional BN/SDS PAGE. Native gels were run, and upon completion whole lanes were
excised from the gel for incubation in 1xNuPAGE LDS sample buffer (Invitrogen; 62mmol/L
Tris, 0.5% w/v LDS, 2.5% glycerol, 0.13mmol/L EDTA, 0.55mmol/L Coomassie G250 and
0.04mmol/L phenol red, pH 8.5) with 1% w/v DTT, at 24°C for 20min and then at 37°C for
10min. Strips were placed on a 4-12% NuPAGE gel (1.5mm 2D well, Invitrogen) and overlaid
with 0.5% agarose. The gels were run using MES running buffer (50mmol/L MES, 50mmol/L
Tris–base, 0.1% w/v SDS, 1.0mmol/L EDTA, pH 7.3) at 50V (30min), 100V (1h) and 150V
(30min). Gels were fixed in 50% v/v methanol, 10% v/v acetic acid and silver stained2. Spots of
interest were excised and prepared for MS as described below.
Western blotting. BN-PAGE gels were incubated in transfer buffer (25mmol/L bicine, 25mmol/L
bis-Tris and 1mmol/L EDTA, pH 7.2) for 10min prior to being transferred to PVDF (Millipore,
45μm) via the Mini-Transblot Cell (Bio-Rad) for 1h at 100V. Membranes were blocked with
Western Blocking Reagent (Roche) in TBS-t (20mmol/L Tris, 150mmol/L NaCl and 0.1% v/v
Tween, pH 7.5) at 4°C for 18h. Blots were then incubated with primary antibody (concentrations
listed below) for 1h at room temperature, washed for 30min with TBS-t, incubated with
secondary antibody for 1h and then washed for 1h with TBS-t (0.2M Tris, 1.5M NaCl, 0.1% v/v
Tween-20, pH 7.5). Primary antibodies were ATP synthase β subunit (a gift to RJ from Mike
Yaffe, UCSD) (rabbit, 1:10,000) and ATP synthase alpha subunit from MitoSciences (mouse,
1:10,000). Secondary antibodies were alkaline phosphatase conjugated donkey anti-rabbit and
alkaline phosphatase conjugated goat anti-mouse, both from Jackson ImmunoResearch (1:10,000
dilution).
In-solution ATPase assays. ATP hydrolysis assays were performed on the isolated F1Fo ATP
synthase complex.5 Briefly, 5ug of the isolated complex was incubated in 50mmol/L Tris-SO4,
pH 8.5, 4mmol/L MgSO4 and 10mmol/L ATP (Sigma) for 2h at 37°C, and the reaction stopped
by the addition of 10% w/v TCA. 100μL of the mixture was incubated with 0.5% w/v TCA,
0.5% w/v ammonium molybdate and 0.01% w/v aminonaphtholsulfonic acid (prepared in 15%
w/v NaHSO3 and 6% w/v Na2SO3) at 24°C for 20 minutes, then analyzed at 595nm, to quantify
the free Pi content. The ATPase reaction was performed in duplicate for each of the three
separate isolations from each yeast strain. The measurement of the free Pi was performed in
duplicate for each of the ATPase reactions.
In-gel ATPase Assay. ATP hydrolysis was measured following 1D BN-PAGE as described in 6
using the local concentration of free phosphate created by the ATPase reaction to precipitate lead
on the site of ATP hydrolysis. Gels were incubated in 35mmol/L Tris, 270mmol/L glycine,
14mmol/L MgSO4, pH 8.0 directly after BN-PAGE for 2h at 24°C, and then transferred into the
reaction buffer (35mmol/L Tris, 270mmol/L glycine, 14mmol/L MgSO4, pH 8 with 0.2% (w/v)
lead nitrate and 8mmol/L ATP) and incubated at 24°C for 2h. After being washed in ddH2O for
10min, the gels were scanned and the lead precipitates quantified based on densitometry
(Progenesis, Nonlinear Dynamics).
Mass Spectrometry. Each protein band was analyzed following trypsin or chymotrypsin digestion
in order to maximize the number of proteins detected. All samples were pre-cleaned with C18
Omix tips (Varian) according to manufacture’s protocol. The 2D gel spot peptides were
resuspended in 50% ACN, 0.1% TFA and plated with matrix cyano-4-hydroxy-trans-cinnamic
acid to stainless steel plates. MS spectra were acquired on the 4800 MALDI TOF/TOF Analyzer
(ABI) using 1000 shots/spectra and a laser power between 4300–4700 units. The MS spectra
were processed using a signal to noise (S/N) threshold of 5:1, and the 10 largest peaks were
selected for MS/MS. MS/MS was acquired from largest to smallest for each spot, using 1000–
4000 laser shots; shots were allowed to accumulate until five peaks were greater than 70:1 S/N, or
until 4000 shots were reached. MS/MS spectra were processed using a 3:1 S/N cutoff. Mass lists
were generated based on the processing thresholds. 1D gel bands were analyzed using the LTQ
(ThermoFinnigan) with a C18 column (75μm column hand-packed with YMC ODS-AQ 5μm
particle size, 120A pore size) in gradient mode (inject at 8.5-30% 0.1% formic acid/90%
acetonitrile (30min), 60% 0.1% formic acid/90% acetonitrile (18 minutes) and to 100% 0.1%
formic acid/90% acetonitrile (22min)) with a flow rate of 300 nL/min. The electrospray voltage
was 2.2 kV, and precursor scans were taken from 350–1800 m/z and the top eight ions picked for
MS/MS.
