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J. Biol. Chem., Vol. 275, Issue 42, 32931-32939, October 20, 2000

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Mitochondrial F₀F₁ ATP Synthase

SUBUNIT REGIONS ON THE F₁ MOTOR SHIELDED BY F₀, FUNCTIONAL SIGNIFICANCE, AND EVIDENCE FOR AN INVOLVEMENT OF THE UNIQUE F₀ SUBUNIT F₆^*

Young Hee Ko, Joanne Hullihen, Sangjin Hong, and Peter L. Pedersen

From the Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

Received for publication, May 23, 2000, and in revised form, June 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F₀F₁) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F₁ within F₀F₁, all six N-terminal regions derived from  +  subunits are shielded, indicating that one or more F₀ subunits forms a "cap." Second, at the bottom of F₁ within F₀F₁, the N-terminal region of the single  subunit and the C-terminal regions of all three  subunits are shielded also by F₀. Third, and in contrast, part of the  subunit located at the bottom of F₁ is already shielded in F₁, indicating that there is a preferential propensity for interaction with other F₁ subunits, most likely  and . Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F₁ are shielded by F₀, further proteolytic shaving of  and  subunits at these locations eliminates the capacity of F₁ to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F₀ subunit called "F₆," unique to animal ATP synthases, is involved in shielding F₁. The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP synthases represent one of natures' most unique enzyme classes (reviewed in Refs. 1-3). Structurally, these enzymes are observed by electron microscopy (4-12) to consists of four distinct features, a headpiece, a basepiece, a central stalk or "stem region" connecting the headpiece and basepiece, and a "second stalk" extending from the basepiece to the top of the headpiece (4-10). Animal ATP synthases contain, in addition, what appears to be a fifth structural feature, a ring-like disc or "collar" surrounding the central stalk (11). Biochemically, however, ATP synthases separate most readily into only two units. One is a water-soluble component, F₁, containing five subunit types in the ratio ₃₃, whereas the other is a detergent-soluble component, F₀, containing three subunit types (a, b, c) in bacteria and at least 10 more in animal systems (1-3). A regulatory protein, IF₁, is found also in isolated mitochondrial ATP synthases. A variety of studies involving biochemica1 (13-25), electron microscopic (4-12), and x-ray crystallographic (26-30) approaches, have shown that the headpiece and central stalk are derived primarily from F₁ subunits and the basepiece and second stalk are derived primarily from F₀ subunits. In contrast, the subunit source of the ring-like disc or "collar" surrounding the central stalk in animal ATP synthases (11) remains unknown.

Nature's rationale for providing ATP synthases with this unique molecular architecture has become apparent only in recent studies where evidence that these fascinating enzymes contain two motors has been mounting (31-33). One of the proposed motors is F₁, which is driven by ATP binding and/or hydrolysis (31), whereas the other is contained within F₀ and driven by a proton gradient (33). During ATP synthesis, the proposed motor within F₀, composed of 10-12 subunit c molecules (30, 34) and a single subunit a, is believed to drive the F₁ motor in reverse (35) via a central rotor, the F₁- subunit. This subunit extends from a ring of subunit c molecules in the F₀ basepiece (19), through the central stalk region, and finally through the center of F₁ where it interacts differently with each of the three pairs (26-29). During one 360° rotation of the  subunit, the binding of ADP and inorganic phosphate, the synthesis of ATP, and the release of this ATP are believed to occur on each pair as proposed by the "binding change mechanism" (2, 36). During this process, the  subunit in bacteria, or its  subunit equivalent in animals, is also believed to rotate (32), presumably at the bottom of F₁ within the central stalk region.

Although much has been learned about the structure and function of ATP synthases as summarized above, a three-dimensional structure has not been obtained for the complete enzyme (F₀F₁) from any source. In fact, of the 16 different subunit types within the mitochondrial ATP synthase of animal cells, only two and a half of these (, , and about half of ) have been solved at atomic resolution (26, 28). Consequently, there are still many important questions that remain unanswered about structure/function relationships in ATP synthases, and how, within these remarkably complex machines, energy coupling between the proposed proton gradient-driven motor within F₀ is coupled to the reversal of the ATP-driven F₁ motor. Specifically, one important question relates to the identity of exterior subunit regions at the top and bottom of F₁ that are shielded by F₀ in the complete ATP synthase, because these regions may include F₀/F₁ contact sites essential for energy coupling during ATP synthesis. Although information about these regions, particularly at or near the top of F₁, is gradually being generated for the Escherichia coli and chloroplast enzymes (12, 23), little information is available for animal ATP synthases.

