Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cation pumps

Haiyan Chua, Estela Puchulu-Campanellaa, Jacob A. Galanb, W. Andy Taob, Philip S. Lowa,1, and Joseph F. Hoffmanc,1

Departments of aChemistry and bBiochemistry, Purdue University, West Lafayette, IN 47907 and cDepartment of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520

Contributed by Joseph F. Hoffman, June 4, 2012 (sent for review April 24, 2012) The type of metabolic compartmentalization that occurs in red The hallmark test of whether or not ATP is in the confined blood cells differs from the types that exist in most eukaryotic pool is to evaluate the ability of either pump to form its respec- cells, such as intracellular organelles. In red blood cells (ghosts), tive phosphointermediate (E-P) on utilization of [γ-32P]-ATP in ATP is sequestered within the cytoskeletal–membrane complex. the presence of either Na+ or Ca2+ (7, 9, 10). If nonradioactive These pools of ATP are known to directly fuel both the Na+/K+ ATP is first entrapped within the pool, then neither of the pumps’ and Ca2+ pumps. ATP can be entrapped within these pools either 32P-labeled E-P is seen until the unlabeled pools of ATP are by incubation with bulk ATP or by operation of the phosphoglyc- emptied. In the case of IOVs, instead of E-Ps, ATP pool-de- erate kinase and pyruvate kinase reactions to enzymatically gen- pendent 22Na+ or 45Ca2+ uptake (transport) by their respective erate ATP. When the pool is filled with nascent ATP, metabolic pumps can be measured (8, 9). + + 2+ labeling of the Na /K or Ca pump phosphoproteins (ENa-P and In recent studies reported in intact RBCs, the changes in γ 32 ECa-P, respectively) from bulk [ - P]-ATP is prevented until the nucleotide metabolism (i.e., concentrations of ATP, ADP, and pool is emptied by various means. Importantly, the pool also can AMP) were measured under conditions of marked stimulation be filled with the fluorescent ATP analog trinitrophenol ATP, as of the Ca2+ pump (11). The patterns of changes in nucleotide

well as with a photoactivatable ATP analog, 8-azido-ATP (N3-ATP). concentrations were consistent with the requirement for nucle- PHYSIOLOGY fl Using the uorescent ATP, we show that ATP accumulates and otide sequestration within the cytoskeletal–membrane complex, fi then disappears from the membrane as the ATP pools are lled distinct from the nucleotides contained within the cytoplasm. and subsequently emptied, respectively. By loading N3-ATP into Thus, these results complement the foregoing evidence for the membrane pool, we demonstrate that membrane fi ’ membrane pools of ATP. Importantly, that study was the rst to that contribute to the pool s architecture can be photolabeled. identify ATP pools in intact cells, where previously their pres- With the aid of an antibody to N3-ATP, we identify these labeled ence could only be inferred (12). proteins by immunoblotting and characterize their derived pepti- fi