Protein Identification. MS/MS data from the 4800 TOF/TOF (ABI) were analyzed in Mascot
Sequence Query tool (Matrix Sciences) using the SwissProt database and the following criteria:
Species: S. Cerevisiae; variable modifications: carbamidomethyl; oxidation (methionine)
maximum missed cleavages: 1; peptide tolerance: ± 0.5Da; MS/MS tolerance: ± 0.8 Da. MS/MS
data from the LTQ LC/MS/MS (Thermo) were analyzed using a Sorcerer 2™-SEQUEST®, with
post-search analysis performed using Scaffold (Proteome Software). All raw data peak extraction
was performed using Sorcerer 2™-SEQUEST® default settings. Data was searched using the
Swissprot database, using either a full trypsin or full chymotrypsin digestion, with the following
criteria: Species: all species; variable modifications: carbamidomethyl, oxidation (methionine);
peptide mass tolerance: 1.2amu. All MS/MS spectra were manually examined using Scaffold
(Proteome Software).
Online Figure I. Amino acid alignment of rabbit and yeast ATP synthase β subunits. Amino
acid alignment performed using the online tool ClustalW2
(http://www.ebi.ac.uk/Tools/clustalw2/index.html). Underlined and bold residues indicate the
tryptic peptide observed in the phospho-analysis of the ATP synthase β in rabbit heart.
Highlighted residues are the observed phospho-residues in rabbit and the site that was mutated in
the yeast mutant strains.
Online Figure II. Yeast growth assays. Strains were created by transforming the atp2Δ strain
with plasmids containing either the WT ATP2 gene or mutant ATP2 (list of mutant amino acids in
Table 1 and primers indicated in Online Table I). All strains were streaked onto either SD-His or
YEPD+EtBr (ethidium bromide) and incubated at 16, 30 and 37°C. All strains grew at WT levels
30°C and 37°C (data not shown). However, at 16°C T262E did not grow as well as T262A on
SD-His (A) and T262E did not form any colonies on the YEPD+EtBr (B), where the T262A grew
comparably to WT. The double mutants T262A/T318A and T262E/T318E showed the same cold
sensitive growth phenotype on YEPD+EtBr as the single T262A and T262E mutants (C) with the
alanine mutant growth like WT and the phospho-mimic mutant showing no growth.
Online Figure III. Map of 2D BN/SDS-PAGE gels. Numbers correspond with identification data
located in Online Table III.
Online Figure IV. Enrichment of complex V. (A) 1D SDS-PAGE of sucrose fractions from WT
mitochondria. The red box indicates the fractions that were pooled for ATPase assays. (B)
Representative gel of isolated ATP synthase fraction used for ATPase assays in solution.
References
1.
2.
3.
4.
5.
6.
Hanson BJ, Schulenberg B, Patton WF, Capaldi RA. A novel subfractionation approach
for mitochondrial proteins: a three-dimensional mitochondrial proteome map.
Electrophoresis. 2001;22:950-9.
Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins
silver-stained polyacrylamide gels. Anal Chem. 1996;68:850-8.
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia
P, Zardi L, Righetti PG. Blue silver: a very sensitive colloidal Coomassie G-250 staining
for proteome analysis. Electrophoresis. 2004;25:1327-33.
Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein
complexes in enzymatically active form. Anal Biochem. 1991;199:223-31.
Tzagoloff A. Oligomycin-sensitive ATPase of Saccharomyces cerevisiae. Methods
Enzymol. 1979;55:351-8.
Bisetto E, Di Pancrazio F, Simula MP, Mavelli I, Lippe G. Mammalian ATPsynthase
monomer versus dimer profiled by blue native PAGE and activity stain. Electrophoresis.
2007;28:3178-85.
Online Table I. Primers used for site directed mutagenesis
Phospho-site
Primers
T58A
5’-GCTCAACATTTGGGTGAAAACGCCGTCAGAACCATTGCTATGG-3’
5’-CCATAGCAATGGTTCTGACGGCGTTTTCACCCAAATGTTGAGC-3’
T58E
5’-GCTCAACATTTGGGTGAAAACGAGGTCAGAACCATTGCTATGG-3’
5’-CCATAGCAATGGTTCTGACCTCGTTTTCACCCAAATGTTGAGC-3’
S213A
5’-CATTAACTTGGAAGGTGAAGCCAAGGTCGCCTTAGTGTTCGG-3’
5’-CCGAACACTAAGGCGACCTTGGCTTCACCTTCCAAGTTAATG-3’
S213D
5’-CATTAACTTGGAAGGTGAAGACAAGGTCGCCTTAGTGTTCGG-3’
5’-CCGAACACTAAGGCGACCTTGTCTTCACCTTCCAAGTTAATG-3’
T262A
5’-CGACAATATCTTTAGATTTGCCCAAGCTGGTTCAGAAGTCTCTGC-3’
5’-GCAGAGACTTCTGAACCAGCTTGGGCAAATCTAAAGATATTGTCG-3’
T262E
5’-CGACAATATCTTTAGATTTGAGCAAGCTGGTTCAGAAGTCTCTGC-3’
5’-GCAGAGACTTCTGAACCAGCTTGCTCAAATCTAAAGATATTGTCG-3’
T318A
5’-GTTCCAGCCGATGATTTAGCCGATCCTGCTCCTGCCAC-3’
5’-GTGGCAGGAGCAGGATCGGCTAAATCATCGGCTGGAAC-3’
T318E
5’-GTTCCAGCCGATGATTTAGAGGATCCTGCTCCTGCCAC-3’
5’-GTGGCAGGAGCAGGATCCTCTAAATCATCGGCTGGAAC-3’
Online Table II. BN-PAGE band quantification.
