Functional and Structural Consequences Assessed in a Model System
Lesley A. Kane, Matthew J. Youngman, Robert E. Jensen, Jennifer E. Van Eyk
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 F1component 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
phosphomi-metic 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 F1FoATP synthase complex, suggesting the possibility that several of the
phosphorylations observed in the rabbit heart can have structural and functional consequences to the F1FoATP
synthase complex. (Circ Res. 2010;106:504-513.)
Key Words: mitochondria 䡲 ATP synthase 䡲 phosphorylation 䡲 preconditioning
P
reconditioning (PC) is a phenomenon by which physio-logical and pharmacophysio-logical interventions protect the heart from damage during future ischemic episodes (for review see1). Mitochondria have long been implicated in thisprotective phenotype.2,3In studying the link between PC and
the mitochondria, our group observed phosphorylation of the F1FoATP synthase complex subunit (ATP) in response to adenosine mediated PC.4 There have been several
observa-tions connecting modulation of the ATP synthase complex to PC, including its specific downregulation to preserve ATP pools during ischemia.5–7The 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 partic-ipate in control by kinase cascades and protein phosphoryla-tion (for reviews, see elsewhere8 –11). Several groups have
shown that the F1FoATP synthase complex can be
phosphory-lated on subunits ␣,12–14 ,12,14,15 ␦,16 ,12 ␥,12,14,17 4,12
OSCP,12,14 c,14and g,12 in a broad range of species.
Phos-phorylation has been correlated to dimerization of the ATP synthase complex in yeast (subunit g)12 and in heart (␥
subunit),17but there is currently no evidence that
phosphory-lation of ATP regulates any aspect of the complex. Regu-lation of this complex could come at a variety of points: transcription, translation, import, assembly, or direct func-tional regulation. The F1FoATP 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
Eu-karyotic F1FoATP 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-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
tion of the enzyme,20and 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 nonphosphorylat-able (alanine) or phosphomimetic (aspartic acid and glutamic acid) residues. The acidic residue mutations incorporate a charged residue to mimic phosphorylation. The alanine mu-tations act as a control mutation by ensuring backbone spacing is retained, without the possibility of phosphoryla-tion. 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 dephos-phorylation 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 forma-tion), because several phosphomimetic mutants had dramatic effects.
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.
Mitochondria and ATP Synthase Isolation
Crude mitochondria were isolated from yeast homogenates22 and
purified using sucrose gradients.22,23 ATP synthase was isolated
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.25Two-dimensional BN/SDS-PAGE
was also performed to analyze complex subunit composition.25See 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 silver26or colloidal Coomassie.27
Western Blotting
Gels were transferred and blotted as described in the Online Data Supplement.
ATPase Assays
In-solution ATPase assays were performed on sucrose gradient isolated complex.28In-gel ATPase analyses were performed
follow-ing BN-PAGE.19See the Online Data Supplement.
Tandem MS
Protein bands from 1D BN-PAGE and 2D BN/SDS-PAGE were cut and prepared for digestion.29See the Online Data Supplement for
data acquisition and protein identification details.
Three-Dimensional Structure
The␣33hexamer of the S cerevisiae ATP synthase from Protein Data Bank (http://www.pdb.org) 2HLD structure30 was modeled
using DeepView/Swiss-PdbViewer v3.7.
Non-standard Abbreviations and Acronyms
ATP ATP synthase subunit
BN-PAGE blue native polyacrylamide gel electrophoresis
DIG digitonin
EtBr ethidium bromide
LM lauryl maltoside
MS mass spectrometry
MS/MS tandem mass spectrometry
mtDNA mitochondrial DNA
PC preconditioning
WT wild type
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
Phosphomimetic 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.
Kane et al ATP Synthase Subunit Phosphorylation 505
Results
Phosphomimetic MutationsFour of the 5 phosphorylated residues observed in the rabbit heart protein are conserved in the yeast protein (see align-ment, 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 matrix-facing 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 dis-played 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,31mitochondria from all strains were
solu-bilized 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 observa-tions32–35 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 excep-tion being the LM-solubilized T262E/T318E, which is
re-Figure 1. The amino acid residues of interest mapped onto the 3D structure of the␣/ hexamer of S cerevisiae (Protein Data Bank
no. 2HLD).30Amino 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.