The present study not only con rms, by the use of different CHEMISTRY des by mass spectrometry. These analyses show that the specific methods, the primary hallmarks for characterizing the membrane peptides that corral the entrapped ATP derive from sequences pool of ATP in ghosts as mentioned above, but also extends the within β-, ankyrin, band 3, and GAPDH. analysis by identifying some of the cytoskeletal components that confine the pooled ATP. One method used to label the latter confocal microscopy | Western blots | membrane peptides components involved an antibody raised against 8-azido-ATP (N3- ATP), a photoactivatable ATP analog (13). N3-ATP (obtained he advent of techniques to isolate and characterize intracel- from Affinity Photoprobes) was entrapped within the ATP pool in Tlular organelles has made it possible to establish that meta- – the dark. On exposure to UV irradiation, the N3-ATP labeled bolic pathways and their associated intermediates are commonly proteins were detected on Western blot analysis after solubiliza- localized to membrane-enclosed compartments (1, 2). Although tion and gel electrophoresis. Another method involved the use of mammalian red blood cells (RBCs) have no intracellular mem- 8-azido-[α-32P]-ATP with subsequent identification of the labeled branes or organelles, previous work has documented that the proteins by phosphoimaging. In other experiments, N -ATP–la- + + 2+ 3 ATP that fuels the Na /K (ATP1A1) and Ca (ATP2B1) beled pool proteins were identified by mass spectrometry (MS). pumps resides within a structurally distinct compartment that Using these methodologies, we have unequivocally identified constitutes the preferred source of ATP for both types of pumps β-spectrin, ankyrin, glyceraldehyde-3-phosphate dehydrogenase, (3–6). This membrane-associated ATP pool can be filled with and band 3 as components that corral the ATP pools present in exogenous ATP in either hemoglobin-free porous ghosts or in- the cytoskeletal–membrane complex in RBC ghosts. side-outside membrane vesicles (IOVs) in ways that allow control of the contents of the ATP pool. One method of filling this pool is Results to incubate the ghosts or IOVs with bulk ATP; another is to run Effects of Pool ATP on Pulse-Labeling of Na+ and Ca2+ Pump E-Ps. reactions with the membrane-bound ATP synthesizing enzymes Central to the experiments described below, we felt it neces- phosphoglycerate kinase (PGK) and pyruvate kinase (PK) in the sary to repeat the basic characteristics of loading and emptying forward direction (7–9). In all of these methods, the ghosts and the ghost pool of ATP as described above, because that work had IOVs are washed free of loading substrates, leaving only the ATP entrapped within the membrane-associated pools to energize the pumps. The ATP thus sequestered is unavailable for reaction with Author contributions: H.C., P.S.L., and J.F.H. designed research; H.C., E.P.-C., and J.A.G. performed research; H.C., W.A.T., P.S.L., and J.F.H. analyzed data; and H.C., W.A.T., P.S.L., added hexokinase plus glucose (8, 9). Moreover, not only do the and J.F.H. wrote the paper. Na+ and Ca2+ pumps use the same pools of ATP, but the pools The authors declare no conflict of interest. can be emptied either by running either pump forward or running 1To whom correspondence may be addressed. E-mail: [email protected] or joseph.hoffman@ the PGK reaction backward (7, 9). Pool ATP alone also has been yale.edu. + 2+ shown to support a variety of Na and Ca pump functions once This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. thought to be energized by bulk ATP (9). 1073/pnas.1209014109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1209014109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 been carried out in a different laboratory with different analyt- a substrate for the Na+ pump (14, 15), but it does act like ATP, ical methods. Hemoglobin-free porous (frozen-thawed) RBC inhibiting the pumps by ∼76% when incorporated into the ghosts were prepared from freshly drawn blood as described membrane pools (using the same protocol as with ATP). Evidence previously (9). These ghosts are permeable to all added con- that TNP-ATP can fill the ATP pools derives from the fact that stituents. Of essential importance, after all pool manipulations ghosts incubated with TNP-ATP cannot phosphorylate the Na+/ and incubations, the ghosts were thoroughly washed to remove K+ pump with [γ-32P]-ATP until the pool is emptied of TNP-ATP. all nonpool solutes (including ATP and its derivatives) from Thus, the relative values of the 32P-labeled pump E-Ps measured inside and outside of the ghosts. Assay for pulse-labeling of the on a 20-s exposure to [γ-32P]-ATP were 1.05 ± 0.03 pmol 32P/mg ± 32 ENa-P and ECa-P were carried out by incubation at 0 °C with ∼2 after preincubation without ATP, 0.13 0.01 pmol P/mg μM[γ-32P]-ATP for 20 s. All reactions were stopped by exposure protein after preincubation with ATP, and 0.35 ± 0.04 pmol 32P/ to trichloroacetic acid (TCA) (Fig. 1). The precipitates were mg protein after preincubation with TNP-ATP. Moreover, when washed, solubilized in SDS, and then separated by SDS/PAGE TNP-ADP was preincubated with the ghosts and converted to electrophoresis. The relative radioactivity of the separated bands TNP-ATP by running the PGK reaction forward, the level of the was assessed by phosphoimaging (Cyclone Plus Storage System; 32P-labeled pump phosphoimtermediate was 0.42 ± 0.01 pmol PerkinElmer). This method differs from previously used meth- 32P/mg protein. ods (9) in which total TCA precipitates were counted, thus in- Fig. 2A shows a fluorescent image of porous ghosts when the cluding interference from other possible labeled constituents. pools are filled with TNP-ATP. The TNP-ATP incorporated into The phosphoimages give precise resolution of the E-Ps of the membrane pools has a punctate appearance, localized on the Na+ and Ca2+ pumps (Fig. 1 A and B). Thus, it is clear that when membrane, with the interior of the ghosts empty. When the pool the membrane pools contain unlabeled ATP, pulse-labeling of of TNP-ATP is emptied by running the PGK reaction backward 32 Materials and Methods fl both the ENa-P and ECa-P with [γ- P]-ATP is inhibited until the ( ), the relative uorescence is markedly ATP pools have been emptied. The results presented in Fig. 1B decreased (Fig. 2B). Fig. 2 C and D shows control experiments in confirm that the Na+ and Ca2+ pumps use the same pools of which the PGK reaction is dependent on its full complement of ATP (9). substrates, including NADH and 3-phosphoglycerate (PGA), to remove the pool of TNP-ATP. In Fig. 2C, one of the substrates Visualization of ATP Pool Filling and Emptying Using a Fluorescent (PGA) needed to run the reaction backward is omitted, and in ATP Analog. The fluorescent analog used was 2′ (or 3′)-O-(2,4,6- Fig. 2D, both substrates (PGA and NADH) required in the re- trinitrophenyl) (TNP)-ATP or TNP-ADP. TNP-ATP is not action are omitted. In these two controls, membrane-associated