Lauryl Maltoside
Digitonin
F1Fo
monomer
F1
F1Fo dimer
WT
45.54±7.22
28.92±5.1
29.22± 4.92
T58A
50.18± 5.3
26.54± 4.7
56.00± 10.8*
T58E
51.79± 9.04
19.18± 4.4*
27.51± 5.1
S213A
45.15± 5.4
39.73± 6.3*
52.54± 10.1*
S213D
46.16± 7.9
34.07± 6.1
57.47± 13.6*
T262A
50.32± 7.52
26.79± 1.7
36.82±6.63
T262E
48.91± 10.6
0±0*
37.30± 8.8
T318A
55.91± 11.3
11.14± 1.2*
44.59± 10.45*
T318E
44.11±10.3
6.52± 1.1*
32.76± 6.37
T262A/T318A
45.87±11.8
8.79± 2.7*
47.91± 8.8*
T262E/T318E
32.59±8.4*
0±0*
27.57± 6.1
Values are averages, in arbitrary units based on densitometry, and
standard deviations. For Lauryl maltoside values are derived from
ATPβ western blots and are normalized based on total protein stain
lane intensity (n=5). For Digitonin values are derived from
Coomassie stain of the BN-PAGE gels and are also normalized
based on total lane intensity (n=3). * = p<0.05 compared to WT,
values in bold = p<0.05 comparing A to E strain.
Online Table III. 4800 MSMS data for identification of 2D BNP spots. For gel map see Online Figure III.
Spot # Protein name
Pyruvate dehydrogenase E1 component
subunit alpha
1
Pyruvate dehydrogenase E1 component
subunit beta
2
Protein
accession
number
Protein
molecular
weight (Da)
Protein Score
(based on MS
and MS/MS)
Peptide sequence
Mascot
Ion Score
P16387
46313
61
GPLVLEYETYR
42
P32473
40029
87
EALNSAMAEELDR
VLVPYSAEDAR
38
21
3
Dihydrolipoyllysine-residue
succinyltransferase component of 2oxoglutarate dehydrogenase complex
P19262
50399
211
4
ATP synthase alpha chain
P07251
58572
81
AQEPPVASNSFTPFPR
DIPAVNGAIEGDQIVYR
NAESLSVLDIENEIVR
EAYPGDVFYLHSR
64
58
53
36
4
ATP synthase beta chain
P00830
54760
94
5
6
Cytochrome b-c1 complex subunit 2
ATP synthase gamma chain
P38077
40453
34329
96
85
IINVIGEPIDER
VVDLLAPYAR
VALVFGQMNEPPGAR
SAEDQLYAITFR
FEIDTDANVPR
YSILYNR
29
37
21
37
55
21
7
8
ATP synthase oligomycin sensitivity
conferral protein
Heat shock protein 60
P09457
P19882
22800
60714
61
161
9
ATP synthase subunit beta
P00830
54760
139
NSSIDAAFQSLQK
NVLIEQPFGPPK
LIDEYGDDFAK
VVDLLAPYAR
IINVIGEPIDER
AHGGFSVFTGVGER
61
74
59
44
40
46
10
Isocitrate dehydrogenase [NAD] subunit
2
P28241
39715
119
TTYENVDLVLIR
YAFEYAR
62
48
10
11
Isocitrate dehydrogenase [NAD] subunit
1
ATP synthase gamma chain
P28834
P38077
39300
34329
80
109
DYAVFEPGSR
TIEQSPSFGKFEIDTDANVPR
44
109
12
Potassium-activated aldehyde
dehydrogenase
P46367
56688
315
13
Malate dehydrogenase
P17505
35628
225
14
Superoxide dismutase [Mn]
P00447
25758
159
15
ATP synthase subunit alpha
P07251
58572
101
16
ATP synthase subunit alpha
P07251
58572
267
17
ATP synthase subunit beta
P00830
54760
184
18
ATP synthase subunit beta
P00830
54760
350
19
ATP synthase subunit beta
P00830
54760
198
20
ATP synthase subunit alpha
P07251
58572
237
FIEEFK
NEGATLITGGER
SPNIVFADAELK
EEIFGPVVTVTK
GYFIKPTVFGDVK
DDLFAINASIVR
DTDMVLIPAGVPR
DVIEPSFVDSPLFK
FISEVENTDPTQER
MIAIQQNIK
AIDEQFGSLDELIK
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
LFLAQYR
SNHNELLTEIR
AQPTEVSSILEER
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
EVAAFAQFGSDLDASTK
VVDLLAPYAR
IINVIGEPIDER
VALVFGQMNEPPGAR
LVLEVAQHLGENTVR
VVDLLAPYAR