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 F1portion 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 condi-tions (based on densitometry, n⫽3 each, Online Table II). In
particular, T58E has much less of the F1band 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 ab-sence of this F1band 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 F1Focomplex, 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 solubiliza-tion) shows that the F1Fo(700-kDa) band of the WT, T262A,
Figure 2. BN-PAGE of isolated WT and
mutant mitochondria. A, One-dimensional SDS-PAGE (4% to 20%), denaturing/re-ducing gel. The total protein stain shows equal protein loading and subunit con-tent of all lanes (except atp2⌬, which lacks the subunit). Total protein stain of BN-PAGE gels shows equal protein con-tent 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, respec-tively. Bands are labeled according to the complex that is present; F1Fodimers, F1Fo
monomers, the F1portion of the complex,
and␣/ lower-molecular-weight bands. (For intensity data values, see Online Table II.)
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
Subunit Dimer Band Digitonin F1FoBand F1Band Lauryl Maltoside 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 f 2 j 2
Kane et al ATP Synthase Subunit Phosphorylation 507
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 F1band (480 kDa) in the WT and T262A strains is missing the Focomponent OSCP subunit (Figure 3A). For the T262E strain, in which there is no detectable F1band 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 F1subunits␣, , 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 chymo-trypsin independently. Several subunits of both the F1and 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 nonphos-phorylatable mutations was performed on complexes isolated from mitochondria by sucrose gradient centrifugation.24
Mi-tochondria 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⫾0.51mol Pi/mg per minute [S213E]) did not differ from WT (3.28⫾0.56mol 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⫾0.38mol 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
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 Foand/or F1subunits at the correct location in the BN-PAGE. (For MS identification data, see Online Table III.)
(T262E) had no detectable activity. It is of note that the double mutant of T262E/T318E (0.14⫾0.27mol 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 F1FoATP 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 F1Fodimer 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 muta-tions 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 com-plex 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 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 indi-cate 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.
Figure 5. In-gel ATPase assays. In-gel
ATPase assays were performed on BN-PAGE of digitonin solubilized mito-chondria from all strains. Left, Represen-tative 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).
Kane et al ATP Synthase Subunit Phosphorylation 509
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,36the
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 F
1 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 F1component assembly or function that can be stabilized by the Foportion of the complex.
F1FoATP 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 addi-tional observation of the F1Fo complex dimer. It is possible that the independent F1 portion may be a product of the detergent extraction31and 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 phos-phorylations (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). How-ever, there is a striking difference in the amount of free F1 complex between WT and some mutant strains. The phos-phomimetic mutants T262E, T318E, and T262E/T318E have reduced quantities of the F1complex (Figure 2C and 2E). It is probable that the F1portion 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 F1is essential in the absence of the Fo.
Figure 6. Schema of ATP synthase
structures and the effects of the phos-phomimetic 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.
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 inter-ference, suggesting unknown biophysical aspects of this amino acid residue. All of the other sites that caused a decrease in the quantity of the F1component 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.40The T58 residue is
also located within a region involved in subunit interactions, the-barrel domain.41,42Although T318 and T58 have less F
1 by BN-PAGE and known subunit interactions, they did not show defects in growth assays, indicating that any functional effect of this F1destabilization in the accessible T58 or buried T318 mutants is incomplete.
F1FoATP Synthase Complex Dimer Assembly
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,35improve efficiency of the
enzyme,43 and even affect mitochondrial membrane
poten-tial44(for review, see elsewhere45). Phosphorylation of ATP
synthase complex subunits, g subunit in yeast12and␥ subunit
in bovine,17have 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.
F1FoATP Synthase Complex Function
The amino acid residues of ATP involved in the binding of nucleotide/phosphate and catalysis have been defined in yeast.30None 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 phosphory-lated amino acid residues exhibited a large difference be-tween 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 dis-cussed 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
dis-rupted by the T58E mutation (or by phosphorylation), causing an inefficient complex and producing the lower ATPase rate.
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, phosphory-lation 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.
Implications of ATP Phosphorylation
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 single-site 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 phosphoryla-tion of ATP can have important implications to the structure and function of the F1FoATP 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 af-fected dimer formation and decreased ATPase function, and the T262E mutant ablated ATPase function. The matrix-facing, 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,
Kane et al ATP Synthase Subunit Phosphorylation 511
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 regu-lation of this phenomenon are still unknown and must be uncovered to gain a full understanding of the consequences of ATP phosphorylation.
Acknowledgments
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.
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.
References
1. Liem DA, Honda HM, Zhang J, Woo D, Ping P. Past and present course of cardioprotection against ischemia-reperfusion injury. J Appl Physiol. 2007;103:2129 –2136.
