The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification*

Andrew N. Stephens, Muhammad A. Khan, Xavier RoucouDagger, Phillip Nagley, and Rodney J. Devenish§

From the Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Victoria 3800, Australia

Received for publication, January 29, 2003, and in revised form, February 25, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F1-F0 interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F1F0-ATP synthase.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mitochondrial F1F0-ATP synthase (mtATPase)1 consists of a membrane-extrinsic sector (F1) linked to a membrane-embedded proton channel (F0) by two protein stalks (1). Synthesis/hydrolysis of ATP occurs on the structurally well characterized F1 sector comprised of five subunits alpha 3-beta 3-gamma 1-delta 1-epsilon 1 (2-4) and is coupled to proton flux through the F0 sector. The crystal structure of an incomplete mtATPase from yeast determined by Stock et al. (3) showed that the gamma -delta -epsilon subunits form a central stalk that is closely associated with a ring of 10 copies of membrane-embedded subunit 9. Together these proteins constitute the "rotor" of mtATPase, which has been conclusively demonstrated to rotate during ATP synthesis/hydrolysis relative to the other subunits of the complex (5-13).

By contrast to that of F1 no high-resolution structure of the F0 sector is available. In yeast mitochondria F0 is comprised of at least 12 different polypeptides: OSCP, b, d, e, f, g, h, i/j, and k that are nuclearly encoded, and subunits 6, 8, and 9 that are mitochondrially encoded (1). F0 has two important functions. First, some F0 subunits form a "stator" stalk anchored in the membrane, which during coupled ATP synthesis/hydrolysis prevents futile rotation of mtATPase subunits relative to the rotor. This structure has been visualized in ATP synthases from several organisms (14-16). In the structurally less complex ATP synthase of Escherichia coli the stator stalk is composed of subunit delta  and two copies of subunit b (17). However, only a single copy of subunit b is present in the mtATPase of the yeast Saccharomyces cerevisiae (18); thus, in conjunction with OSCP (the mitochondrial homolog of subunit delta ), other F0 subunits contribute to form the stator stalk. Second, movement of protons through the membrane-embedded channel of F0 allows conversion of the proton gradient generated by respiratory chain activity into chemical energy, in the form of ATP made by phosphorylation of ADP on F1. Based on molecular genetic and biochemical studies in yeast the assembly and stability of the membrane-embedded proton channel of mtATPase depends on the presence of the three mitochondrially encoded protein subunits 6, 8, and 9 (19). Subunits 6 and 9 are the homologs of bacterial ATP synthase subunits a and c, respectively, and have well defined roles in proton channel function (20). Subunit 8 is not found in bacteria but is an additional subunit present in the mtATPase of mammals and fungi. The absence of all but one of these F0 subunits from the yeast F1-c10 crystal structure (3) highlights the need for additional biochemical approaches aimed at elucidating the structure and function of F0 subunits.

In yeast subunit 8 (Y8) is a 48-amino acid intrinsic membrane protein essential for the assembly and function of mtATPase (1). Whereas divergent in amino acid composition, Y8 and its other eukaryotic homologs exhibit three highly conserved regions: an N-terminal MPQL motif, a central hydrophobic domain (CHD), and a C-terminal positively charged region (21-23). Allotopic expression (24), whereby a mitochondrial gene is recoded for nuclear expression with subsequent delivery of the protein back to mitochondria, has enabled our laboratory to undertake a detailed molecular genetic approach to investigate the structure and function of Y8. Detailed studies on genetically modified Y8 variants, allotopically expressed in yeast lacking endogenous Y8, have suggested that the N-terminal motif of Y8 plays a functional role in mtATPase (25-27) while the positively charged amino acid region at the C terminus of Y8 is involved in both the assembly and function of the F0 sector (27-29). A surprising feature of Y8 is the functional accommodation of charged amino acid residues within the CHD (30-33). Analysis by Roucou et al. (33) has shown that variants bearing either positive or negatively charged amino acids in the CHD display structural destabilization of the F1-F0 interaction but nevertheless retain function.

Cysteine scanning mutagenesis has been used extensively to analyze membrane proteins in their native state and provides a powerful alternative for examining the molecular neighborhood surrounding membrane proteins (34-36). In a previous study, analysis of allotopically expressed Y8 variants having a cysteine substitution at the N- or C-terminal residue of Y8 demonstrated that the CHD spans the inner mitochondrial membrane with Nout-Cin orientation (37). Here we have combined cysteine-scanning mutagenesis with allotopic expression to construct a series of single cysteine replacements encompassing the entire length of the Y8 protein (excluding three residues within the C-terminal positively charged region whose replacement would abrogate function (28)). Without exception each cysteine replacement was tolerated and all of the expressed Y8 variants restored mtATPase function in cells lacking endogenous Y8, demonstrating that none of the substituted amino acid residues of Y8 are essential for either ATP synthesis/hydrolysis activity or proton flow. Specific labeling experiments using the thiol-modifying chemical reagent fluorescein 5-maleimide (FM) showed that the membrane-spanning CHD of Y8 encompasses residues 15 to 35. By assessing the accessibility of introduced cysteine residues to FM in partially dissociated mtATPase complexes, the C terminus of Y8 was found to be occluded by other mtATPase subunits, especially in a small region close to Val-40 demonstrated to play an important role in maintaining the structural interaction between the F1 and F0 sectors. In addition, site-directed cross-linking experiments demonstrated extensive interactions between Y8 and subunits d, f, b, and 6. These results lead us to propose a significant role for Y8 as part of the stator stalk of mtATPase.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Y8 Cysteine Replacements-- The gene cassettes N9L-D/Y8-1 and N9L-D/Y8-1-FLAG, yeast expression vector pPD72, and yeast strains YM2 and FTC2 were as described previously (37). Briefly, each gene cassette encodes a full-length wild-type Y8 protein bearing a 7-amino acid extension at the N terminus (YSSEISS, numbered from -7 to -1), which is retained following import of Y8 into mitochondria and processing of the N9L mitochondrial import sequence by matrix protease. The N9L-D/Y8-1-FLAG construct expresses the same protein bearing a C-terminal FLAG epitope (DYKDDDDK). Cells expressing the N9L-D/Y8-1 cassette are denoted YM2, those expressing N9L-D/Y8-1-FLAG are denoted FTC2.