32 Fig. 1. Effect of loading the ATP pool in ghosts with nonradioactive ATP on the ability to detect [γ- P]-labeled ENa-P and ECa-P by phosphoimaging. The ATP pool in ghosts was filled with nonradioactive ATP and then either emptied or left filled, as indicated. After addition of [γ-32P]-ATP and the desired cations, the

ENa-P and ECa-P were detected by SDS/PAGE, followed by phosphoimaging. The relative intensities of these bands reflect the ability to label the ENa-P and 32 ECa-P with a 20-s pulse of 2 μM[γ- P]-ATP. (A) Lanes 1 and 2 show labeling of the EMg-P and ENa-P under conditions with all ATP in the pools removed by previous washing. Lanes 3 and 4 show the same experiment as in lanes 1 and 2, except with the membrane pools filled with unlabeled ATP before the addition of [γ-32P]-ATP. Lanes 5 and 6 and lanes 7 and 8 show the E-P values when the pools of ATP have been depleted either by running the Na+/K+ pump forward with Na+ and K+ (lanes 5 and 6) or by running the PGK reaction backward (lanes 7 and 8). Note that when the ATP pools are filled with unlabeled ATP 32 (lanes 3 and 4), ENa-P and EMg-P are of similar intensity; however, if the ATP pools are emptied, thereby allowing [γ P]-ATP to label the pumps, then the 2+ relative intensity of the band representing the ENa-P is greater than its Mg counterpart. Quantitation ratios of the intensities of the bands for each pair of lanes were 2.9 for lanes 1 and 2, 0.88 for lanes 3 and 4, 2.2 for lanes 5 and 6, and 1.6 for lanes 7 and 8, with an SEM of ±3% for n =2.(B) These lanes show the

same band intensity relationships as in A for both the ENa-P (lower bands) and the ECa-P (upper bands), even when both types of E-Ps are labeled in the same incubation. Note that when the pools are filled with ATP (lanes 5–8), labeling of the E-Ps with [γ-32P]-ATP is inhibited. Moreover, when the ATP pool is emptied by running the Na+/K+ pump forward (lanes 1–4), the calcium pump also can be labeled with [γ-32P]-ATP, indicating that the Na+/K+ and Ca2+ pumps share the same ATP pools. The phosphoimages shown here are representative of multiple experiments.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209014109 Chu et al. Downloaded by guest on October 2, 2021 Fig. 3. Identification of ghost proteins labeled with pool-associated N3-ATP. Here the ghosts were filled or emptied of N3-ATP, as described in Materials and Methods. After washing, the membranes were photolyzed and sepa- rated by SDS/PAGE. Equal amounts of ghost proteins (30 μg) were loaded in all lanes. Lanes 1–7 were transferred to nitrocellulose and immunoblotted

with an antibody to N3-ATP, whereas lane 8 was stained with Coomassie blue. Lane 1 shows pools filled with N3-ATP; lanes 2 and 3, pools first filled + with N3-ATP and then emptied by either running the Na pump forward or running PGK reaction backward, respectively. In lane 4, pools are filled with

N3-ATP in the presence of excess unlabeled ATP to block any specific ATP- binding sites. Lanes 5–7 show the specificity of the N3-ATP antibody, dem- Fig. 2. Dynamic imaging by confocal microscopy of the loading and emp- onstrating that it labels only BSA photolyzed with N3-ATP (lane 6), not un- tying of membrane pools of ATP using the fluorescent ATP analog TNP-ATP.