IINVIGEPIDER
AHGGFSVFTGVGER
VALTGLTIAEYFR
VALVFGQMNEPPGAR
LVLEVAQHLGENTVR
IPSAVGYQPTLATDMGLLQER
VVDLLAPYA
IINVIGEPIDER
VALVFGQMNEPPGAR
LVLEVAQHLGENTVR
IPSAVGYQPTLATDMGLLQER
LFLAQYR
26
21
90
39
61
41
40
32
33
40
111
44
24
20
36
22
63
29
31
35
22
25
38
34
30
60
20
35
58
83
39
25
28
34
28
22
20
21
ATP synthase subunit beta
ATP synthase subunit beta
P00830
P00830
54760
54760
67
331
21
ATP synthase subunit alpha
P07251
58572
128
22
ATP synthase subunit beta
P00830
54760
403
22
ATP synthase subunit alpha
P07251
58572
267
23
ATP synthase gamma chain
P38077
34329
241
24
ATP synthase gamma chain
P38077
34329
115
25
ATP synthase oligomycin sensitivity
conferral protein
P09457
22800
236
25
ATP synthase subunit b
P05626
26993
214
26
ATP synthase subunit beta
P00830
54760
429
SNHNELLTEIR
IGEFESSFLSYLK
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
AHGGFSVFTGVGER
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
FTQAGSEVSALLGR
LVLEVAQHLGENTVR
LFLAQYR
SNHNELLTEIR
TGNIVDVPVGPGLLGR
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
FTQAGSEVSALLGR
LVLEVAQHLGENTVR
QLSLLLR
LFLAQYR
AVDALVPIGR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
YSILYNR
FEIDTDANVPR
DAPTFQESALIADK
YSILYNR
FEIDTDANVPR
55
40
66
26
36
33
36
85
62
90
31
65
25
53
40
99
81
99
30
44
70
66
43
56
98
55
59
46
NLDGYVVNLLK
NSSIDAAFQSLQK
LFGVEGTYATALYQAAAK
AVLDSWVR
ANSIINAIPGNNILTK
VLQQSISEIEQLLSK
VVDLLAPYAR
IINVIGEPIDER
62
62
99
63
52
58
22
20
26
ATP synthase subunit alpha
P07251
58572
27
ATP synthase subunit beta
P00830
54760
231
27
ATP synthase subunit alpha
P07251
58572
216
28
ATP synthase gamma chain
P38077
34329
416
29
ATP synthase gamma chain
P38077
34329
333
30
ATP synthase gamma chain
P38077
34329
380
31
32
ATP synthase oligomycin sensitivity
conferral protein
ATP synthase subunit beta
P09457
P00830
22800
54760
116
547
32
ATP synthase subunit alpha
P07251
58572
243
AHGGFSVFTGVGER
FTQAGSEVSALLGR
VALVFGQMNEPPGAR
IPSAVGYQPTLATDMGLLQER
LFLAQYR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
VVDLLAPYAR
IINVIGEPIDER
AHGGFSVFTGVGER
FTQAGSEVSALLGR
LFLAQYR
SNHNELLTEIR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
YSILYNR
FEIDTDANVPR
DAPTFQESALIADK
TIEQSPSFGKFEIDTDANVPR
YSILYNR
FEIDTDANVPR
TIEQSPSFGKFEIDTDANVPR
YSILYNR
FEIDTDANVPR
DAPTFQESALIADK
TIEQSPSFGKFEIDTDANVPR
90
81
56
142
22
63
40
38
28
88
68
32
72
61
43
57
94
75
168
56
94
128
57
89
73
147
LFGVEGTYATALYQAAAK
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
FTQAGSEVSALLGR
VALVFGQMNEPPGAR
IPSAVGYQPTLATDMGLLQER
LFLAQYR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
108
73
53
112
89
54
128
44
69
50
33
ATP synthase subunit beta
P00830
54760
422
33
ATP synthase subunit alpha
P07251
58572
140
34
ATP synthase subunit beta
P00830
54760
240
34
ATP synthase subunit alpha
P07251
58572
131
35
ATP synthase gamma chain
P38077
34329
202
36
ATP synthase gamma chain
P38077
34329
211
37
ATP synthase subunit d
P30902
19797
164
37
ATP synthase oligomycin sensitivity
conferral protein
P09457
22800
157
37
ATP synthase subunit b
P05626
26993
106
38
ATP synthase subunit f
Q06405
11305
90
39
ATP synthase subunit beta
P00830