Site-directed mutagenesis was carried out on the N9L-D/Y8-1 gene cassette as described (37) to generate 46 single cysteine replacements in the expressed Y8 proteins. The Y8(M1C) FLAG variant was constructed by ligation of a BamHI/KpnI fragment containing the N9L-D sequence and the first 15 nucleotides of the synthetic Y8 gene bearing the M1C mutation into a similarly digested pUC9 vector containing the FTC2 variant. The (M1C) FLAG cassette was then excised by BamHI/NotI restriction digestion and ligated into the pPD72 vector as described (37).

A DNA sequence encoding an HA epitope (YPYDVPDYA) was introduced into the N9L-D/Y8-1 cassette using two rounds of PCR mutagenesis. The expressed protein is designated NHAY8. First, the upstream and downstream flanking primer pair, 5'-CAGGAAACAGCTATGACC-3' and 5'-GGAACGTCGTATGGGTAAGACGAGATCTCGGAAGAGTAGGC-3', respectively, were used to introduce nucleotides into the N9L-D/Y8-1 cassette specifying the amino acid sequence YPYDV inserted between the YSSEISS sequence that was retained following import of allotopically expressed Y8 into mitochondria and the N terminus of the Y8 peptide. A second round of PCR using the flanking primer pair 5'-CAGGAAACAGCTATGACC-3' and 5'-GGCTCGAGGAGGCGTAGTCAGGAACGTCGTATGGGTAAGACGAGATC-3' was then used to incorporate adjacent nucleotides into the N9L-D/Y8-1 cassette specifying amino acids PDYA, to form the complete HA epitope, along with three additional serine residues between this epitope and the Y8 passenger protein. Thus, the N9L-D/NHAY8-1 cassette encodes a protein that contains the N9L-D-YSSEISS sequence (as for YM2) followed by the sequence YPYDVPDYA-SSS-subunit 8, with the original matrix protease cleavage site maintained. A BamHI/XhoI restriction fragment bearing the nucleotide sequence encoding N9L-D/NHA was ligated into similarly digested pUC9 containing the gene cassette encoding selected N9L-D/Y8-1 cysteine variants to create constructs NHAY8(L4C), NHAY8(F7C), NHAY8(M10C), NHAY8(F44C), and NHAY8(L48C). The fidelity of all constructs was checked by DNA sequencing.

Expression of Cysteine Variants in Yeast and Tests for Restoration of Respiratory Function-- Yeast strain M31 (MATalpha ade1 his6)[aap1-] lacking endogenous Y8 has been described previously (30). M31 cells were transformed by the method of Klebe et al. (38) with the pPD72 expression vector that carries a functional ADE1 gene (28). Transformants displaying the Ade+ phenotype were selected and restoration of respiratory-competent phenotype by the expressed Y8 cysteine variants was assessed by testing growth on medium containing ethanol as the sole carbon source. Generation times, ATP hydrolysis activity in isolated mitochondria, and its sensitivity to oligomycin were measured as described previously (39). Comparisons (Student's t test) were made against strain YM2 (28), allotopically expressing unmodified Y8.

Thiol-specific Labeling of Mitochondrial Proteins and Analysis of Protein Fluorescence-- Thiol-specific labeling of mitochondrial proteins by FM was carried out for 4 h on mitochondria osmotically lysed by exposure to a hypotonic solution (denoted lysate) as described (37). Isolated proteolipids were separated by Tricine SDS-PAGE (18% polyacrylamide) according to the method of Schagger and von Jagow (40). Analysis of protein fluorescence was as described (37). For Y8 cysteine variants that remained unmodified under these conditions, labeling by FM was repeated as above in the presence of 1% SDS prior to proteolipid extraction.

Cross-linking of Mitochondrial Proteins-- Cross-linking of mitochondrial proteins using the heterobifunctional reagents p-azidophenacyl bromide (APA-Br) and N-(4-p-azidosalicylamidobutyl)-3'-(pyridyldithio)-propionamide (APDP) was as described, except that mitochondrial membranes equivalent to 0.5 mg of protein were used (37). Cross-linking using CuCl2 was performed as follows. Mitochondrial membranes (equivalent to 0.5 mg of protein) were pelleted at 131,440 × g for 15 min at 4 °C and resuspended in 200 µl of buffer C (50 mM Tris-HCl, 2 mM MgCl2, pH 7.5). CuCl2 was added to 1.5 mM (from a 25 mM stock solution, prepared fresh) and the samples were incubated on ice for 2 h. EDTA was then added to 10 mM to chelate the remaining Cu2+ and the samples were incubated for 5 min. Mitochondrial membranes were then pelleted at 131,440 × g for 15 min at 4 °C and the pellet resuspended in 125 µl of 4% SDS (w/v) and 125 µl of dissociation buffer (125 mM Tris-HCl, 2% SDS (w/v), 50% glycerol (v/v), 0.02% bromphenol blue (w/v), pH 6.7). To reduce samples 1 µl of 14.3 mM 2-mercaptoethanol was added and the samples were incubated on ice for 30 min, then at 65 °C for 10 min prior to analysis by SDS-PAGE and immunoblotting.

Removal of the F1 Sector from Mitochondrial Membranes-- Removal of the F1 sector from mitochondrial membranes was achieved using the modified method of McEnery et al. (41). Mitochondrial membranes (equivalent to 2 mg of protein) were pelleted at 131,440 × g for 15 min at 4 °C, and then resuspended in 0.8 ml of PAB buffer (0.15 M K2HPO4, 25 mM EDTA, 100 mM 2-mercaptoethanol, pH 7.9). After 5 min incubation on ice, the mitochondrial membranes were centrifuged as before and the pellet resuspended in 1.0 ml of GPAB buffer (0.15 M K2HPO4, 25 mM EDTA, 100 mM 2-mercaptoethanol, 3.0 M guanidine-HCl, pH 7.9). Following incubation on ice for 13 min, membranes were pelleted as before and washed twice in PA buffer (150 mM K2HPO4, 25 mM EDTA, pH 7.9) by resuspension and centrifugation. For fluorescent labeling experiments membrane samples were then resuspended in 0.2 ml of buffer A and labeling performed as described above. Samples not intended for labeling studies were prepared for denaturing glycine SDS-PAGE as described (37).