labeled BSA (lane 5) or unlabeled RBC ghosts (lane 7). Lane 8 shows a PHYSIOLOGY ATP pools in porous ghosts were either loaded or loaded and then emptied Coomassie blue stain of ghost proteins after separation by SDS/PAGE. The of TNP-ATP by running the PGK reaction backward. Ghosts were then ob- proteins tentatively identified are indicated with lettered lines. Possible fi served by confocal microscopy. (A) Ghosts with their ATP pools lled with protein candidates for the labeled bands are A, β-spectrin and/or ankyrin; B, TNP-ATP. Because a large ensemble of ghosts is shown, the plane of focus 2+ Ca pump (Mr ∼140 kDa) or fragments of spectrin/ankyrin; C, α-subunit of passes through the center of most ghosts, showing the fluorescent ATP only + + the Na /K pump (Mr ∼110 kDa); D, band 3; E, ; and F, GAPDH. on the cell periphery where the plane of focus coincides with the membrane. However, in a few ghosts, the plane of focus passes through part of the membrane, revealing pool ATP in the mid region of the cell. The fact that exposure to UV irradiation by running the Na+ pump forward in ghosts exhibit only punctate fluorescence in a ring on the cell periphery + + CHEMISTRY – the dark (with Na and K ). Importantly, labeling of all mem- indicates that the TNP-ATP loaded pools are localized to the membrane (12). M ∼ (B) The entrapped TNP-ATP is labile and susceptible to removal by running brane proteins except actin ( r 42 kDa) was prevented by this the PGK reaction backward. (C and D) Controls, with one of the substrates procedure, suggesting that all of the labeled proteins except (PGA) needed to run the PGK reaction backward omitted (C) and both possibly actin participate in fencing of the ATP pools. Lane 3 is fl substrates (PGA and NADH) omitted (D). In D,the uorescent images of the a similar control to that shown lane 2, but with the pool N3-ATP loaded ghosts are stable even after continued incubation in the absence emptied by running the PGK reaction backward in the dark (with of both substrates. These images are similar to ones as studied by Hoffman NADH and PGA) before exposure to UV irradiation. Lane 4 et al. (12). shows a very different control, in which both nonspecific and pool-specific ATP binding sites were blocked by coincubation fluorescence is similar to that shown in Fig. 2A. These results with a 20-fold excess of unlabeled ATP. In this latter case, demonstrate the dynamic nature of the filling and emptying of photolabeling of the actin band was prevented as well. Inhibition the ATP pools. of actin labeling in this latter result was obviously expected, given that actin contains an ATP-binding site that will bind ATP even in the absence of a membrane-associated pool (16, 17). Based on Identification of Pool Corral Components by Photolabeling with N3- ATP. To identify the membrane proteins that form the compart- a comparison with the staining pattern of RBC membrane pro- ment containing the entrapped ATP, the ATP pool was filled teins (lane 8), the foregoing data collectively suggest that membrane/cytoskeletal-associated ATP pool components might with N3-ATP, and photolabeling of pool corral components was β 2+ + + initiated by UV illumination. To identify each photolabeled include -spectrin, ankyrin, the Ca pump, the Na /K pump, band 3, and GAPDH. component, an antibody was raised against N3-ATP, as described Materials and Methods in , and used in immunoblots to stain those Analysis of ATP-Associated Membrane and Cytoskeletal Proteins proteins photolabeled with N3-ATP. As shown in Fig. 3, the anti– 32 Labeled with [α- P]-N3-ATP. To validate the foregoing antibody N -ATP antibody readily detected BSA photolyzed in the pres- 3 staining of ATP pool components photolabeled with N3-ATP, we ence of N -ATP (lane 6), but not unlabeled BSA (lane 5) or 32 3 turned to analysis of ghost proteins photolabeled with [α- P]-N3- unlabeled RBC ghosts (lane 7). ATP after using, following an analogous protocol, the radio- – Fig. 3, lane 1, shows Western blots of N3-ATP labeled pro- labeled form of the ATP analog to either fill or empty the ATP fi teins after the pools were lled with N3-ATP and the entrapped pools of ATP. Thus, autoradiographs were analyzed instead of N3-ATP was photoactivated. As shown in lane 1, proteins that Western blots. Fig. 4, lanes 1–4, shows autoradiographs of the β M ∼ 2+ migrate near -spectrin and/or ankyrin ( r 250 kDa), the Ca labeled proteins after separation by SDS/PAGE, and lane 5 shows M ∼ pump ( r 140 kDa), or fragments of spectrin/ankyrin, the a Coomassie blue-stained control. Examination of lane 1 reveals + + 2+ α-subunit of Na /K pump (Mr ∼110 kDa), band 3, actin, and that proteins that migrate near spectrin, ankyrin, the Ca pump, GAPDH are labeled by this protocol. Lane 2 shows a control the α-subunit of Na+/K+ pump, band 3, and actin are all radio- 32 experiment in which the pool N3-ATP was removed before labeled by [α- P]-N3-ATP. The ghosts in lane 2 were emptied in