54760
481
VVDLLAPYAR
IINVIGEPIDER
AHGGFSVFTGVGER
FTQAGSEVSALLGR
VALVFGQMNEPPGAR
IPSAVGYQPTLATDMGLLQER
LFLAQYR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
FTQAGSEVSALLGR
VALVFGQMNEPPGAR
LFLAQYR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
YSILYNR
FEIDTDANVPR
TIEQSPSFGKFEIDTDANVPR
YSILYNR
FEIDTDANVPR
TIEQSPSFGKFEIDTDANVPR
QLLELQSQPTEVDFSHYR
DLQSTLDNIQSARPFDELTVDDLTK
45
26
100
68
45
121
28
60
43
25
29
63
68
40
21
68
32
23
44
115
24
26
136
104
27
LGHLLLNPALSLK
LFGVEGTYATALYQAAAK
AVLDSWVR
VLQQSISEIEQLLSK
SLPQGPAPAIK
IANVVHFYK
VVDLLAPYAR
AHGGFSVFTGVGER
VALVFGQMNEPPGAR
LVLEVAQHLGENTVR
IPSAVGYQPTLATDMGLLQER
44
93
40
56
37
43
39
102
42
118
128
39
ATP synthase subunit alpha
P07251
58572
216
40
ATP synthase subunit alpha
P07251
58572
362
40
ATP synthase subunit beta
P00830
54760
339
41
ATP synthase subunit beta
P00830
54760
513
41
ATP synthase subunit alpha
P07251
58572
242
42
ATP synthase gamma chain
P38077
34329
240
43
ATP synthase gamma chain
P38077
34329
551
44
ATP synthase oligomycin sensitivity
conferral protein
P09457
22800
447
LFLAQYR
GIRPAINVGLSVSR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
LFLAQYR
SNHNELLTEIR
GIRPAINVGLSVSR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
LVLEVAQHLGENTVR
VVDLLAPYAR
AHGGFSVFTGVGER
VALVFGQMNEPPGAR
LVLEVAQHLGENTVR
IPSAVGYQPTLATDMGLLQER
LFLAQYR
GIRPAINVGLSVSR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
YSILYNR
FEIDTDANVPR
TIEQSPSFGKFEIDTDANVPR
YSILYNR
FEIDTDANVPR
KMDEAEQLFYK
DAPTFQESALIADK
HLNDQPNADIVTIGDK
TIEQSPSFGKFEIDTDANVPR
32
28
71
45
36
99
51
69
51
39
43
106
110
50
106
51
104
128
28
47
89
45
31
41
154
28
63
50
71
103
133
NLDGYVVNLLK
NSVIDAIVETHK
LGHLLLNPALSLK
NSSIDAAFQSLQK
IASDFGVLNDAHNGLLK
33
70
58
53
49
45
ATP synthase subunit f
Q06405
11305
92
46
ATP synthase epsilon chain
P21306
6738
196
47
ATP synthase subunit beta
P00830
54760
321
47
ATP synthase subunit alpha
P07251
58572
310
48
ATP synthase subunit alpha
P07251
58572
420
48
ATP synthase subunit beta
P00830
54760
248
49
ATP synthase gamma chain
P38077
34329
223
50
ATP synthase gamma chain
P38077
34329
524
51
ATP synthase subunit delta
Q12165
17010
225
52
ATP synthase oligomycin sensitivity
conferral protein
P09457
22800
234
LFGVEGTYATALYQAAAK
SLPQGPAPAIK
IANVVHFYK
SQTDAFYTQYK
SSLKTELQTASVLNR
AGISYAAYLNVAAQAIR
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
LVLEVAQHLGENTVR
LFLAQYR
SNHNELLTEIR
GIRPAINVGLSVSR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
LFLAQYR
AVDALVPIGR
SNHNELLTEIR
GIRPAINVGLSVSR
EAYPGDVFYLHSR
TGNIVDVPVGPGLLGR
VVDLLAPYAR
VLDTGGPISVPVGR
AHGGFSVFTGVGER
YSILYNR
FEIDTDANVPR
TIEQSPSFGKFEIDTDANVPR
YSILYNR
FEIDTDANVPR
KMDEAEQLFYK
DAPTFQESALIADK
HLNDQPNADIVTIGDK
TIEQSPSFGKFEIDTDANVPR
EAAEAAIQVEVLENLQSVLK
LQFALPHETLYSGSEVTQVNLPAK
134
38
44
28
36
82
51
27
108
102
31
58
51
85
48
31
67
99
34
81
51
69
28
112
22
47
132
31
31
40
67
99
154
104
110
NSSIDAAFQSLQK
87
53
ATP synthase epsilon chain
P21306
6738
192
LFGVEGTYATALYQAAAK
TELQTASVLNR
SQTDAFYTQYK
AGISYAAYLNVAAQAIR
134
36
51
44
Online Table IV. MSMS data for the identification of proteins from 1D BNP bands solubilized in 1.3% lauryl maltoside.