Isolation of Monomeric MtATPase and in Situ ATP Hydrolysis-- Monomeric mtATPase complexes were isolated using clear native gel electrophoresis according to the method of Arnold et al. (42), but with the omission of Serva Blue G dye from the sample. Mitochondrial membranes (equivalent to 100 µg of protein) were pelleted at 131,440 × g for 15 min at 4 °C and resuspended in 20 µl of native gel sample buffer (50 mM NaCl, 2 mM aminohexanoic acid, 1 mM EDTA, 50 mM imidazole, 5 mM phenylmethylsulfonyl fluoride). Lauryl maltoside was added to a final concentration of 2.86% (w/v) and the samples (total sample volume 26 µl) were incubated on ice for 20 min. Insoluble material was pelleted at 131,440 × g for 15 min at 4 °C, and 20 µl of the sample was applied to a 4-13% polyacrylamide gradient gel (dimensions 13 × 10 × 0.075 cm). Electrophoresis was carried out for 3 h with an initial current of 15 mA and voltage increasing to a maximum of 500 V. On completion of electrophoresis, gels were transferred to small plastic containers and in situ ATP hydrolase activity was assessed using the method of Yoshida et al. (43). Gels were photographed, then fixed in a solution of methanol (50%) (v/v), acetic acid (10%) (w/v) for 30 min, and then stained in acetic acid (10%) (v/v), Serva Blue G dye (0.025%) (w/v) overnight. Gels were destained in acetic acid (10%) (v/v) for at least 2 h with several changes of solution until protein bands were distinctly blue against a clear background. Coomassie-stained gels were analyzed using a ProXPRESS multiwavelength fluorimager (PerkinElmer Life Sciences) and Phoretix 2D analysis software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom).

Second Dimension Protein Analysis by SDS-PAGE-- Denaturing second dimension gel electrophoresis analysis of isolated mtATPase complexes was performed as follows. Monomeric mtATPase complexes isolated using native gel electrophoresis and stained with Serva Blue G dye were carefully excised from the gel and each polyacrylamide fragment rinsed in 1 ml of distilled water in a 1.5-ml snap-cap tube. After a 60-s centrifugation at 20,798 × g, the water was aspirated off and 50 µl of SDS (5%) (w/v) was added to the tube. The gel fragment was then finely ground using a thin glass rod and centrifuged at 20,798 × g for 1 min. Grinding and centrifugation were repeated and then 50 µl of 2× dissociation buffer (125 mM Tris-HCl, 2% SDS (w/v), 50% glycerol (v/v), 0.02% bromphenol blue (w/v), 2% 2-mercaptoethanol (v/v), pH 6.7) was added to the SDS-gel slurry and the samples were heated to 65 °C for 5 min. Following incubation at 4 °C overnight, samples were stored at -20 °C until use. Prior to electrophoresis, the samples were heated to 65 °C for 5 min and then centrifuged at 20,798 × g for 5 min. Immediately following centrifugation 20 µl of the sample was removed, taking care to avoid any fragments of acrylamide gel present in the tube. Samples were electrophoresed (glycine SDS-PAGE) in a 15% polyacrylamide denaturing gel and transferred to PVDF membrane for immunoblotting.

Immunoblotting-- Membranes prepared as above were probed with mouse monoclonal antibodies against either the FLAG (diluted 1:380) or HA epitopes (diluted 1:2000) (Sigma), or yeast mtATPase subunits alpha  (diluted 1:5000), beta  (diluted 1:7000), or b (diluted 1:5000) from our library of mtATPase-specific antibodies (44). Rabbit polyclonal antisera were also employed against yeast mtATPase subunits gamma , d, or OSCP (diluted 1:1000) (44), f, 6, or h (diluted 1:10000, kindly provided by Dr. J. Velours), and e, g, i/j, or k (diluted 1:500, kindly provided by Dr. R. A. Stuart). Secondary antibodies and detection were as described (37). Antibody binding was quantified using ImageQuant software (Amersham Biosciences).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Single Cysteine Mutants-- A total of 46 single cysteine replacements were made in the Y8 protein at positions ranging from the amino acid directly preceding the N-terminal methionine of allotopically expressed Y8 (position -1) to the most C-terminal residue (position 48). The lysine at position 37 and two arginines at positions 42 and 47 were not substituted as each of these residues contributes to the C-terminal positively charged region required for assembly of Y8 into mtATPase (28). To test the functionality in vivo of Y8 cysteine variants, M31(aap1-) cells expressing each single cysteine variant were plated onto medium containing ethanol as the sole carbon source. Without exception, expression of each variant was able to restore respiratory function. However, cells expressing either the Y39C or V40C Y8 variants displayed apparently slower growth on solid medium than did control strain YM2 at 37 °C, yet displayed normal growth at either 18 or 28 °C. To further assess functionality of the Y8 variants at the whole cell level, the generation time of each Y8 variant-expressing strain was assessed in liquid medium containing ethanol as the sole carbon source. Of the 46 cysteine variants tested, only three had a significant effect (Student's t test) on whole cell growth rate. Expression of the L38C variant resulted in a significant decrease (p < 0.01) in generation time compared with strain YM2 expressing unmodified Y8 (Table I). The reason for this increased rate of growth is unclear and has not been examined further. Because yeast should have an excess capacity for ATP production, increased ATP synthesis should not promote growth. By contrast, expression of Y8 variants Y39C or V40C, respectively, displayed a significant increase in generation time compared with that of strain YM2 (p < 0.01 in each case) (Table I).


                              
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Table I
Growth properties and mitochondrial ATP hydrolase activities of cells expressing Y8 variants
Mitochondria were isolated from cells grown at 28 °C with 2% ethanol (v/v) as the carbon source. ATP hydrolysis rates were measured in triplicate from at least two independent experiments. Where indicated, oligomycin was included at 100 µg mg-1 mitochondrial protein. Whole cell generation times represent the mean ± S.D. of triplicate assays.

Next, performance of the mtATPase was assessed by measuring ATPase activity. In each case, the ATP hydrolysis rate was similar to that measured for mitochondria isolated from strain YM2. In the presence of the inhibitor oligomycin, however, mitochondria isolated from cells expressing Y8 variants Y39C or V40C showed faster ATP hydrolysis rates (p < 0.01 and p < 0.001, respectively) than mitochondria isolated from strain YM2 (Table I). The decrease in sensitivity to oligomycin displayed by these two cysteine variants compared with the wild type reflects less efficient coupling of ATP hydrolysis on the F1 sector with proton pumping across the membrane in the F0 sector. Collectively, these results indicate expression of the individual Y8 cysteine substitutions is generally well tolerated at the level of whole cell growth, with an effect apparent only when cysteine substitutions are made in a small region of the C-terminal domain of Y8.