In studies reported below, we used a variety of approaches to identify exterior subunit regions at the top and bottom of rat liver F₁ that are shielded by F₀. We then assessed the requirement of these regions for coupling a proton gradient to ATP synthesis. In addition, we obtained evidence for the involvement of an F₀ subunit unique to animal ATP synthases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Rats (Harlan Sprague-Dawley^CD, white males) were obtained from Charles River Breeding Laboratories. ATP, MgCl₂, ²EDTA, DTT,¹ tricine, Sephadex G-25 (coarse), soybean trypsin inhibitor, oligomycin, and DCCD were from Sigma. SDS, acrylamide, and bisacrylamide were from Bio-Rad, PVDF membranes from Millipore, and Western blot reagents from Amersham Pharmacia Biotech. Modified trypsin (sequencing grade), pyroglutamate aminopeptidase, hexokinase, and glucose-6-phosphate dehydrogenase were from Roche Molecular Biochemicals. The detergent CHAPS was obtained from Anatrace, whereas sucrose, urea, and potassium phosphate were from J.T. Baker. Coomassie Blue dye binding reagent for protein determination was from Pierce, AEBSF was from Calbiochem, and 3,5-dimethoxy-4-hydroxy-cinnamic acid and -cyano-4-hydroxy-cinnamic acid were from Aldrich. Centriprep filtration units for protein concentration were from Amicon. Venturicidin was from BDH Chemicals. The antibodies to F₀ components were generous gifts from Drs. Y. Hatefi and Akemi Matsuno Yagi. All other products were of the highest purity or grade commercially available.

Methods

Purification of Mitochondrial ATP Synthase (F₀F₁)-- This complex was purified from rat liver mitochondria by a modification of a previously described method developed in this laboratory (6). A washed submitochondrial particle fraction, referred to previously as 3X membranes (6), was stored at ~15 mg/ml and 20 °C in TA buffer (50 mM Tricine, 1 mM ATP, 25 mM EDTA, 0.5 mM DTT, and 5% ethylene glycol, pH 7.9), thawed, and solubilized at 4 mg/ml in 0.6% CHAPS for l h on ice with occasional stirring. After centrifugation for 1 h at 48,000 rpm at 4 °C in a 70.1-Ti rotor in a Beckman Optima LE 80K ultracentrifuge to remove unsolubilized material, the supernatant was concentrated using Amicon's Centriprep filtration unit (molecular mass cutoff = 50 kDa). The concentrate was diluted with TA buffer to give a CHAPS concentration of 0.1% and frozen in dry ice and ethanol, and then stored at 20 °C for 3 h. The sample (~20 ml) was then thawed, and 5-ml aliquots (2.0-2.5 mg/ml) were placed on 20 ml of 25% sucrose in TA buffer without CHAPS and centrifuged 12 h and 45 min at 4 °C in the same centrifuge described above but using the SW-28 rotor. The pellet, F₀F₁ (7-10 mg/ml), judged to be >90% pure, was stored at 80 °C in TA buffer. The specific activity was 14-15 µmol of ATP hydrolyzed × min¹ × mg¹ protein based on the ATPase and protein assays described below.

Purification of the F₁ Moiety of Mitochondrial ATP Synthase-- F₁ with a specific activity of about 25 µmol × min¹ × mg¹ in TrisCl buffer was purified from isolated rat liver mitochondria exactly as described by Catterall and Pedersen (37) with one modification. The terminal Sephadex G-200 step was replaced by chromatography on a 2.1- × 13-cm Sephadex G-25 column packed on top of a 1.5-cm layer of sea sand and a 1-cm layer of glass wool. It was then stored or prepared for use as described by Ko et al. (38).

Preparation of Inner Mitochondrial Membrane Vesicles and F₁-depleted Vesicles, and Reconstitution of F₁ with the F₁-depleted Vesicles-- All steps were carried out exactly as described previously by Pedersen and Hullihen (39).

Treatment of F₀F₁ with Pyroglutamate Aminopeptidase-- To remove the N-terminally blocked group on the  subunit, F₀F₁ (1 mg/ml) was placed in a 200 µl of digestion buffer containing 100 mM sodium P_i, pH 8.0, 10 mM EDTA, 5 mM DTT, 5% glycerol, 0.6% CHAPS, and pyroglutamate aminopeptidase (0.019 mg/ml). The enzyme was then incubated in this mixture first for 12 h at 4 °C and then for 2 h at 25 °C, after which the entire reaction mixture was added to SDS sample buffer and analyzed by SDS-PAGE. The  subunit band was then subjected to N-terminal sequence analysis exactly as described below.

Treatment of F₁ and F₀F₁ with Trypsin-- F₁ and F₀F₁ (each 160 µg) were subjected to digestion for various incubation times at 25 °C in a final volume of 0.1 ml containing 250 mM potassium P_i, pH 7.5, 5 mM EDTA, and 10 µg of trypsin. Trypsin inhibitor (50 µg), or AEBSF at a final concentration of 5 mM, was then added at various incubation times to stop the reaction. Following the addition of ammonium sulfate to the mixture to give 65% saturation, the resultant suspension was centrifuged at 13, 000 rpm in a Sorvall SS 34 rotor for 1 h at 25 °C.

Assay for ATPase Activity-- ATPase activity was assayed exactly as described previously (37) by a spectrophotometric procedure in which ADP formed was coupled to the pyruvate kinase and lactic dehydrogenase reactions.

Assay for ATP Synthesis-- ATP synthesis was monitored spectrophotometrically at 340 nm and 25 °C by coupling the production of ATP to the reduction of NADP⁺ via the hexokinase and glucose-6-phosphate dehydrogenase reactions. The assay mixture contained, in a final volume of 1 ml, 10 mM potassium P_i, 10 mM succinate, 50 mM Tris acetate, 1 mM glucose, 1 mM NADP⁺, 2.0 mM MgCl₂, 2 mg/ml bovine albumin, 200 mM sucrose, 20 IU hexokinase, 0.25 IU glucose-6-phosphate dehydrogenase, and 100-200 µg of inner mitochondrial membrane vesicles. The reaction was started by the addition of 1 mM ADP.