Chu et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 Peptides of β-spectrin, ankyrin, band 3, and actin (Table 1) were found to contain the extra mass attributed to N3-ATP la- beling. The ATP labeling on β-spectrin is at the extreme COOH terminus, near the tetramerization site and just beyond the ankyrin- binding site (18, 19). Ankyrin is labeled in its ZU-5 domain, within the spectrin-binding sequence of ankyrin (20, 21). Band 3 is labeled specifically at a glycolytic enzyme-binding site that can associate with GAPDH, aldolase, phosphofructokinase, and lactate dehydrogenase. Actin is labeled on amino acids proximal to its known N3-ATP–binding site (17). GAPDH seen on the immunoblots of labeled proteins (Fig. 3) was not detected in our MS/MS analyses. However, for unknown reasons, only 9% of GAPDH peptides in fresh preparations of trypsin-digested ghosts could be detected by this procedure (Table S1), suggesting 32 Fig. 4. Labeling of ATP pool-associated membrane proteins with [α- P]-N3- that GAPDH peptides may be photolabeled, as indicated in Fig. ATP. This was accomplished following the same protocol used for N3-ATP. 3, but not readily detected by MS. Although proteins corre- Labeled proteins were separated by SDS/PAGE and analyzed with a Cyclone sponding to the molecular weights of the catalytic subunits of phosphoimager. Lanes 1–4 show phosphoimages of ghost proteins after sep- both the Na+/K+ and Ca2+ pumps were seen in the immunoblots aration. Lane 5 is the Coomassie blue-stained counterpart. The proteins ten- of N -ATP–labeled proteins, neither of these polypeptides was tatively identified are indicated with arrows. Possible protein candidates for 3 2+ identified by MS. the labeled bands are: A, β-spectrin and/or ankyrin; B, Ca pump (Mr ∼140 α + + kDa) or fragments of spectrin/ankyrin; C, -subunit of the Na /K pump Discussion (Mr ∼110 kDa); D, band 3; and E, actin. Previous work has established that ATP is sequestered within compartments residing in the membrane– complex in 32 + + the dark of entrapped [α- P]-N3-ATP by running the Na /K human RBC ghosts. The ATP so entrapped serves as the prefer- pump forward with Na+ plus K+ before exposure UV irradiation. ential and proximal substrate for both the Na+/K+ and Ca2+ Lanes 3 and 4 are controls in which the pool was filled (lane 3) pumps. The main results reported in this paper identify several of with or emptied of (lane 4) the radiolabeled ATP analog, but with the membrane and cytoskeletal components that serve to corral neither lane exposed to UV irradiation. It is evident that, except the local pools of ATP, including β-spectrin, ankyrin, GAPDH, for actin, the only membrane proteins labeled are those exposed and band 3 (Figs. 3 and 4 and Table 1). The methods used to fi fi to the conditions described for lane 1. identify these proteins involve rst lling the pool with N3-ATP, then after photolysis, identifying the proteins in SDS poly- – MS Identification of Specific Peptides of ATP Pool-Associated acrylamide gels or isolating the N3-ATP labeled peptides and fi Membrane Proteins Labeled with N3-ATP. We first evaluated determining their identities by MS. Clearly, we have not identi ed whether the liquid chromatography–tandem MS (LC-MS/MS) all of the elements involved in the corral, including some unknown protocol used (Materials and Methods) would provide the nec- protein bands labeled in Figs. 3 and 4, but not detected by MS. Moreover, we detected no peptides from either the Na+/K+ or essary sensitivity to detect the peptides of ghost proteins. For this 2+ purpose, a crude trypsin digest of ghost proteins was loaded onto Ca pump on our MS analyses, even though polypeptides cor- responding to their respective molecular weights (∼110 kDa for a C18 column of an Eksigent Ultra2D NanoLC system, which + + ∼ 2+ was coupled inline to a hybrid dual-cell linear trap-orbitrap mass the Na /K pump and 140 kDa for the Ca pump) are labeled with the N -ATP in Figs. 3 and 4. We presume that this inability to spectrometer (LTQ-Orbitrap Velos; Thermo Fisher) for peptide 3 detect the two pumps by MS derives from (i) the poor capacity of sequencing. Peptides from all ghost membrane/cytoskeletal their peptides to ionize in the mass spectrometer, as demonstrated proteins were detected (Table S1). by our inability to detect any peptides from either pump in MS To determine which ghost proteins might be involved in cor- fi ii fi analyses of unmodi ed membranes (Table S1); ( ) their relatively ralling the ATP entrapped within the membrane pools, we rst low copy numbers in the ATP pool complex; or (iii) the stringent photolabeled the pool-associated elements with N3-ATP as de- threshold that we set for inclusion of an N3-ATP–labeled peptide scribed earlier (Fig. 3). We then isolated the ATP-derivitized in our MS dataset. fi peptides by af nity chromatography with anti-ATP antibody- Several aspects of our results merit further comment. First, it coated beads and/or a polymer-based metal ion capturing (Poly- is clear that the entrapped ATP cannot be tightly bound, because MAC) reagent, as described in Materials and Methods. To detect strongly immobilized ATP cannot serve as a substrate for either fi + + 2+ N3-ATP labeled peptides by MS, we found it necessary to rst the glycolytic enzymes, PGK and PK, or the Na /K or Ca treat the samples with alkaline phosphatase to remove negatively pump. Nevertheless, it is likely that the entrapped ATP, esti- charged phosphates from the labeled peptides. This step gave us mated as 100–600 molecules per pool (7), resides in a hydro- the ability to identify specific peptides labeled with N3-ATP when phobic environment, given that the fluorescence of TNP-ATP is the ATP pool was first filled with the photoactivatable ATP. largely quenched in aqueous solution, but becomes more intense