Physical Destabilization of the F1F0 Complex by Y39C and V40C Variants-- To investigate whether the Y39C and V40C substitutions resulted in structural changes within mtATPase, monomeric enzyme complexes were isolated from mitochondria by clear native gel electrophoresis. The gels were first assayed for ATP hydrolysis activity in both the presence and absence of oligomycin, and then stained with Coomassie Blue to examine any difference in the profile of the isolated complexes.

In each sample ATP hydrolase activity could be detected as a discrete band (Fig. 1A, lanes 1-3) demonstrating that the enzyme was isolated in an intact and functional state. In the presence of oligomycin ATP hydrolase activity was completely inhibited in enzyme isolated from strain YM2, expressing unmodified Y8 (Fig. 1A, lane 4). By contrast, a very small amount of residual activity was seen for mtATPases isolated from cells expressing either the Y39C or V40C variants (Fig. 1A, lanes 5 and 6). This clearly demonstrates that while the isolated mtATPases are both intact and coupled, in mtATPases assembled with either the Y39C or V40C variants the structure of the complex has become sufficiently destabilized such that it is less sensitive to oligomycin in the in situ assay. Whereas not excluding the possibility of alteration to the binding of oligomycin the results indicate that there exists a heterogenous population of mtATPases in Y39C or V40C mitochondria, some of which are insensitive to oligomycin inhibition.


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Fig. 1.   Structural perturbation of the F1-F0 interaction by the Y8 substitutions Y39C and V40C. Intact, monomeric mtATPase complexes were isolated by native gel electrophoresis (4-13% polyacrylamide gradient). Prior to staining, the ATPase activity of complexes (panel A) was demonstrated in situ in the absence (lanes 1-3) or presence (lanes 4-6) of oligomycin. The polyacrylamide gels were then stained with Coomassie Blue (panel B), and the identity of proteins comprising each band was established by second dimension SDS-PAGE (15% acrylamide) and immunoblotting analysis (not shown). The source of mitochondria is indicated at the top of each panel.

Next, the gels were stained with Coomassie Blue to demonstrate the presence of intact mtATPases. In each case, a single protein band was stained at a position corresponding exactly to that of the oligomycin-sensitive, ATPase-active band observed after in situ assay (Fig. 1B, lanes 1-6). Immunoblotting analysis confirmed the identity of this band as intact mtATPase because several subunits belonging to both the F1 (subunits alpha , beta  and gamma ) and F0 (subunits b, d, f, OSCP, and 6) sectors were identified (data not shown). In each of the Y39C and V40C variants a second prominent stained band of faster mobility was detected (Fig. 1B, lanes 2-3 and 5-6), which was either absent or below the limit of detection in the YM2 sample (Fig. 1B, lanes 1 and 4). Immunoblotting analysis demonstrated the presence of both the alpha  and beta  subunits in each of these bands but not any F0 subunits tested (data not shown); thus, this band represents a dissociated F1 portion of mtATPase.

Strategy for Determination of Solvent-exposed Regions of Y8-- We next sought to determine the extent of the membrane-embedded and extra membranous regions of Y8. Our approach was to determine the local side chain environment using FM to specifically label the cysteine residues introduced into Y8. FM is a small, non-polar compound that is highly reactive with thiol groups in an aqueous environment (45). It is also sufficiently hydrophobic to partially penetrate into hydrophobic environments and even to cross the mitochondrial membrane at high concentrations (37, 46). Importantly, FM is unable to react with a thiol group that is either: 1) very close to and facing the boundary of the lipid bilayer; or 2) embedded within it, because generation of a reactive thiolate anion at, or in, the membrane is impeded by the low dielectric constant of that environment (47, 48). Thus, FM should react readily with any available thiol group in an aqueous environment, less readily in a hydrophobic environment, and not at all within a lipid environment. Accordingly, our expectation was that as the cysteine residue introduced into Y8 was located closer to the aqueous-lipid interface, its reactivity with FM would steadily decrease until no reaction is observed because of the proximity of the lipid bilayer. Such decreased reactivity would be reflected as a decreased intensity of fluorescence that was detectable following labeling of membrane preparations containing Y8 and analysis of the partially purified proteins separated by SDS-PAGE. Use of this strategy potentially enables accurate delineation of the boundaries of the lipid-aqueous interface for both the N- and C-terminal domains of Y8.

Accessibility of Introduced Cysteine Residues in Y8-- To assess the exposure of cysteine residues to the aqueous phase, isolated osmotically lysed mitochondria were incubated in the presence of 1 mM FM for 4 h. After removal of excess FM, proteolipids were extracted from the membranes and separated by Tricine SDS-PAGE. The gel was scanned for FM fluorescence (upper panel in Fig. 2, A and B) and then silver-stained (lower panel in Fig. 2, A and B). The stained gel was then overlaid onto the fluorescent image to correlate stained proteins with the fluorescent signal obtained. Y8 variants S-1C and L48C have previously been demonstrated to react strongly with FM in a mitochondrial lysate (37) and effectively act as positive controls for labeling of the N and C termini of Y8, respectively. Cysteine residues introduced at positions -1 through 14 at the N terminus of Y8 were all accessible and available for modification by FM (Fig. 2A, lanes 2-16) as were cysteines introduced at positions 36 through 48 in the C terminus (Fig. 2B, lanes 18-27). As expected, no fluorescence was observed for unmodified Y8 (Fig. 2A, lane 1). These cysteine residues are therefore, a priori, exposed to the aqueous phase and available for modification in a mitochondrial lysate. While decreasing the period of FM incubation to 1 h resulted in a reduction of the intensity of labeling for all of the reactive residues, neither extended incubation times (up to 8 h) nor increased concentrations of FM (2 or 4 mM) resulted in detectable modification of introduced cysteine residues at positions 15 through 35, although Y8 could still be detected by silver staining (data not shown). These non-reactive residues could, however, be modified in the presence of SDS (data not shown) suggesting that they can indeed be modified but are not accessible to FM in a mitochondrial lysate, as expected for residues sequestered within the lipid bilayer. Thus, we conclude that the first 14 and last 13 amino acids at the N and C termini of Y8, respectively, are exposed to the aqueous phase, extrinsic to the lipid bilayer, and that residues 15 through 35 are embedded in the inner mitochondrial membrane.