SDS-PAGE-- This was carried out as indicated either by the method of Laemmli (40) or the more sensitive method of Schägger and von Jagow (41) for separating proteins in the molecular mass range of from 1 to 100 kDa.

Western Analysis-- After conducting SDS-PAGE, the proteins on the gel were transferred electrophoretically at 4 °C in 10 mM CAPS, 10% methanol transfer buffer, pH 11, onto a PVDF membrane (1 h at 100 V and 0.2 amp). The membrane was then blocked for l h with 2% bovine albumin plus 5% non-fat dry milk in phosphate-buffered saline T buffer (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, 0.1% Tween 20, pH 7.5), incubated for 1 h at 23 °C with polyclonal antibodies as indicated in the figure legends, and then further incubated for 1 h at 23 °C with secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG). The immunoreactive bands were detected by an enhanced chemiluminescence (ECL) system.

Mass Spectral Analysis-- Mass spectra were acquired on samples of 10-20 pmol in a PerSeptive Biosystems Voyager MALDI-TOF-DE mass spectrometer using a nitrogen laser (wavelength 337 nm). The matrix was either 3,5-dimethoxy-4-hydroxy-cinnamic acid or -cyano-4-hydroxy-cinnamic acid. Positive ion mass spectra were analyzed using PerSeptive GRAMS software version 3.01c.

N-terminal Sequence Analysis-- F₁ or F₀ subunits were transferred from SDS-PAGE gels onto PVDF membranes by electroblotting. Transfer conditions were for 1 h in 10 mM CAPS buffer, 10% methanol, pH 11, at 4 °C. The peptides were then excised and subjected to N-terminal sequencing (42) using an Applied Biosystems 475A protein sequencing system (43).

Determination of Protein-- Three different methods were used to determine protein, Lowry et al. (44), biuret (45), and Coomassie dye (Pierce). In all cases bovine albumin was used as the standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Objectives and Systems Employed-- The first objective of this study was to identify exterior regions on F₁ that are shielded by F₀ within a fully active, complete ATP synthase (F₀F₁) preparation from an animal mitochondrial source. The regions on F₁ of most interest (Fig. 1A) were its top where the N-terminal regions of  and  subunits reside (26-28) and its bottom where the C-terminal regions of these same subunits (26-28) reside together with part of the  subunit (26, 28), the  subunit (25, 30), and likely also the  subunit. The second objective was to assess the importance of these regions for coupling an electrochemical proton gradient to ATP synthesis. This information is generally lacking for animal ATP synthases, which are much more complex in their F₀ subunit composition than related enzymes from E. coli and chloroplasts. The third objective was to identify at least one F₀ subunit unique to the animal enzyme involved in shielding F₁, because such a subunit would represent a candidate for part of the stator or second stalk believed to stabilize the F₁ motor. To complete these objectives, it was essential to have at hand, in addition to our extensively studied rat liver F₁ preparation (25, 28, 37-39), an intact, highly purified preparation of the complete ATP synthase from the same source. This was accomplished by introducing several modifications (see "Methods") into the CHAPS-based procedure previously developed in this laboratory (6). The resultant preparation exhibits a specific activity of 14-15 µmol of ATP hydrolyzed × min¹ × mg¹ protein, contains all 16 subunits (Fig. 1B) previously reported for the bovine heart ATP synthase (20), and is markedly inhibited by oligomycin, DCCD, and venturicidin (Fig. 1C), agents known to bind to F₀ (46, 47).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   A, structure of rat liver F1 at 2.8 Å (28) depicting those regions examined in this study for possible shielding by subunits of F0. The ribbon representation of the F1 structure was constructed using the atomic coordinates of the rat liver enzyme (1mab (28)) using the program Quanta. The -helical, -sheet, and loop structures are colored in green, yellow, and purple, respectively. Nucleotides are shown in red. Regions examined in this study are located at the top and bottom of F1. These include the N-terminal regions of the  and  subunits (top) and the C-terminal regions of these same subunits (bottom), together with the central stalk ("stem") region (bottom). The central stalk region includes two sections, one that projects about 20 Å out of the F1 headpiece as revealed by x-ray crystallography (28) and shown in the figure, and another section (not shown) that is not yet resolvable by x-ray crystallography and is predicted to extend about another 30 Å (30). Regions of the  subunit that were resolved by crystallography include residues 4-48, 209-273, and 77-90 (28). The  subunit (not shown) is believed to be located also within the region of the central stalk, both on the basis of biochemical studies (25) and on the basis of the recent structure at 3.9-Å resolution of a yeast F1-(subunit c)10 complex (30). B, SDS-PAGE gels of F1 and F0F1 preparations used in this study. F1 and F0F1 were purified and subjected to SDS-PAGE as described under "Methods." The presence of all known F0F1 subunits (16) was confirmed by N-terminal sequence analyses ("Methods") and/or by Western analyses ("Methods") using specific antibodies. C, results of ATPase assays demonstrating that the F0F1 preparation used in this study is inhibited by agents known to act at the level of F0. ATPase assays were carried out in a 1-ml system as indicated under "Methods." In all cases, 14 µg of F0F1 was used to initiate the assay, and where shown the following inhibitors were added: oligomycin (2.5 µg), DCCD (5 µg), or venturicidin (5 µg).