MS/MS
sample
name
F1Fo Band
Protein name
ATP synthase alpha
chain
ATP synthase beta
chain
Protein
accession
numbers
Protein
molecular
weight (Da)
Protein
identification
probability
Number of
unique
peptides
Peptide sequence
Next
Previous amino
amino acid acid
Best Peptide
identification
probability
Best
SEQUEST
XCorr
score
gi|584807
58,601.40
100.00%
16 APGILPR
AQPTEVSSILEER
AVDALVPIGR
EVAAFAQFGSDLDASTK
GVSDEANLNETGR
HALIVYDDLSK
LFLAQYR
PAINVGLSVSR
RSVHEPVQTGLK
SNHNELLTEIR
STVAQLVQTLEQHDAMK
SVHEPVQTGLK
TAVALDTILNQK
TGNIVDVPVGPGLLGR
VLAVGDGIAR
VVDALGNPIDGK
K
K
K
R
K
K
K
R
R
K
R
R
K
R
R
R
R
I
G
Q
V
Q
E
V
A
E
Y
A
R
V
V
G
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
2.01
2.99
2.81
5.66
4.64
3.59
2.84
3.03
3.89
3.58
5.7
2.26
3.45
3.49
2.41
3.68
gi|114575
54,906.80
100.00%
17 AHGGYSVFTGVGER
ETGVINLEGESK
FLSQPFAVAEVFTGIPGK
FTQAGSEVSALLGR
GISELGIYPAVDPLDSK
IGLFGGAGVGK
IPSAVGYQPTLATDMGLLQER
KPIHADPPSFAEQSTSAEILETGIK
LKDTVASFK
LLDAAVVGQEHYDVASK
LVLEVAQHLGENTVR
TVFIQELINNIAK
VALTGLTIAEYFR
VALVFGQMNEPPGAR
VLDTGGPISVPVGR
VQETLQTYK
VVDLLAPYAR
K
K
R
R
R
K
R
R
R
R
K
K
R
K
K
K
K
T
V
L
I
S
T
I
V
A
V
T
A
D
A
E
S
G
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
2.98
2.8
3.97
4.81
3.06
3.04
5.09
4.56
2.33
4.99
4.92
2.65
3.31
3.79
3.51
2.89
3.07
ATP synthase D chain gi|399076
F1 Band
19,792.20
99.90%
3 ITGSTATQLSSFK
PFDELTVDDLTK
QLQVIESFEK
R
R
K
K
I
H
95.00%
95.00%
95.00%
3.02
4.46
2.44
3 NGTAASEPTPITK
SQTDAFYTQYK
TELQTASVLNR
K
R
K
N
S
95.00%
95.00%
95.00%
3.39
2.39
3.59
ATP synthase epsilon
chain
gi|416683
6,724.80
99.90%
ATP synthase gamma
chain
gi|584817
34,334.20
100.00%
8 DAPTFQESALIADK
ELIVAITSDK
FEIDTDANVPR
GLCGSIHSQLAK
HLNDQPNADIVTIGDK
NLDVEATETGAPK
TIEQSPSFGK
YSILYNR
K
K
K
K
R
K
K
R
L
G
D
A
I
E
F
T
95.00%
95.00%
95.00%
94.70%
95.00%
95.00%
95.00%
95.00%
2.65
2.62
3.42
2.22
5.08
4.51
2.07
2.32
ATP synthase
oligomycin sensitivity
conferral protein
gi|114685
22,797.30
100.00%
9 GGLIVELGDK
GTVTSAEPLDPK
IASDFGVLNDAHNGLLK
LENVVKPEIK
LFGVEGTYATALYQAAAK
NLDGYVVNLLK
NSSIDAAFQSLQK
NSVIDAIVETHK
TVDLSISTK
K
K
K
K
R
K
K
R
K
T
S
G
G
N
V
V
N
I
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
3.59
2.69
4.93
3.15
4.53
2.37
4.75
4.38
2.58
ATP synthase subunit
4
gi|114589
26,992.60
100.00%
6 ANSIINAIPGNNILTK
DRIDSVSQLQNVAETTK
IDSVSQLQNVAETTK
KVSDVLNASR
VSDVLNASR
YLAPAYK
K
K
R
K
K
K
T
V
V
N
N
D
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
3.29
5.43
4.97
2.95
3.68
1.48
ATP synthase alpha
chain
gi|584807
58,601.40
100.00%
K
K
K
R
K
K
R
I
G
Q
V
L
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
2.25
3.22
1.9
4.31
3.75
5.46
20 APGILPR
AQPTEVSSILEER
AVDALVPIGR
EVAAFAQFGSDLDASTK
GIRPAINVGLSVSR
GMALNLEPGQVGIVLFGSDR
GVSDEANLNETGR
HALIVYDDLSK
IKGVSDEANLNETGR
KLYCVYVAVGQK
LFLAQYR
RSVHEPVQTGLK
SNHNELLTEIR
STVAQLVQTLEQHDAMK
SVHEPVQTGLK
TAVALDTILNQK
TGNIVDVPVGPGLLGR
VLAVGDGIAR
VVDALGNPIDGK
VVDALGNPIDGKGPIDAAGR
ATP synthase beta
chain
gi|114575
54,906.80
100.00%
ATP synthase epsilon
chain
gi|416683
6,724.80
99.90%
ATP synthase gamma
chain
gi|584817
34,334.20
100.00%
K
K
R
K
K
R
K
R
R
K
R
R
R
R
V
Q
V
R
E
A
E
Y
A
R
V
V
G
S
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
3.92
3.64
4.26
3.95
2.54
4.12
3.57
5.29
3.3
3.69
4.12
2.24
3.42
4.26
K
R
K
R
R
K
R
R
R
R
R
K
R
K
K
K
K
K
T
V
V
I
S
T
G
I
V
A
V
T
G
A
A
E
S
A
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
94.70%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
3.18
3.71
2.73
3.87
4.03
2.53
2.35
2.98
4.61
2.36
4.7
4.97
3.46
2.71
3.06
4.66
2.34
4.81
3 NGTAASEPTPITK
SQTDAFYTQYK
TELQTASVLNR
K
R
K
N
S
95.00%
95.00%
95.00%
2.39
3.69
2.13
8 DAPTFQESALIADK
ELIVAITSDK
GLCGSIHSQLAK
HLNDQPNADIVTIGDK
K
K
K
R
L
G
A
I
95.00%
95.00%
95.00%
95.00%
3.64
2.52
1.9
5.17
18 AHGGFSVFTGVGER
EMKETGVINLEGESK
ETGVINLEGESK
FTQAGSEVSALLGR
GISELGIYPAVDPLDSK
IGLFGGAGVGK
IINVIGEPIDER
IPSAVGYQPTLATDMGLLQER
KPIHADPPSFAEQSTSAEILETGIK
LKDTVASFK
LLDAAVVGQEHYDVASK
LVLEVAQHLGENTVR
TIAMDGTEGLVR
TVFIQELINNIAK
VALVFGQMNEPPGAR
VLDTGGPISVPVGR
VQETLQTYK
YDHLPENAFYMVGGIEDVVAK
KMDEAEQLFYK
NLDVEATETGAPK
TIEQSPSFGK
YSILYNR
K
K
K
R
N
E
F
T
95.00%
95.00%
95.00%
95.00%
3.94
4.71
2.22
1.74
Online Table V. MSMS data for the identification of proteins from 1D BNP bands solubilized in 2% digitonin.