Table 1. N3-ATP–labeled peptides of proteins that entrap the membrane-associated pool of ATP Protein description Accession no. Peptide sequence

Spectrin β chain P11277 2124-SSWESLQPEPSHPY-2137 Ankyrin P16157 961-TPPPLAEEEGLSDR-974 Ankyrin P16157 1069-LCQDYDTIGPEGGSLK-1084 Band 3 P02730 361-GLDLNGGPDDPLQQTGQL-378 Actin P68133 121-QIMFETFNVPAMYVAIQAVLSLYASGR-147

The letters in bold type in each sequence represent the amino acids directly labeled with N3-ATP.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1209014109 Chu et al. Downloaded by guest on October 2, 2021 data from our laboratory suggest that PK, like many other gly- colytic enzymes, binds to band 3, no information exists on the location of PGK on the membrane. Nevertheless, association of PGK with the membrane appears likely, given the report that an antibody to PGK inhibits the activity of purified PGK, but not PGK bound to the RBC membrane (30), indicating that PGK could be tightly associated with pool ATP in the RBC membrane. Finally, it is not known what other functions might be associ- ated with the RBC membrane ATP pool. Possible candidates include providing fuel for an ATP-dependent glucose transporter (31), energizing a flippase (32), or supplying the ATP for the deoxygenation-promoted ATP release pathway (33, 34). With regard to the latter, we have preliminary evidence that TNP-ATP entrapped in the ATP pool of resealed ghosts is released to the outside of the ghosts on deoxygenation. Materials and Methods Loading and Emptying the Membrane Pools of ATP in Porous Ghosts. Two fi fi Fig. 5. Possible arrangement of membrane components that form the ATP incubations, where indicated, were needed, the rst to ll the pools and the pools that fuel the cation pumps. Membrane components that were pho- second to empty them of entrapped ATP. Thus, whether or not the pools were empty could be determined by a pulse chase experiment with 2 μM[γ32-P]- tolabeled with N3-ATP and identified by MS are shown in color, and other membrane components implicated in the pool’s architecture but not iden- ATP for 20 s at 0 °C, as described above. All incubations and ATP type pool manipulations were carried out using the methods and solutions described tified by MS are shown in gray. The locations of the major N3-ATP–labeled peptides in ankyrin (residues 961–974), band 3 (residues 361–378), and previously (7, 9). β-spectrin (residues 2124–2137) are marked with an asterisk. The first incubation was carried out for 30 min at 37 °C in solution (A), containing 10 mM Tris (pH 7.5), 40 mM NaCl, 2 mM MgCl2, and 0.25 mM EDTA. In addition, for those ghosts in which the pools were to be loaded with ATP, solution (A) also contained 1.5 mM ATP. The ghosts were washed as solvent polarity declines (14). The strong fluorescence of PHYSIOLOGY TNP-ATP entrapped in the ghost membrane pool (Fig. 2) is (always with 17 mM Tris, pH 7.5) and then exposed to a second incubation consistent with a hydrophobic ATP compartment. for another 15 min at 37 °C in solution (A), but this time the buffer also contained either 10 mM KCl (to run the Na+/K+ pump forward) or solution Our results also raise the question of how the corrals that (B) (5 mM MgSO4, 17.5 mM NaHCO3, 20 mM cysteine, 50 mM glycine, 5 mM harbor the ATP pool relate to the known structures of the 3-phosphoglyceric acid, and 0.25 mM NADH; pH 7.0), to run the phospho- – – membrane cytoskeletal complexes (22 24). The major RBC glycerate (PGK) reaction backward. In other experiments, the ATP pools also membrane complexes are of two types, a junctional complex and could be filled by running the PGK reaction forward. This was done using an ankyrin complex, with some overlap in the constituents of the solution (C), containing 0.83 mM Triose-P, 0.415 mM NAD, 0.25 mM ADP, CHEMISTRY two (25, 26). The prime distinctions between