2. Murphy E, Steenbergen C. Preconditioning: the mitochondrial con-nection. Annu Rev Physiol. 2007;69:51– 67.
3. Halestrap AP, Clarke SJ, Khaliulin I. The role of mitochondria in pro-tection of the heart by preconditioning. Biochim Biophys Acta. 2007; 1767:1007–1031.
4. Arrell DK, Elliott ST, Kane LA, Guo Y, Ko YH, Pedersen PL, Robinson J, Murata M, Murphy AM, Marban E, Van Eyk JE. Proteomic analysis of pharmacological preconditioning: novel protein targets converge to mito-chondrial metabolism pathways. Circ Res. 2006;99:706 –714. 5. Ala-Rami A, Ylitalo KV, Hassinen IE. Ischaemic preconditioning and a
mitochondrial KATP channel opener both produce cardioprotection accompanied by F1F0-ATPase inhibition in early ischaemia. Basic Res
Cardiol. 2003;98:250 –258.
6. Ylitalo K, Ala-Rami A, Vuorinen K, Peuhkurinen K, Lepojarvi M, Kaukoranta P, Kiviluoma K, Hassinen I. Reversible ischemic inhibition of F(1)F(0)-ATPase in rat and human myocardium. Biochim Biophys
Acta. 2001;1504:329 –339.
7. Penna C, Pagliaro P, Rastaldo R, Di Pancrazio F, Lippe G, Gattullo D, Mancardi D, Samaja M, Losano G, Mavelli I. F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat. Am J Physiol Heart Circ Physiol. 2004;287:H2192–H2200.
8. Huttemann M, Lee I, Samavati L, Yu H, Doan JW. Regulation of mitochondrial oxidative phosphorylation through cell signaling. Biochim
Biophys Acta. 2007;1773:1701–1720.
9. Gibson BW. The human mitochondrial proteome: oxidative stress, protein modifications and oxidative phosphorylation. Int J Biochem Cell
Biol. 2005;37:927–934.
10. Horbinski C, Chu CT. Kinase signaling cascades in the mitochondrion: a matter of life or death. Free Radic Biol Med. 2005;38:2–11.
11. Pagliarini DJ, Dixon JE. Mitochondrial modulation: reversible phosphor-ylation takes center stage? Trends Biochem Sci. 2006;31:26 –34. 12. Reinders J, Wagner K, Zahedi RP, Stojanovski D, Eyrich B, van der Laan
M, Rehling P, Sickmann A, Pfanner N, Meisinger C. Profiling
phospho-proteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase. Mol Cell Proteomics. 2007;6:1896 –1906. 13. Vosseller K, Hansen KC, Chalkley RJ, Trinidad JC, Wells L, Hart GW, Burlingame AL. Quantitative analysis of both protein expression and serine/threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics. 2005;5:388 –398.
14. Hopper RK, Carroll S, Aponte AM, Johnson DT, French S, Shen RF, Witzmann FA, Harris RA, Balaban RS. Mitochondrial matrix phospho-proteome: effect of extra mitochondrial calcium. Biochemistry. 2006;45: 2524 –2536.
15. Hojlund K, Wrzesinski K, Larsen PM, Fey SJ, Roepstorff P, Handberg A, Dela F, Vinten J, McCormack JG, Reynet C, Beck-Nielsen H. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes. J Biol
Chem. 2003;278:10436 –10442.
16. Ko YH, Pan W, Inoue C, Pedersen PL. Signal transduction to mito-chondrial ATP synthase: evidence that PDGF-dependent phosphorylation of the delta-subunit occurs in several cell lines, involves tyrosine, and is modulated by lysophosphatidic acid. Mitochondrion. 2002;1:339 –348. 17. Di Pancrazio F, Bisetto E, Alverdi V, Mavelli I, Esposito G, Lippe G.
Differential steady-state tyrosine phosphorylation of two oligomeric forms of mitochondrial F0F1ATPsynthase: a structural proteomic analy-sis. Proteomics. 2006;6:921–926.
18. Ackerman SH, Tzagoloff A. Function, structure, and biogenesis of mito-chondrial ATP synthase. Prog Nucleic Acid Res Mol Biol. 2005;80: 95–133.
19. 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 –3185.
20. Mueller DM. Partial assembly of the yeast mitochondrial ATP synthase.
J Bioenerg Biomembr. 2000;32:391– 400.
21. Adams A, Gottschling DE, Kaiser CA, Stearns T. Methods in Yeast
Genetics. Plainview, NY: Cold Spring Harbor Laboratory Press; 1997.