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Fig. 2.   Reactivity of introduced cysteine residues in Y8 with FM. Isolated mitochondria were osmotically lysed and treated with the thiol-specific reagent FM. Proteolipids were extracted and separated by Tricine SDS-PAGE (18% polyacrylamide) and analyzed by fluorescence (F) and direct silver staining (S) (images aligned with respect to the geometry of the original gel). The position of the cysteine replacement in Y8 is indicated. Panel A, N-terminal cysteine replacements at positions -1 through 14 (lanes 2-16); panel B, C-terminal cysteine replacements at positions 35 through 48 (lanes 17-27). Unmodified Y8 is designated YM2 (panel A, lane 1). In each of panels A and B, the fluorescent image is located directly above the corresponding silver-stained region of the gel containing Y8. Panel C, immunoblotting of C-terminal FLAG-tagged Y8 variants either unmodified (FTC2, lane 1) or bearing an N-terminal substituted cysteine residue (M1C, lane 2) and detected using anti-FLAG antibodies.

Y8 variants bearing cysteine replacements at residue positions -1 through 5 (Fig. 2A, lanes 2-7) and labeled with FM apparently migrated more slowly in SDS-PAGE than did unmodified Y8 (Fig. 2A, lane 1) or any of the other labeled Y8 cysteine variants. Variants S-1C and M1C co-migrated with an unidentified, non-fluorescent band of ~8-9 kDa (Fig. 2A, lanes 2 and 3, lower panel), whereas the labeled Y8 variants P2C through V5C each migrated with an apparent mobility slightly slower than that of unmodified Y8 (Fig. 2A, lanes 4-7, lower panel). The apparent alteration to molecular weight was not due to binding of the FM moiety, as the size shift was observed after silver staining regardless of the presence or absence of FM (data not shown). For each of the S-1C and M1C variants, a second broad silver-stained band of apparent equal molecular weight to unmodified Y8 (see Fig. 2A, lanes 1-3, lower panel) was identified, suggesting the presence of two distinct species of Y8 in these samples. Moreover, immunoblotting of the Y8(M1C)-FLAG variant (bearing a C-terminal fused FLAG epitope) also showed the presence of two Y8 species (Y8 and Y8*), one having mobility comparable with that seen for FTC2 mitochondria expressing cysteine-less Y8-FLAG and one of slower mobility, respectively (Fig. 2C, lanes 1 and 2). To assess the relative accessibilities of cysteines at the N and C termini of Y8, the intensity of labeling by FM for each Y8 variant, P2C through T14C and L36C through L48C, was analyzed and compared with the intensity of the silver-stained protein (Fig. 3). The data for Y8 variants S-1C and M1C were not analyzed because of the apparent presence of two distinct proteolytically processed variants.


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Fig. 3.   Relative accessibility of residues at the N and C termini of Y8. The intensity of FM fluorescence ("fluorescent intensity") for each reactive cysteine replacement at either the N terminus (panel A) or C terminus (panel B) of Y8 was quantified and compared with the intensity of silver staining ("protein intensity") for each sample. Fluorescent intensity was divided by protein intensity to relate the amount of fluorescent signal from FM to the amount of protein present, and the values for each individual residue averaged from at least three independent experiments to generate the term "accessibility." The position of the cysteine substitution in Y8 is indicated. Unmodified Y8, corresponding to background levels of fluorescence, is designated YM2.

At the N terminus of Y8 a gradient of accessibility to FM was evident proceeding from the N terminus toward residue 14 (Fig. 3A). Cysteines at positions -1 through 9 displayed strong reactivity with FM compared with cysteines at positions 10 through 14 (Fig. 3A; see also Fig. 2A, lanes 1-16). As stated above, neither extended incubation with FM nor higher FM concentrations increased the reactivity of residues beyond position 10. Moreover, conditions involving a shorter period of incubation (1 h) with FM, during which the Y8 variant P2C was maximally labeled, resulted in decreased fluorescent intensity for labeled cysteines beyond position 10 and a complete absence of labeling for Y8 variants L13C and T14C (data not shown). These results demonstrate that cysteines at positions -1 through 9 are in a highly exposed location, extending into the intermembrane space of the mitochondrion. Residues beyond position 10 are located closer to the lipid bilayer, where the environment becomes less favorable for reaction between FM and the introduced thiol moiety. Therefore, residues at positions 13 and 14 are very close to the lipid boundary but when substituted by cysteine are still reactive with FM, thus demonstrating their location on the outer face of the inner mitochondrial membrane.

At the C terminus of Y8 a gradient of decreasing accessibility was again evident from residue position 48 through 36 (Fig. 3B). As for the N-terminal residues, increased concentrations of FM or extended periods of incubation failed to increase the reactivity of any residue at the C terminus of Y8. Moreover, shorter incubation times (1 h) again resulted in an overall decrease in detectable FM fluorescence and a loss of fluorescent signal from variants L38C and L36C (data not shown). Residues at positions 38 and 36 therefore are likely to lie in close proximity to the lipid bilayer, whereas residue 35 is located within, or faces the lipid bilayer and is unavailable for modification because a substituted cysteine at this position is not reactive with FM. It was also noted that, whereas cysteines at positions 48 through 36 are reactive with FM and thus exposed to the aqueous phase, they are comparatively less reactive than are most residues at the N terminus of Y8 (compare Fig. 3, A and B). Because the C terminus of Y8 is located in the mitochondrial matrix (37, 49) it seems likely that the apparent reduction in reactivity may be because of steric occlusion by other mtATPase subunits.

Effect of Partial Removal of the F1 Sector on Accessibility of the C terminus of Y8-- If the comparatively reduced reactivity of C-terminal versus N-terminal cysteine substitutions observed is because of steric occlusion of Y8 by other mtATPase subunits, the removal of the F1 sector from membranes may increase their accessibility to FM. To demonstrate removal of the F1 sector of mtATPase from mitochondrial membranes, mitochondria isolated from strain FTC2 (expressing wild type Y8 with a C-terminal FLAG epitope) were treated with guanidine HCl and the remaining protein complement assessed by SDS-PAGE and immunoblotting. Membranes were probed with monoclonal antibodies against mtATPase subunit beta  as a marker for F1-sector proteins and monoclonal antibodies against the FLAG epitope to demonstrate the presence of Y8 in the membrane preparations. After treating isolated mitochondrial membranes with guanidine HCl the amount of detectable subunit beta  was reduced by 45% compared with untreated mitochondria (Fig. 4, lanes 1 and 2). Degradation of subunit beta  was evident as cross-reactive protein bands of higher apparent mobility in each sample. Y8 remained associated with membranes after treatment (Fig. 4, lane 4) and its detection was enhanced using anti-FLAG antibodies compared with untreated mitochondria (Fig. 4, lane 3). Thus, guanidine HCl treatment of the mitochondrial membrane was able to partially remove at least the beta  subunit of the F1 sector of mtATPase while not causing depletion of membrane-embedded Y8.