the two complexes 5.0 mM MgSO4, 50.0 mM Na2HPO4/NaH2PO4, and 132 mM glycine. The lie in the fact that the former contains actin, adducin, and protein ghosts were always washed to remove all bulk substrates before attempts to + + 2+ 32 4.1, whereas the latter contains ankyrin. Our results clearly pulse-label the Na /K pump or Ca pump with [γ- P]-ATP. After pulse-la- beling, the reaction was stopped by adding a solution containing 2.5% TCA, demonstrate that N3-ATP labels ankyrin in its ZU5 domain, which contains the binding site for β-spectrin (20) and lies adja- 1mMATP,and1mMK3PO4. The precipitate was washed with this solution before preparation for analysis by phosphoimaging. cent to the ankyrin-binding site for band 3 (27, 28). N3-ATP also β fl For the results shown in Fig. 1B, the experiments were performed by labels -spectrin in a exible region near the site responsible for fi αβ loading the pool with ATP as before, by rst incubating at 37 °C for 30 min in association of -spectrin dimers to tetramers and not too distant modified solution (A) containing 1.5 mM ATP, 15 mM Tris (pH 7.5), 12 μM β – from the ankyrin-binding site on -spectrin (18 20). In addition, MgCl2, 50 mM NaCl, or 50 mM NMDG. The second incubation, to unload the + N3-ATP labels band 3 near the junction of its cytoplasmic and pooled ATP, was carried out as described above for running the Na pump membrane-spanning domains within a peptide recently found to forward with Na+ plus K+. The ghosts were then washed before pulse-la- include a second binding site for GAPDH. Because this peptide beling with [γ-32P]-ATP to measure Na+ as well as Ca2+ phosphoproteins, as resides close to the docking site of ankyrin on band 3 (27, 28), and described previously. because ankyrin is known to bind the cytoplasmic domain of the + Fluorescence Microscopy. The ghost’s ATP pools were either loaded with TNP- Na pump (29), it can be argued that all of the prominent N3- ATP–labeling sites on the membrane except actin appear to re- ATP or loaded, then emptied of TNP-ATP following the same protocol as described for the loading manipulations with ATP. The ghosts were washed side within or near the ankyrin–band 3 complex, as depicted in and imaged with an Olympus FluoView FV1000 inverted confocal micro- Fig. 5. scope with a 60× oil-immersion objective. The fact that N3-ATP labels actin, which resides at the junc- ∼ tional complex at least 30 nm from the ankyrin complex, war- Production and Use of a Polyclonal Antibody to N3-ATP. Keyhole limpet he- rants further discussion. Although an actin-associated ATP pool mocyanin photolabeled with N3-ATP (13) was used as an antigen. UV pho- cannot be unequivocally excluded by our data, the fact that la- tolysis was carried out in PBS buffer (pH 7.4) at room temperature. The beling of actin was not prevented by emptying N3-ATP from the antigen suspended in Gold Adjuvant Reagent (Sigma-Aldrich) was injected pool by running either the Na+ pump forward or the PGK re- into rabbits at regular intervals over a 4-mo period. The separated serum action backward (Fig. 3, lanes 2 and 3) suggests that actin la- was stored at −80 °C until use. Specificity of the antibody was determined by Western blot analysis (Fig. 3). beling is not mediated by N3-ATP within the pool. We suggest instead that N3-ATP bound to the ATP-binding site on actin is responsible for this prominent labeling. This conclusion is sup- Photolabeling and Detection of Pool-Related Protein Components by N3-ATP. The ghost’s ATP pools were loaded with N3-ATP in the dark following the ported by the observation that addition of excess unlabeled ATP same protocol as for ATP. The ghosts were washed before exposure to UV competitively blocks N3-ATP labeling of actin in porous ghosts irradiation, which resulted in photolabeling of the proteins residing adjacent