Evidence That the N-terminal Regions of the  and  Subunits Located at the Top of F₁ Are Shielded by One or More F₀ Subunits in the Complete ATP Synthase-- Here, studies focused on the N-terminal regions of the three  and three  subunits as they are known from the atomic resolution structures of bovine heart and rat liver F₁ (Fig. 1A) to project from the top of the enzyme (26, 28). To determine whether one or more of the six N-terminal regions derived from these subunits are shielded by F₀, we compared the extent to which F₁, both alone and as part of the complete ATP synthase complex (F₀F₁), is shaved by endogenous proteases during purification. This necessitated carrying out N-terminal sequence analysis on both the  and  subunits of preparations of rat liver F₁ and F₀F₁ purified as described under "Methods." Therefore, following purification, these subunits were transferred from SDS-PAGE gels onto PVDF membranes by electroblotting, excised, and subjected to sequence analyses, also as described under "Methods." In these experiments, the N-terminal regions of  subunits derived from the F₀F₁ preparation could be sequenced only following prior treatment with the enzyme pyroglutamate aminopeptidase (48), which removes from the blocked N termini pyrrolidone carboxylic acid (Fig. 2A, inset), a cyclic form of glutamine.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   A and B, N-terminal sequences obtained for the  and  subunits within purified preparations of F0F1. The sequence of the  subunit was obtained following removal of the cyclic glutamine residue (A, inset) blocking the N terminus. Removal of the cyclic glutamine residue, preparation of samples for N-terminal sequence analyses, and the sequencing method used are described under "Methods." C and D, N-terminal sequences obtained for the  and  subunits within purified preparations of F1. See "Methods" for procedural details. E, summary of A-D. The summary diagram emphasizes that, when F1 is isolated in the absence of F0, endogenous proteases cleave 2 amino acids, Q and K from the N-terminal region of  subunits and a 6 amino acid peptide, AAQSSA, from the N-terminal region of  subunits.

The results of these experiments revealed that the N-terminal regions of all  and  subunits remain intact in F₀F₁ during its purification from mitochondria, because the amino acid sequences obtained (Fig. 2, A and B) correspond to those predicted for the mature sequences of these subunits. In contrast, the same subunits are shaved during the purification of F₁ (Fig. 2, C and D). Thus, two amino acids, Q and K, are shaved from  subunits, whereas the 6-amino acid peptide, AAQSSA, is shaved from  subunits (Fig. 2E). The degree of shaving remained the same in a number of F₁ preparations examined. In all cases the N-terminal sequence data obtained was homogeneous, thus ruling out the possibility of differences among the N-terminal regions of each of the three  subunits and each of the three  subunits. These results indicate that, when within the complete ATP synthase, all six N-terminal regions of  +  subunits located at the top of F₁ are shielded during purification from specific proteases endogenous to the mitochondria. Moreover, they strongly implicate one or more F₀ subunits as comprising this shield, which appears to "cap" the top of F₁ rather than being confined to a limited region. Consistent with these conclusions, in experiments not reported here, treatment of purified F₀F₁ with trypsin for 1.5 h was unable to cleave the N-terminal regions of either the F₁  or  subunits despite the presence of potential cleavage sites (R and/or K) in both cases.

Evidence That, of the Three Remaining Subunits (, , and ), Only the N-terminal Region of , Located at the Bottom of F₁, Is Shielded by One or More F₀ Subunits in the Complete ATP Synthase-- A comparison of amino acid sequencing data presented in Figs. 3A and 3B, and summarized in 3G, shows that the N-terminal region of the  subunit has not been shaved by proteases during isolation of F₁. Rather, its N-terminal sequence is identical to that found in purified F₀F₁, and to the known sequence for this subunit. This is an expected result, which serves as a control. Thus, it is known from the atomic resolution structures of bovine heart and rat liver F₁ preparations (26, 28) that the N-terminal region of the  subunit is tucked deep inside the central cavity of the F₁ headpiece where it is well shielded. In contrast, sequencing data for the  subunit presented in Figs. 3C and 3D, and summarized in 3G, show that in F₁ purified alone a 4-amino acid peptide, AQAA, is shaved from its N-terminal region. However, when the  subunit is purified as part of the complete F₀F₁ complex, the AQAA peptide is retained. Finally, a comparison of N-terminal sequencing data presented in Figs. 3E and 3F, and summarized also in Fig. 3G, show that the  subunit, just as the  subunit, has not been shaved during isolation, indicating that it may be shielded also within F₁. To date, little is known about the precise location and role of the  subunit in mitochondrial ATP synthases, although it has been reported to subfractionate with a complex (49) and to bind to the  subunit (50).

View larger version (56K): [in this window] [in a new window]   Fig. 3.   A and B, N-terminal sequences obtained for the  subunit within purified preparations of F0F1 (A) and F1 (B). Preparation of samples for N-terminal sequence analyses and the sequencing method used are described under "Methods." C and D, N-terminal sequences obtained for the  subunit within purified preparations of F0F1 (C) and F1 (D). Preparation of samples for N-terminal sequence analyses and the sequencing method used are described under "Methods." E and F, N-terminal sequences obtained for the  subunit within purified preparations of F0F1 (E) and F1 (F). Preparations of samples for N-terminal sequence analyses and the sequencing method used are described under "Methods." G, summary of A-F. The summary diagram emphasizes that the N-terminal regions of the  and  subunits are not altered by endogenous proteases during purification of either F0F1 or F1, whereas the 4-amino acid peptide AQAA is cleaved from the N-terminal region of the  subunit in F1 but not F0F1.