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Fig. 4.   Partial removal of the F1 sector of mtATPase by treatment with guanidine HCl. Mitochondria isolated from strain FTC2 expressing C-terminal FLAG-tagged Y8 were incubated with guanidine HCl (G-HCl). The proteins were separated by SDS-PAGE (15% acrylamide) and transferred to PVDF membranes. Membranes were probed with antibodies against the mtATPase subunit beta  (lanes 1 and 2) as a marker for the presence of the F1 sector, or antibodies against the FLAG epitope (lanes 3 and 4) to demonstrate the presence of Y8 in the membrane. The positions of size standards are indicated at the left of the figure.

Mitochondria isolated from yeast expressing the Y8 cysteine variants S46C, V40C, L36C, I35C, and YM2 were lysed and incubated with guanidine HCl prior to incubation with FM. Accessibility of residue 46 was assessed because it is adjacent to Leu-48 but displays only ~50% of the relative accessibility, and that of residue 40 as it marks the boundary of a slight decrease in relative accessibility at the C terminus that extends from residue positions 40 through 46 (Fig. 3B). Cysteine residues at positions 36 and 35 were chosen as they are reactive or not with FM in the mitochondrial lysate, respectively, and are presumed to denote the membrane boundary of Y8. After guanidine HCl treatment the reactivity of cysteine residues at positions 40 and 46 increased substantially compared with untreated controls (Fig. 5, lanes 7-10). The reactivity of the Y8 variant L36C, previously localized to the membrane boundary, was not visibly improved by prior incubation of membranes with guanidine HCl (Fig. 5, lanes 5 and 6) suggesting that its low reactivity was because of the proximity of the lipid bilayer and not steric occlusion of FM binding by other mtATPase subunits. Residue 35, localized within the lipid bilayer, remained unreactive to FM after guanidine HCl treatment (Fig. 5, lanes 3 and 4) indicating that the position of Y8 within the membrane remained unchanged. As expected, no fluorescence was ever detected for YM2 (Fig. 5, lanes 1 and 2).


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Fig. 5.   Reactivity of selected introduced cysteine residues in the C terminus of Y8 after removal of the F1 sector by guanidine HCl treatment. Mitochondrial membranes either treated with guanidine HCl (Gu HCl) (+) or not (-) were incubated with FM. Proteolipids were extracted and separated by Tricine SDS-PAGE (18% polyacrylamide) and analyzed by fluorescence (F) and direct silver staining (S) (images aligned with respect to the geometry of the original gel). The position of the cysteine replacement in Y8 is indicated. Sources of mitochondria were: YM2, expressing unmodified Y8 (lanes 1 and 2); Y8(I35C) (lanes 3 and 4); Y8(L36C) (lanes 5 and 6); Y8(V40C) (lanes 7 and 8); Y8(S46C) (lanes 9 and 10).

These results demonstrate first that the reactivity of cysteine substitutions V40C and S46C can be substantially increased by at least partial removal of the F1 sector from the membrane, whereas reactivity of the L36C substitution cannot be improved in this manner. Second, the increased reactivity displayed by the V40C and S46C substitutions is not because of the removal of phospholipids surrounding the introduced cysteines nor any alteration to the position of the membrane-embedded domain of Y8 because the reactivity of cysteines at positions 35 and 36 remained unchanged. Thus, the reactivity of cysteines at positions 40 and 46 is to some degree determined by the partial occlusion of the C terminus of Y8 by other subunits of mtATPase.

Cross-linking of Y8 to mtATPase Subunits b, d, f, and 6-- Previously, we have demonstrated cross-linking between the matrix-located C terminus of Y8 and each of subunits d and f, and the N terminus of Y8 to subunit f in the intermembrane space (37). Here we have attempted to define more fully interactions involving Y8 with other mtATPase subunits by using additional cysteine substitution mutants in cross-linking experiments. For these studies, an HA epitope was fused to the N terminus of the Y8 protein to facilitate detection of cross-linked products. Cells expressing the HA-tagged Y8 variants demonstrated normal respiratory growth compared with strain YM2, and the epitope-tagged Y8 was detected in isolated, functional mtATPase complexes (data not shown). Mitochondria prepared from selected strains expressing HA-tagged Y8 cysteine variants were incubated with APDP or APA-Br and the cross-linked products analyzed by SDS-PAGE and immunoblotting. APDP is capable of cross-linking over a distance of 21 Å, whereas APA-Br can cross-link proteins up to a distance of 9 Å (37), thus the presence or absence of cross-linked products reflects both distance constraints between the cross-linked partners and accessibility of the reagents to the introduced cysteine. At each of the N and C termini of Y8, several cross-linked products were detected using antibodies against the HA epitope in Y8 (Fig. 6, A and B). No cross-linking was ever detected for wild-type Y8 (Fig. 6, A and B) demonstrating that each of the cross-linked products observed involved the unique introduced thiol in each of the Y8 variants.


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Fig. 6.   Cross-linking of the N and C termini of Y8 using APDP or APA-Br. Mitochondria isolated from cells expressing NHAY8 cysteine variants were incubated with either APDP or APA-Br, the excess cross-linker removed, and the samples were exposed to UV irradiation. Mitochondrial membranes were then dissolved, proteins were separated by SDS-PAGE (15% acrylamide) and transferred to PVDF membranes. Cross-linked products (bands I thorough VIII) were detected using anti-HA antibodies. The presence (+) or absence (-) of either APDP or APA-Br is indicated. Sources of mitochondria were as follows. Panel A, NHAY8(L4C) (lanes 1-2 and 9-10), NHAY8(F7C) (lanes 3-4 and 11-12), and NHAY8(M10C) (lanes 5-6 and 13-14). Panel B, NHAY8(F44C) (lanes 1-2 and 7-8) and NHAY8(L48C) (lanes 3-4 and 9-10). Cysteine-less Y8 (panel A, lanes 7-8 and 15-16, or panel B, lanes 5-6 and 11-12) is designated WT. The positions of size markers are indicated at the left of the figure.