(Fig. 3, lane 4). to the entrapped N3-ATP. The ghosts were solubilized with 2% SDS, and Another problem concerns the locations of the two enzymes protein concentration was determined using the Micro BCA Assay Kit

(PGK and PK) that provide ATP for the pool. Although recent (Thermo Scientific). Ghost proteins were separated by SDS/PAGE, with the N3-

Chu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 ATP–labeled proteins detected by Western blot analysis using the anti–N3- Orbitrap Velos; Thermo Fisher) interfaced with an Eksigent Ultra 2D NanoLC HPLC ATP antibody and the Thermo Scientific Enhanced Chemiluminescence Kit. system. Peptide samples were dissolved in 8 μL of 0.1% formic acid and injected into a capillary column (75 μm i.d. and 12 cm bed length) packed with 5 μmof Radiolabeling and Detection of ATP Pool-Associated Protein Components. C18 Magic beads resin (Michrom). Peptides were then eluted at 300 nL/min using These experiments were carried out using the same protocol described a0–100% acetonitrile gradient over 90 min. The electrospray ionization emitter – above for N3-ATP labeled protein components. After separation by SDS/ tip was generated on the column with a laser puller (model P-2000; Sutter 32 PAGE, the [ P]-labeled proteins were detected by phosphoimaging. Instruments). The mass spectrometer was operated in the data-dependent mode in which a full-scan MS (from m/z 300–1,700 with a resolution of 60,000 at m/z fi – Puri cation of N3-ATP Photolabeled Peptides. ATP pool components of ghosts 400) was followed by 20 MS/MS scans of the most abundant ions. Ions with were photolabeled with N3-ATP, as described above, with 2% C12E8 (Affy- a charge state of +1 were excluded. The mass exclusion time was 90 s. metrix) in 100 mM Tris-HCl (pH 7.5) used to solubilize the ghosts. The sam- ples were then diluted 20 times with 100 mM Tris-HCl and precleared with Peptide Identification. The LTQ-Orbitrap raw files were searched directly rabbit preimmune IgG immobilized on Protein A/G PLUS-Agarose (Santa against a Homo sapiens database with no redundant entries (67,250 entries; Cruz Biotechnology) following the manufacturer’s protocol. N -ATP–labeled 3 human International Protein Index v.3.64) using the SEQUEST algorithm proteins were purified by immobilized anti–N -ATP antibody on Affi-Gel Hz 3 on Proteome Discoverer version 1.2 (Thermo Fisher) or Sorcerer IDA server hydrazide gel (Bio-Rad), then trypsin-digested to prepare a mixture of la- fi beled and unlabeled peptides. PolyMAC reagent functionalized with Ti (IV) version 2.5.6 (Sage-N Research). Proteome Discoverer created DTA les from the raw data with a minimum ion threshold of 15 and absolute intensity ions (Tymora Analytical) was used to separate N3-ATP–labeled peptides from unlabeled peptides as described previously (35). Alkaline phosphatase (New threshold of 50. Peptide precursor mass tolerance was set at 10 ppm, and fi England BioLabs) was added to remove the 5′-phosphate from N3-ATP, and MS/MS tolerance was set at 0.8 Da. Search criteria included a static modi - then removed from the peptide mixture using a 10-kDa cutoff protein cation of cysteine residues of +57.0214 Da, a variable modification concentrator (Millipore). Peptide samples were desalted using a Sep-Pak C18 of +15.9949 Da to include potential oxidation of methionine residues. N3- desalting column (Waters) before MS analysis. In some studies, total mem- ATP–photolabeled peptides were identified based on the additional mass

brane protein extracts were first digested with trypsin (35), and then N3- associated with the N-adenosine adduct (+280.092 Da) or the N-adenine ATP–labeled peptides were isolated as described above. adduct (+148.050 Da). False discovery rates were set for 1%.

LC-MS/MS Analysis. Tryptic peptides were analyzed by LC-MS/MS on a high- ACKNOWLEDGMENTS. This work was supported by National Institutes of resolution hybrid duel-cell linear ion trap orbitrap mass spectrometer (LTQ- Health Grants R01 GM24417 (to P.S.L.) and R01 GM088317 (to W.A.T.).

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