The studies described here, taken together with the above studies, indicate that, within the complete liver mitochondrial ATP synthase, regions of F₀ located at both the top and bottom of F₁ shield the N-terminal regions of three of its subunit types (, , and ), whereas the remaining two subunit types ( and ) are shielded or tightly folded within F₁.

Limited Treatment of Isolated F₁ with Trypsin Results in Further Shaving of Its  and  Subunits While Leaving the Smaller Subunits , , and  Unaltered-- Results presented in Fig. 4 (A and B) show that treatment of isolated F₁ with trypsin for as long as 90 min has no effect on the staining intensities or mobilities of the , , and  subunits of F₁ in SDS-PAGE gels but does noticeably affect the mobility of the  subunit. Two different gel systems were used in these experiments. One contained 7.5% acrylamide (Fig. 4A), which separates best the , , and  subunits but not the  and  subunits, which migrate with the dye front. The second contained 15% acrylamide (Fig. 4B), which separates the , , and  subunits better than the  and  subunits.

View larger version (57K): [in this window] [in a new window]   Fig. 4.   A and B, SDS-PAGE pattern of F1 following limited treatment with trypsin. F1 was previously treated with trypsin exactly as described under "Methods" for the times indicated at the top of the gel and then subjected to SDS-PAGE in gels containing 7.5% (A) or 15% (B) polyacrylamide. The 7.5% gels resolved best the , , and  subunits of F1 but not the smaller subunits  and , which migrated with the dye front. In contrast the 15% gels resolved the subunits , , and  better than the  and  subunits. C and D, N-terminal sequences obtained for the  subunits (C) and the  subunits (D) following treatment of F1 with trypsin for l h. The summary diagram at the bottom of C shows that trypsin shaves 13 amino acids more off the N-terminal region of the  subunit than the 2 amino acids (Q and K) shaved off by endogenous proteases during purification (Fig. 2E). The summary diagram at the bottom of D shows that trypsin shaves only 3 amino acids more off the N-terminal region of the  subunit than the 6 (AAQSSA) shaved off by endogenous proteases during purification (Fig. 2E).

When all five F₁ subunits from the SDS-PAGE gels were subjected to N-terminal sequence analyses, it was found that a 13-amino acid peptide (TGTAEMSSILEER) had been cleaved from the N-terminal region of the  subunit (Fig. 4C) and a 3-amino acid peptide (APK) from the N-terminal region of the  subunit (Fig. 4D). In data not presented here, the N-terminal regions of the , , and  subunits were found to be unaltered. Because the decrease in mass of the  subunit appeared by inspection of SDS-PAGE gels (Fig. 4, A and B) to be greater than the mass of the 13-amino acid peptide cleaved from its N terminus, this suggested that trypsin may have cleaved also at the C terminus. For this reason we subjected both isolated F₁ and trypsin-treated F₁ to MALDI-TOF-DE mass spectral analysis as described under "Methods." Consistent with SDS-PAGE and N-terminal sequence analyses, these studies confirmed that the mass of the , , and  subunits were unaltered and showed that the mass of each  subunit had decreased by only 341 Da (Fig. 5A), as expected from the loss of the APK peptide. In contrast, however, the mass of each  subunit decreased by 4425 Da, of which only 1406 Da could be accounted for by the 13-amino acid peptide cleaved from the N-terminal region (Fig. 4C). Significantly, the remaining decrease in mass, which must come from the C-terminal region, corresponds most closely to a 26-amino acid -helix resulting from trypsin cleavage between amino acid Arg-484 and Ser-485 (Fig. 5B).

View larger version (34K): [in this window] [in a new window]   Fig. 5.   A, summary of molecular masses obtained for F1 subunits before and after limited treatment with trypsin. F1 was subjected to limited treatment with trypsin for 1 h exactly as described under "Methods" and then analyzed in a MALDI-TOF-DE mass spectrometer, also as described under "Methods." The masses obtained from two different experiments varied less than 1%. B, predicted trypsin cleavage site within the C-terminal region of the  subunit. Taking into consideration the large total mass loss of the  subunit, and the known loss of mass from its N-terminal region, the mass loss arising from its C-terminal region was calculated and found to correspond precisely to loss of the 26-amino acid peptide shown (see text for discussion). C, lack of effect of trypsin on the mobility of the  and  subunits within purified F0F1. F0F1 was treated for up to 1.5 h with trypsin as described under "Methods" and then subjected to SDS-PAGE, also as described under "Methods."

These results, while demonstrating that limited trypsin treatment of isolated F₁ causes further shaving of the exterior N-terminal regions of both the  and  subunits located at the top of the molecule, and the exterior C-terminal helix of the  subunit located at its bottom, also show that all three of the smaller subunits (, , and ) are resistant to limited trypsin treatment. Significantly, in other studies it was shown that trypsin treatment of the complete ATP synthase (F₀F₁), in contrast to treatment of F₁, had no detectable effect on the electrophoretic mobility of  subunits in SDS-PAGE (Fig. 5C), indicating that F₀ shields the C-terminal regions of all three of these subunits near the bottom of the F₁ headpiece.