Immunoblotting analysis using antibodies against other mtATPase subunits was undertaken to identify partners involved in cross-links to Y8. Each of the ~22- and ~24-kDa adducts formed using APDP was cross-reactive with antibodies against mtATPase subunit f (Fig. 7A). The apparent anomalous migration of the Y8-subunit f cross-linked product is similar to that observed in experiments involving cross-linking of subunit b to d (50). This effect is presumably caused by alternate locations of the cross-linking between the two proteins, leading to differences in their hydrodynamic volumes during SDS-PAGE and thus reflected as a difference in apparent mobility in the gel (50). Identical results were obtained when cross-linking was performed using APA-Br (not shown), except that the ~22-kDa Y8-subunit f cross-link was absent in the L4C sample but present in the M10C sample; also, both of the ~22- and ~24-kDa Y8-subunit f adducts were simultaneously present in the F7C, F44C, and L48C samples when the shorter cross-linker was used (Fig. 6). Presumably this reflects steric constraints on the ability of each cross-linker to form a product when bound at different positions in Y8.


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Fig. 7.   Identification of cross-linked products involving Y8. Mitochondrial proteins isolated from cells expressing NHAY8 cysteine variants were separated by SDS-PAGE (15% acrylamide) and transferred to PVDF membranes. Cross-linked products detected previously (Fig. 6, A and B) were probed using antibodies against mtATPase subunits f (panel A), b (panel B), d (panel C), or 6 (panel D). The presence (+) or absence (-) of cross-linker, source of mitochondria, and position of size markers are indicated.

The ~40-kDa adduct formed from Y8 F7C, M10C, and F44C variants using APDP was cross-reactive with antibodies against mtATPase subunit b (Fig. 7B). Identical results were obtained using APA-Br (not shown), except that the ~40-kDa Y8-subunit b product was not formed from the F7C or M10C replacements (Fig. 6). The ~30-kDa adduct formed from each of the F44C and L48C variants using APDP was cross-reactive with antibodies against mtATPase subunit d (Fig. 7C). Identical results were obtained when cross-linking was performed using APA-Br (not shown).

The ~35-kDa adduct formed from the Y8 F7C and M10C replacements was cross-reactive with antibodies against mtATPase subunit 6 (Fig. 7D). Identical results were obtained when cross-linking was performed using APA-Br (not shown), except that the ~35-kDa Y8-subunit 6 product was not formed from the F7C variant (Fig. 6). Also, this Y8-subunit 6 adduct was formed in response to UV irradiation alone in the M10C sample (Fig. 7D, lane 3) suggesting it may represent oxidation of the unique thiol group on Y8.

Disulfide Formation between Y8 F7C or M10C and Subunit 6 C23-- The UV-induced Y8-subunit 6 adduct could be reduced in the presence of 2-mercaptoethanol (Fig. 6A, lane 13) suggesting it may involve a disulfide bridge. Incubation of mitochondria with CuCl2 strongly induced formation of the ~35-kDa product in each of the F7C and M10C variants but not in the wild-type sample (Fig. 8A, lanes 1-3). Subsequent incubation with 2-mercaptoethanol completely reduced the disulfide bridge (Fig. 8A, lanes 4-6). This CuCl2-induced product was confirmed as a Y8-subunit 6 cross-link using antibodies against mtATPase subunit 6 (Fig. 8B).


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Fig. 8.   Disulfide bridge formation between NHAY8 cysteine variants (F7C) and (M10C) with the endogenous Cys-23 of subunit 6. Mitochondria isolated from strains expressing NHAY8 variants (F7C or (M10C) were incubated with CuCl2. Mitochondrial membranes were dissolved, proteins were separated by SDS-PAGE (15% polyacrylamide) and transferred to PVDF membranes. Cross-linked products were detected as previously described using antibodies against the HA epitope (panel A), or mtATPase subunit 6 (panel B). The presence (+) or absence (-) of cross-linker, source of mitochondria, and position of size markers are indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Membrane Topography of Subunit 8-- Y8 is an intrinsic membrane protein whose N terminus is in the intermembrane space and C terminus in the mitochondrial matrix (37). In this work, application of FM affinity labeling enabled identification of the membrane-intrinsic, as opposed to membrane-extrinsic, regions of Y8 in its assembled form within the mtATPase complex. Thus, a 14-amino acid region at the N terminus of Y8, and a 13-amino acid region at its C terminus, can be modified by FM in a mitochondrial lysate and are thus located outside the lipid bilayer. Accordingly, Y8 possesses a 21-amino acid transmembrane span, in agreement with hydropathy profiles (21, 23). The membrane-extrinsic regions of Y8 are expected to interact with other mtATPase subunits. Indeed, the C-terminal region of Y8, exposed in the matrix, was partially occluded from FM binding in the intact, membrane-embedded mtATPase. Partial removal of the F1 subunits increased the exposure of this region of Y8 to the aqueous phase, allowing more efficient reaction with FM. Therefore, the C-terminal region is intimately involved with other matrix-located components of mtATPase.

The N terminus of Y8 maintains a highly conserved MPQL motif (23, 51) that has previously been assigned a role in function (23, 27). Cysteine substitutions for any of these 4 conserved amino acids had no demonstrable effect on cell growth or ATP hydrolase activity, suggesting that in yeast none of these residues is individually required for mtATPase function. However, it was noted that substitutions in this region caused aberrant mobility of the respective Y8 cysteine variants, especially for the M1C replacement, during SDS-PAGE. The reason for this phenomenon is not known, but may reflect altered processing of the Y8 pre-protein or modification of Y8 because of the introduction of a cysteine at these positions.

Localization of Subunit 8 in the Complex with Respect to Other mtATPase Subunits-- Y8 plays an essential role in maintenance of the structural integrity of the interaction between F1 and F0, and is thus essential for mtATPase function (1). Previous work demonstrated interactions between Y8 and mtATPase subunits d and f based on analysis of Y8 variants bearing cysteine substitutions at either their extreme N or C termini (37). Here we have employed site-directed cross-linking to demonstrate contacts between Y8 and subunits d, f, b, and 6. This leads to novel information about the molecular neighborhood of Y8 in yeast mtATPase.