MS/MS sample
name
Protein name
ATP synthase alpha
chain
F1 Band
F1Fo Band
Protein
accession
numbers
Protein
molecular
weight (Da)
Protein
identification
probability
Number of
unique
peptides Peptide sequence
7 AQFGSDLDASTKQTL
ASLKSATESF
ASTKAQPTEVSSIL
AVGDGIARVF
KQNQYSPLATEEQVPL
VAVGQKRSTVAQL
VQTLEQHDAMKY
Best Peptide
identification
probability
Best
SEQUEST
XCorr
score
Previous
amino
acid
Next
amino
acid
F
L
L
L
L
Y
L
V
V
E
G
I
V
S
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
4.37
1.8
3.15
3.07
1.95
2.82
4.14
gi|584807
58,601.40
100.00%
ATP synthase beta
chain
gi|114575
54,906.80
100.00%
10 DAAVVGQEHY
GGAGVGKTVF
GMDELSEEDKL
INNIAKAHGGF
LDAAVVGQEHY
LGRIPSAVGY
NALEIKTPQGKL
RFTQAGSEVSAL
VLEVAQHL
VRLKDTVASF
L
F
L
L
L
L
L
F
L
L
D
I
T
S
D
Q
V
L
G
K
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
2.66
2.28
3.89
2.4
3.27
1.86
2.27
2.9
2.24
2.58
ATP synthase alpha
chain
gi|584807
58,601.40
100.00%
12 AQFGSDLDASTKQTL
ASLKSATESF
ASTKAQPTEVSSIL
AVGDGIARVF
IIGDRQTGKTAVAL
IVYDDLSKQAVAY
KQNQYSPLATEEQVPL
SVSRVGSAAQVKAL
TALPVIETQGGDVSAY
TQLLKQNQY
VAVGQKRSTVAQL
VQTLEQHDAMKY
F
L
L
L
L
L
L
L
L
L
Y
L
V
V
E
G
D
R
I
K
I
S
V
S
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
4.31
2.21
3.86
2.89
2.88
2.36
3.43
3.04
2.35
2.79
3.23
4.02
ATP synthase beta
chain
gi|114575
54,906.80
100.00%
7 DAAVVGQEHY
DTGGPISVPVGRETL
GGAGVGKTVF
L
L
F
D
G
I
95.00%
95.00%
95.00%
2.67
3.88
2.35
LGRIPSAVGY
NALEIKTPQGKL
RFTQAGSEVSAL
VRLKDTVASF
Dimer Band
L
L
F
L
Q
V
L
K
95.00%
95.00%
95.00%
95.00%
2.11
2.38
3.51
2.22
ATP synthase epsilon
chain
gi|416683
6,724.80
99.90%
3 LNVAAQAIRSSL
NRSQTDAFYTQY
TQYKNGTAASEPTPITK
Y
L
Y
K
K
-
95.00%
95.00%
95.00%
3.16
3.15
3.2
ATP synthase gamma
chain
gi|584817
34,334.20
99.00%
2 EIDTDANVPRDLF
TLANQMLTAMAQGY
F
Y
E
A
95.00%
95.00%
2.36
4.21
oligomycin sensitivity
conferral protein
gi|114685
22,797.30
99.00%
2 KGTVTSAEPLDPKSF
QAAAKNSSIDAAF
L
Y
K
Q
95.00%
95.00%
3.18
2.75
ATP synthase alpha
chain
gi|584807
58,601.40
100.00%
4 AQFGSDLDASTKQTL
ASTKAQPTEVSSIL
DDLSKQAVAY
TALPVIETQGGDVSAY
F
L
Y
L
V
E
R
I
95.00%
95.00%
95.00%
95.00%
4.26
3.59
1.93
3.62
ATP synthase beta
chain
gi|114575
54,906.80
99.00%
ATP synthase D chain
gi|399076
19,792.20
99.50%
5 DAAVVGQEHY
DTGGPISVPVGRETL
GGAGVGKTVF
LGRIPSAVGY
VPADDLTDPAPATTF
2 ELQSQPTEVDFSHY
RITGSTATQL
L
L
F
L
Y
L
L
D
G
I
Q
A
R
S
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
95.00%
3.19
3.23
3.04
2.3
2.44
2.53
2.49
oligomycin sensitivity
conferral protein
gi|114685
22,797.30
99.00%
2 EKIASDF
QAAAKNSSIDAAF
F
Y
G
Q
95.00%
95.00%
1.67
2.57
RABBIT
RABBIT
ATP2
YEAST
MLSLVGRVASASASGALRGLTPSASLPQAQLLFRAAPAAVQPARDYAAQTSPAPKAGLAT 60
--MVLPRLYTATSRAAFKAAKQSAPL---------LSTSWKRCMASAAQSTP------IT 43
:: *: :*:: .*::. . **.*..:.