Trypsin-treated F₁, Although Active as an ATPase and Capable of Rebinding to F₀ within F₁-depleted Inner Mitochondrial Membranes, Is Incapable of Coupling a Proton Gradient to ATP Synthesis-- Results presented in Fig. 6A show that those conditions under which trypsin further shaves the N-terminal regions of both  and  subunits at the top of rat liver F₁, and the C-terminal region of the  subunits at its bottom (Figs. 4 and 5), are without effect on the capacity of the enzyme to catalyze ATP hydrolysis. Moreover, similar to isolated F₁, trypsin-treated F₁ is able to restore to F₁-depleted inner membrane vesicles (IMVs) the capacity to catalyze high rates of ATP hydrolysis inhibited by oligomycin (Fig. 6B, left) and DCCD (not shown). This shows that trypsin-treated F₁ is capable of binding appropriately to the basepiece of F₀ where the sites of action of oligomycin (subunits c and a) and DCCD (subunit c) are located (46, 47). However, unlike isolated F₁, which is able to restore the capacity of F₁-depleted, actively respiring IMVs to catalyze ATP synthesis inhibited by oligomycin, DCCD (not shown), and the protonophore 2,4-dinitrophenol, trypsin-treated F₁ is not effective in this capacity (Fig. 6B, right). (IMVs of rat liver mitochondria, prior to treatment with urea to deplete F₁ and provide F₁-depleted IMV, exhibit specific activities for ATP hydrolysis in the range of 2000-3000 nmol of ATP hydrolyzed × min¹ × mg¹ protein and specific activities for ATP synthesis in the range of 100-250 nmol × min¹ × mg¹ protein.)

View larger version (35K): [in this window] [in a new window]   Fig. 6.   A, effect of trypsin on the ATPase activity of rat liver F1 as a function of time. F1 was treated with trypsin exactly as described under "Methods" for the times indicated and then assayed for ATPase activity by the spectrophotometric method, also described under "Methods." B, assessment of the capacity of trypsin-treated F1 to bind to F1-depleted inner mitochondrial membrane vesicles and restore ATPase and ATP synthetic activities. Preparation of F1-depleted inner membrane vesicles by treating inner membrane vesicles with 3.2 M urea, and reconstitution of F1 (300 µg) or trypsin-treated F1 (300 µg) with the depleted vesicles (0.5 mg), was carried out exactly as described previously (39). ATPase and ATP synthetic activities were assayed as described under "Methods." Where indicated, oligomycin (2.5 µg/ml), DCCD (5 µg/ml), or 2,4-dinitrophenol (100 µM) was added. Standard deviations are based on three different experiments. (In experiments not presented here, DCCD (5 µg/ml) inhibited by >90% the rates of ATP hydrolysis and ATP synthesis in all cases.)

These results indicate that one or more of the exterior, trypsin-accessible regions on F₁ defined above are critical for coupling a proton gradient to ATP synthesis in the inner mitochondrial membrane, although they are of little or no importance to the enzyme for catalyzing ATP hydrolysis or for binding to F₀.

Evidence for an Involvement of the Unique F₀ Subunit Called F₆ in Shielding F₁-- Having acquired information that regions on rat liver F₁, at its top and bottom, are shielded by F₀, and that one or both of these locations on F₁ may be necessary for coupling a proton gradient to ATP synthesis, our attention turned to the involvement of F₀. Here the major goal was to identify one or more F₀ subunits unique to the animal system involved in shielding F₁ within the complete F₀F₁ ATP synthase. The F₀ subunits named "F₆," "OSCP," and "d" were considered to be likely candidates, because these subunits are not integral membrane proteins (18, 51, 52). The strategy employed took advantage of our knowledge that the N-terminal regions of each  subunit shielded by F₀ at the top of F₁ has its N terminus blocked by pyrrolidone carboxylic acid. Therefore, it was rationalized that, by monitoring the ability of a deblocking enzyme to gain entrance to the N terminus of the blocked  subunit in F₀F_l after the addition of a mild dissociating agent, while monitoring the release of any F₀ subunits, we might be able to identify one or more F₀ subunits involved in shielding F₁.

