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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 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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 Kane et al Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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 ␣33 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 506 Circulation Research February 19, 2010 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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- Kane et al ATP Synthase  Subunit Phosphorylation 507 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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, 508 Circulation Research February 19, 2010 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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 Kane et al ATP Synthase  Subunit Phosphorylation 509 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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). 510 Circulation Research February 19, 2010 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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. Kane et al ATP Synthase  Subunit Phosphorylation 511 Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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. 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 connection. Annu Rev Physiol. 2007;69:51– 67. 3. Halestrap AP, Clarke SJ, Khaliulin I. The role of mitochondria in protection 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 mitochondrial 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. 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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 Proteomics. 2007;6:1690 –1699. ATP Synthase  Subunit Phosphorylation 513 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. Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017 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 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ 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