: .:: : .
***::*:..:. *
RABBIT
RABBIT
ATP2
YEAST
GRIVAVIGAVVDVQFDEG-LPPILNALEVQGRETRLVLEVAQHLGESTVRTIAMDGTEGL 119
GKVTAVIGAIVDVHFEQSELPAILNALEIKTPQGKLVLEVAQHLGENTVRTIAMDGTEGL 103
*::.*****:***:*::. **.******:: : :***********.*************
RABBIT
RABBIT
ATP2
YEAST
VRGQKVLDSGAPIKIPVGPETLGRIMNVIGEPIDERGPIKTKQFAPIHAEAPEFIEMSVE 179
VRGEKVLDTGGPISVPVGRETLGRIINVIGEPIDERGPIKSKLRKPIHADPPSFAEQSTS 163
***:****:*.**.:*** ******:**************:*
****:.*.* * *..
RABBIT
RABBIT
ATP2
YEAST
QEILVTGIKVVDLLAPYAKGGKIGLFGGAGVGKTVLIMELINNVAKAHGGYSVFAGVGER 239
AEILETGIKVVDLLAPYARGGKIGLFGGAGVGKTVFIQELINNIAKAHGGFSVFTGVGER 223
*** *************:****************:* *****:******:***:*****
RABBIT
RABBIT
ATP2
YEAST
TREGNDLYHEMIESGVINLKDATSKVALVYGQMNEPPGARARVALTGLTVAEYFRDQEGQ 299
TREGNDLYREMKETGVINLEGE-SKVALVFGQMNEPPGARARVALTGLTIAEYFRDEEGQ 282
********:** *:*****:. :******:*******************:******:***
RABBIT
RABBIT
ATP2
YEAST
DVLLFIDNIFRFTQAGSEVSALLGRIPSAVGYQPTLATDMGTMQERITTTKKGSITSVQA 359
DVLLFIDNIFRFTQAGSEVSALLGRIPSAVGYQPTLATDMGLLQERITTTKKGSVTSVQA 342
***************************************** :***********:*****
RABBIT
RABBIT
ATP2
YEAST
IYVPADDLTDPAPATTFAHLDATTVLSRAIAELGIYPAVDPLDSTSRIMDPNIVGSEHYD 419
VYVPADDLTDPAPATTFAHLDATTVLSRGISELGIYPAVDPLDSKSRLLDAAVVGQEHYD 402
:***************************.*:*************.**::*. :**.****
RABBIT
RABBIT
ATP2
YEAST
VARGVQKILQDYKSLQDIIAILGMDELSEEDKLTVSRARKIQRFLSQPFQVAEVFTGHMG 479
VASKVQETLQTYKSLQDIIAILGMDELSEQDKLTVERARKIQRFLSQPFAVAEVFTGIPG 462
** **: ** ******************:*****.************* ******* *
RABBIT
RABBIT
ATP2
YEAST
KLVPLKETIKGFQQILGGEYDLLPEQAFYMVG----------------- 511
KLVRLKDTVASFKAVLEGKYDNIPEHAFYMVGGIEDVVAKAEKLAAEAN 511
*** **:*: .*: :* *:** :**:******
Online Figure I
Online
Figure II
A. SD -HIS
T262A
T262E
T262E
T262A
atp2∆
WT
C. YEPD + EtBr
B. YEPD + EtBr
S213D
T262A
T262E
T262A
WT
S213D
T262E
T262A
/T318A
atp2∆
T262E/
T318E
atp2∆
WT
WT
T262E
kDa
66
4
3
8
12
15
16
19
55
1
36
31
2
21
66
55
36
31
21
14
6
6
20 21 22
23
24
25
WT
Online Figure III
13
17
11
7
14
kDa
9
10
5
18
14
26
27
28 29
30
31
T58A
32
33 34
35
37
36
38
T58E
39
40 41
42
44
45
T262A
43
46
47
49
51
48
50
53
52
T262E
A
35%
Onine Figure IV
[Sucrose]
15%
kDa
kDa
66
55
66
55
36
31
21
14
36
31
21
14
6
6
WT
T58A
T58E
S213A
S213D
T262A
T262E
T318A
T318E
T262A/T318A
T262E/T318E
atp2Δ
B