Based on the above strategy and considerable preliminary work, it was found that the deblocking enzyme (pyroglutamate aminopeptidase) gains access to the N terminus of the  subunit after treatment of F₀F₁ with 0.6% CHAPS for 14 h (12 h at 4 °C and then 2 h at 25 °C). Thus, the N-terminal region of the  subunit following SDS-PAGE and electroblotting onto a PVDF membrane could now be sequenced (Fig. 7A), verifying that deblocking had occurred. Significantly, 1 M urea, when included under the above conditions, actually stabilized F₀F₁ and prevented the deblocking enzyme from gaining access to the N terminus of the  subunit (Fig. 7B). When both the supernatant and pellet following treatment of F₀F₁ with CHAPS, but without urea, was analyzed by immunoblotting with an antibody specific for F₆, all of this subunit was recovered in the supernatant (Fig. 7C, Condition 1). In contrast, when 1 M urea was present, F₆ was not detected in the supernatant (Fig. 7C, Condition 2) but remained with the pellet. Experiments conducted in an identical manner but using antibodies specific for OSCP, showed that this subunit is also released into the supernatant (Fig. 7D, Condition 1). However, in this case 1 M urea fails to prevent its release (Fig. 7D, Condition 2). These results indicate that F₆, but not OSCP, plays a primary role in making the top of F₁ inaccessible to the  subunit-deblocking enzyme. Results obtained with an antibody to subunit d not presented here, although less clear, mimicked most closely those obtained with F₆, indicating that subunit d may also shield in part the top of F₁.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   A, N-terminal sequence of the F1  subunit obtained after prior treatment of F0F1 with the detergent CHAPS and then with the deblocking enzyme pyroglutamate amino peptidase. F0F1 (200 µg) was incubated for 12 h at 4 °C and then for 2 h at 25 °C in a 0.2-ml system containing 100 mM sodium Pi, pH 8.0, 10 mM EDTA, 5 mM DTT, 5% glycerol, and 0.6% CHAPS. Following SDS-PAGE, the  subunit band was subjected to N-terminal sequence analysis exactly as described under "Methods." B, absence of N-terminal sequence data for the  subunit upon treating F0F1 as in A but in the presence of urea. All conditions were exactly as in A with the exception that 1 M urea was present. In control studies, urea was shown to be without effect on the activity of the deblocking enzyme. C, Western blots obtained with an antibody to the F0 subunit "F6" following SDS-PAGE of detergent extracts of F0F1 treated as in A or as in B. F0F1 samples were treated exactly as in A with or without urea and then centrifuged at 14,000 rpm for 15 min in a microcentrifuge at 25 °C. The supernatant was removed and boiled for 20 min and then centrifuged at 14,000 rpm for 30 min at 25 °C. The final supernatant was then removed and subjected to SDS-PAGE/Western blot analysis exactly as described under "Methods" using a polyclonal antibody to F6. D, experiments were carried out exactly as described in C using a polyclonal antibody to OSCP rather than F6.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The novel set of studies reported here were undertaken to gain greater insight into the predicted motor-related features of ATP synthases from animal cells. The objectives were 3-fold and focused on (a) identifying exterior regions at the top and bottom of the F₁ motor that are shielded by F₀; (b) determining the importance of these regions for coupling an electrochemical proton gradient via the motor in F₀ to the synthesis of ATP on F₁; and (c) identifying one or more F₀ subunits unique to animal ATP synthases involved in shielding F₁. Each of these objectives was achieved either fully or in part.

Clearly, as revealed by N-terminal sequence analyses (Figs. 1 and 2), exterior regions at both the top and bottom of rat liver F₁ within the complete ATP synthase are shielded from limited proteolysis arising from endogenous proteases during purification. Shielded regions include the N-terminal regions of all  and  subunits, which are known from the atomic resolution structure of rat liver F₁ to reside at its top (28), and the N-terminal region of the single  subunit, which is inferred from biochemical studies to reside at its bottom (25). Treatment of the complete ATP synthase (F₀F₁) with trypsin is also without effect on the N-terminal regions of these same subunits, providing additional support that they are shielded by F₀. These finding, although identifying potential F₀/F₁ contact regions in the complete rat liver ATP synthase, also implicate F₁ as being "capped" at its top by one or more subunits derived from F₀ and shielded at its bottom.

Significantly, the exterior N-terminal regions of F₁, which are shielded from limited proteolysis during isolation of F₀F₁, i.e. the QK dipeptide of the  subunit (Fig. 2E), the AAQSSA hexapeptide of the  subunit (Fig. 2E), and the AQAA tetrapeptide of the  subunit (Fig. 3G), are not required for F₁ to restore ATP synthesis to F₁-depleted inner membrane vesicles (Fig. 6B). However, if the endogenously shaved F₁ preparation is shaved further at both its top and bottom by exogenously added trypsin, the enzyme is unable to catalyze ATP synthesis when reconstituted with F₁-depleted inner membrane vesicles (Fig. 6B). This loss of physiological function occurs despite the fact that the more closely shaved F₁ fully retains both its catalytic ATPase activity (Fig. 6A) and its capacity to bind in a normal manner to F₁-depleted inner membrane vesicles (Fig. 6B). Specifically, trypsin removes from the  subunit's N-terminal region an additional 13 amino acids, TGTAEMSSILEER (Fig. 4C), and from its C-terminal region a 26-amino acid -helix, SDGKISEQSDAKLKEIVTNFLAGFEP (Fig. 5B), while removing also the tripeptide APK from the N-terminal region of the  subunit (Fig. 4D). Taken together, these findings summarized in Fig. 8A indicate that one or more of these trypsin-cleaved exterior regions, which are also shielded by F₀ when within the complete ATP synthase (Figs. 2, 3, and 5), are critical for the capacity of F₁ to couple an electrochemical proton gradient to the synthesis of ATP.

View larger version (56K): [in this window] [in a new window]   Fig. 8.   A, summary of exterior regions of F1 subunits shielded by F0 that are nonessential for mitochondrial ATP synthesis, and those of which one or more is essential. Blue italicized letters designate those exterior amino acid residues that are not essential, while red letters inside dotted boxes designate those of which one or more is essential. B, model of rat liver mitochondrial ATP synthase incorporating conclusions derived from the novel findings reported here. Experimental data reported here are consistent with a model for animal ATP synthases in which (a) one or more F0 subunits completely shields the top of F1 forming a cap; (b) one or more F0 subunits also shields the bottom of the F1 headpiece, perhaps forming a collar; and (c) components of F1, most likely  and , shield the  subunit within the region of the central stalk at the bottom of F1. The data reported are consistent also with a role for the unique F0 subunit F6 in helping shield F1, most likely at its top, although another location cannot be completely ruled out.