22. Daum G, Gasser SM, Schatz G. Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J Biol Chem. 1982;257:13075–13080.
23. Meisinger C, Sommer T, Pfanner N. Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contami-nations. Anal Biochem. 2000;287:339 –342.
24. Hanson BJ, Schulenberg B, Patton WF, Capaldi RA. A novel subfrac-tionation approach for mitochondrial proteins: a three-dimensional mito-chondrial proteome map. Electrophoresis. 2001;22:950 –959.
25. Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal
Biochem. 1991;199:223–231.
26. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996;68:850 – 858.
27. 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;25:1327–1333.
28. Tzagoloff A. Oligomycin-sensitive ATPase of Saccharomyces cerevisiae.
Methods Enzymol. 1979;55:351–358.
29. Kane LA, Yung CK, Agnetti G, Neverova I, Van Eyk JE. Optimization of paper bridge loading for 2-DE analysis in the basic pH region: appli-cation to the mitochondrial subproteome. Proteomics. 2006;6:5683–5687. 30. Kabaleeswaran V, Puri N, Walker JE, Leslie AG, Mueller DM. Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase. EMBO J. 2006;25:5433–5442.
31. Grandier-Vazeille X, Guerin M. Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphoryla-tion complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes. Anal Biochem. 1996;242: 248 –254.
32. Lemaire C, Dujardin G. Preparation of respiratory chain complexes from Saccharomyces cerevisiae wild-type and mutant mitochondria: activity measurement and subunit composition analysis. Methods Mol Biol. 2008; 432:65– 81.
33. Sabar M, Balk J, Leaver CJ. Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native poly-acrylamide gel electrophoresis. Plant J. 2005;44:893–901.
34. Smith CP, Thorsness PE. Formation of an energized inner membrane in mitochondria with a gamma-deficient F1-ATPase. Eukaryot Cell. 2005; 4:2078 –2086.
35. Paumard P, Vaillier J, Coulary B, Schaeffer J, Soubannier V, Mueller DM, Brethes D, di Rago JP, Velours J. The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J. 2002;21: 221–230.
36. Goldring ES, Grossman LI, Krupnick D, Cryer DR, Marmur J. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol. 1970;52:323–335. 37. Chen XJ, Clark-Walker GD. Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae. Mol
Gen Genet. 1999;262:898 –908.
38. Giraud MF, Paumard P, Soubannier V, Vaillier J, Arselin G, Salin B, Schaeffer J, Brethes D, di Rago JP, Velours J. Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Biochim Biophys Acta. 2002;1555: 174 –180.
39. Meyer B, Wittig I, Trifilieff E, Karas M, Schagger H. Identification of two proteins associated with mammalian ATP synthase. Mol Cell
Pro-teomics. 2007;6:1690 –1699.
40. Boltz KW, Frasch WD. Hydrogen bonds between the alpha and beta subunits of the F1-ATPase allow communication between the catalytic site and the interface of the beta catch loop and the gamma subunit.
Biochemistry. 2006;45:11190 –11199.
41. Kagawa Y, Hamamoto T, Endo H. The alpha/beta interfaces of alpha1beta1, alpha3beta3, and F1: domain motions and elastic energy stored during gamma rotation. J Bioenerg Biomembr. 2000;32:471– 484. 42. Bakhtiari N, Lai-Zhang J, Yao B, Mueller DM. Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae.
J Biol Chem. 1999;274:16363–16369.
43. Strauss M, Hofhaus G, Schroder RR, Kuhlbrandt W. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J. 2008; 27:1154 –1160.
44. Bornhovd C, Vogel F, Neupert W, Reichert AS. Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes. J Biol Chem. 2006;281:13990 –13998.
45. Wittig I, Schagger H. Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta. 2009;1787:672– 680.
46. Craig EE, Hood DA. Influence of aging on protein import into cardiac mitochondria. Am J Physiol. 1997;272:H2983–H2988.
Novelty and Significance What Is Known?
● Cardiac preconditioning stimuli can affect mitochondrial function and specifically the F1FoATP synthase enzyme complex.
● Phosphorylation can occur on several subunits of the mitochondrial F1FoATP 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 F1FoATP synthase complex. The study moved a novel proteomic discov-ery that the  subunit was phosphorylated to a system that allowed the functional effect of each modification to be as-sessed. 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 F1FoATP 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. Understand-ing the mechanisms of these phosphorylations provides a new context for mitochondrial involvement in PC.
Kane et al ATP Synthase Subunit Phosphorylation 513