In a mitochondrial lysate, Y8 could be cross-linked to each of subunits b and f in both the matrix and intermembrane space. In the matrix, cross-linking to subunit b was from Y8(F44C) at a distance no greater than 9 Å. Subunit b has two extra membranous regions that project into the matrix between residues 1-26 and 72-209 (50), whereas N-terminal residues 1-66 of subunit f are also extrinsic to the membrane (52). Accordingly, these regions of each subunit are available to interact with Y8. In the intermembrane space, cross-linking occurred from Y8(F7C) and Y8(M10C) at a distance between 9 and 21 Å to subunit b, whose polar loop is exposed from residues 46 to 55 (53, 54). Cross-linking to subunit f, exposed between residues 85 and 94 (52), was from a maximum of 9 Å to a maximum of 21 Å depending on the location of the introduced cysteine (see Fig. 6). Overall, these results demonstrate that Y8 is in close proximity to each of subunits b and f across the membrane and in both compartments of the mitochondrion.

Y8 was also able to cross-link with subunit d in the matrix at no greater than 9 Å. At present the orientation of subunit d is unknown, but cross-linking of subunit d to each of subunits b (50) and i/j (55) in the matrix has also been demonstrated.

A disulfide bridge was formed between Y8(F7C) and Y8(M10C) to subunit 6 in the intermembrane space, demonstrating a direct interaction between these two subunits. Subunit 6 possesses a single endogenous cysteine (Cys-23) in its first extra membranous region and located close to the membrane (53, 56). In addition to the close interaction between subunits 8 and 6, this result emphasizes two points. First, the N termini of each of Y8 and subunit 6 are located in the intermembrane space. Second, the following residues of these proteins all lie close to the membrane boundary: Y8(F7C) and Y8(M10C), and subunit 6 Cys-23. Aspects of the detailed arrangement of other F0 subunits, as understood on the basis of cross-linking analyses here and elsewhere (37, 53-55, 57-59), are shown in Fig. 9.


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Fig. 9.   Schematic diagram suggesting the arrangement of some subunits in the stator stalk of yeast mtATPase. A putative arrangement of subunits in the stator stalk of mtATPase is shown based on data presented here and elsewhere. Subunits b, f, 6, and 8 are shown; the N and C termini are indicated except for subunit d whose topology is not known. Only two of at least five transmembrane stems of subunit 6 are shown. Cross-links are shown as dotted black lines (demonstrated in this study) or open arrows (from other work).

Subunit 8 Is Part of the Stator Stalk of mtATPase-- Several lines of evidence now support the view that Y8 is a part of the stator stalk in yeast mtATPase. Our cysteine scan revealed no significant effects on function for any of the Y8 variants constructed. This suggests Y8 is a structural component of mtATPase, because no amino acid of Y8 directly participates in either ATP synthesis/hydrolysis or proton pumping. A structural role for Y8 is also consistent with the notion that the overall shape of Y8 and broad chemical character of its amino acids (rather than their specific identities) are important for its role in mtATPase function (32, 60). The introduction of charged residues within the CHD by Roucou et al. (33) showed that Y8 is required for maintenance of the interaction between F1 and F0 as well as correct F0 assembly. Here we have defined two residues, Tyr-39 and Val-40, which are similarly important for the integrity of the structural linkages between F1 and F0. Furthermore, occlusion of the C terminus of Y8 by other mtATPase proteins suggests that these effects on stability of the enzyme may be mediated through other mtATPase subunits.

The data presented here show that Y8 maintains close interactions with subunits b, d, f, and 6. Each of these subunits has proposed roles as part of the stator stalk in yeast mtATPase (18, 37, 55). This view is reinforced by the interdependence of each subunit for the assembly of the F0 sector. Each of subunits b, d, f, or 8 is required for the stable integration of subunit 6 into the membrane; the loss of any one of these subunits leads to a functionally uncoupled F1 sector that becomes loosely bound to the membrane (18, 30, 59, 61, 62). Moreover, subunit f is also required for the presence of subunits b, 8, and 9 (59). It has also been suggested that subunit i/j is required for the incorporation of both of subunits 6 and f into the complex (63). The direct interaction between Y8 and the N terminus of subunit 6 implies that these subunits interact directly during their assembly into mtATPases in the mitochondrial membrane.

The stator stalk of bacterial F1F0-ATPase is comprised of two copies of subunit b, which dimerize along a large portion of their length (17, 64). However, only a single copy of subunit b is present in yeast mtATPase (18). If the stator stalks of bacterial, chloroplast, and mitochondrial F1F0-ATPases share a similar overall design, then other proteins should fulfill the role of the missing subunit b in mtATPase (18). Cross-linking studies have demonstrated extensive interactions involving mtATPase subunits b, d, f, and 6 (50, 53-55, 57-59). We now show interactions involving each of these subunits with Y8, highlighting the location of Y8 deeply embedded within, and located close to, each of the major structural components of the stator stalk. Accordingly, we propose that subunits d, f, and Y8 fulfill the role of the missing subunit b in forming the connection between F1 and F0 in yeast mtATPase. Further elucidation of the structural interactions between these subunits will reveal the detailed architecture of the stator stalk in mtATPase.

    ACKNOWLEDGEMENTS

We are grateful to Dr. R. A. Stuart (Marquette University, Milwaukee) for providing antibodies directed against subunits e, g, i/j, and k, and Dr. J. Velours (Institut de Biochimie et Genetique Cellulaires du CNRS, Université Victor Segalen, Bordeaux) for providing antibodies against subunits f, h, and 6.

    FOOTNOTES

* This work was supported by the Australian Research Council.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.

Dagger Present address: Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal QC H3T 1E2, Canada.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia. Tel.: 61-3-9905-3782; Fax: 61-3-9905-3726; E-mail: rod.devenish@med.monash.edu.au.

Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M300967200

    ABBREVIATIONS

The abbreviations used are: mtATPase, mitochondrial F1F0-ATP synthase; APA-Br, p-azidophenacyl bromide; APDP, N-(4-p-azidosalicylamidobutyl)-3'-(pyridyldithio)-propionamide; CHD, central hydrophobic domain; FM, fluorescein 5-maleimide; Y8, yeast mtATPase subunit 8; HA, hemagglutinin; PVDF, polyvinylidene difluoride; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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