Additional results presented here provide new information about the relationship of F₀ in the complete ATP synthase to the central stalk region of F₁, i.e. the region that extends from the bottom of the F₁ headpiece to the basepiece of F₀, a distance of about 50 Å (30). From SDS-PAGE, N-terminal sequencing, and mass spectral studies following treatment with trypsin (Figs. 4 and 5), it is clear that  subunit is already well protected in the central stalk region within F₁. This shield is likely provided, at least in part, by the  subunit of F₁, a view that is supported by previous work in this laboratory on rat liver F₁ (25) and from the low resolution structure of yeast F₁ in complex with a decamer of the subunit c of F₀ (30). The  subunit of F₁, which binds to the  subunit (50), may also comprise, in part, the shield that protects the  subunit within F₁. Because studies reported here implicate F₀ as shielding the N-terminal region of the  subunit (Fig. 3, C and D), it would appear that in the intact ATP synthase complex, one or more components of F₀ may reside sufficiently near the central stalk region to form contacts, but that these contacts may not be directly in contact with the  subunit. In other words, the  subunit in the central stalk region may be doubly shielded, with the first layer involving the  and  subunits of F₁, and the second shield one or more subunits of F₀.

Finally, studies reported here show that those conditions that make the N-terminally blocked residue (Q*) of the  subunit at the top of F₁ accessible in F₀F₁ to a deblocking enzyme also result in the release of the F₀ subunit called F₆ (Fig. 7). Moreover, conditions that prevent the N-terminally blocked residue from becoming accessible to the deblocking enzyme also prevent the release of F₆ (Fig. 7). These findings are consistent with a location, or partial location, of F₆ at the top of F₁ in the complete ATP synthase complex of rat liver, although they do not exclude its interaction or partial interaction at other locations. Significantly, F₆, a small 8.93-kDa protein, required for ATP synthesis in inner mitochondrial membrane vesicles (52), is not found among the F₀ subunits in the simpler ATP synthase preparations derived from E. coli and chloroplasts (1-3). Therefore, the novel finding reported here about F₆ is of special interest, and in future studies may help reveal why the F₀ unit of animal ATP synthases are much more complex than the F₀ units of the E. coli and chloroplast enzymes.

When conclusions from all the studies described here are considered, we envision an overall structural model for the rat liver ATP synthase in which one or more components of F₀ extend from its basepiece to the top of the headpiece of F₁ (Fig. 8B). At the bottom of F₁, components of F₀ are considered to associate closely or "hug" the central stalk region forming a "collar" that shields the C-terminal regions of the three  subunits. At the top of F₁, the extension of this "second stalk" is considered to cover completely the N-terminal regions of all  and  subunits, thus providing a "cap." In addition, the cap may be composed, at least in part, by the F₀ subunit F₆, unique to animal ATP synthases. This model, derived from using a variety of approaches and methodologies, is consistent with current views about ATP synthase structure, which envision the presence of a stator connecting two motors, one within the F₁ headpiece and another within the F₀ basepiece (31-33, 35). In addition, this model and the experimental work on which it is based provide biochemical support for the recent electron microscopic study of bovine heart ATP synthase, showing a collar around the central stalk and a mass at the top of the F₁ (11), and support for a similar study conducted on the E. coli ATP synthase in which a mass was also observed on the top of F₁ (12). Taken together, these studies suggest that earlier models (53-55) depicting the stator or second stalk as interacting with only one pair and failing to extend from the basepiece of F₀ to the top of F₁ may require revision.

Significantly, the extension of the stator to the top of F₁ may have important functional consequences, because not one but all three pairs containing the catalytic sites must be stabilized during ATP synthesis. Moreover, that part of the stator that resides at the top may be important also in facilitating formation of those critical contacts recently suggested to be important in the assembly of the -barrel domains (56).

	ACKNOWLEDGEMENTS

We are grateful to Jodie Franklin for help in obtaining N-terminal sequence and mass spectral data on F₁, and to Drs. Youssef Hatefi and Akemi Matsuno-Yagi for supplying antibodies to F₀ subunits.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA 10951 (to P. L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession numbers: P15999 (), P10719 (), P35435 (), P35434 (), and P29418 ().

To whom correspondence should be addressed: Dept. of Biological Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-955-3827; Fax: 410-614-1944; E-mail: ppederse@welchlink.welch.jhu.edu.

Published, JBC Papers in Press, July 7, 2000, DOI 10.1074/jbc.M004453200

	ABBREVIATIONS

The abbreviations used are: DTT, dithiothreitol; DCCD, N, N'-dicyclohexylcarbodiimide; CHAPS, [3-cholamidopropyl]dimethylammonio-1-propanesulfonate; CAPS, 3-(cyclohexylamino)propanesulfonic acid; TRICINE, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; MALDI-TOF MS, matrix-assisted laser desorption time of flight mass spectrometry; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride; IMV, inner membrane vesicle(s); OSCP, oligomycin sensitivity conferring protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES