Subunit gamma -Green Fluorescent Protein Fusions Are Functionally Incorporated into Mitochondrial F1F0-ATP Synthase, Arguing Against a Rigid Cap Structure at the Top of F1*

Mark PrescottDagger, Szczepan NowakowskiDagger, Paul Gavin, Phillip Nagley, James C. Whisstock, and Rodney J. Devenish§

From the Department of Biochemistry and Molecular Biology, P. O. Box 13D, Monash University, Clayton Campus, Victoria 3800, Australia

Received for publication, May 9, 2002, and in revised form, October 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have investigated the question of the presence of a cap structure located at the top of the F1 alpha 3beta 3 hexamer of the yeast mitochondrial F1F0-ATP synthase complex. Specifically, we sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the alpha 3beta 3 hexamer or alternatively whether the cap is more flexible permitting access to the central shaft space under certain conditions. Thus, we sought to establish whether subunit gamma , an essential component of the F1 central stalk housed within the central shaft space and whose N and C termini would both lie beneath a putative cap, could be fused at its C terminus to green fluorescent protein (GFP) without loss of enzyme function. The GFP moiety serves to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and subunit gamma , as well as being a potential occluding structure in itself. Functional incorporation of subunit gamma -GFP fusions into ATP synthase of yeast cells lacking native subunit gamma  was demonstrated by the ability of intact complexes to hydrolyze ATP and retain sensitivity to oligomycin. Our conclusion is that the putative cap structure cannot be an inflexible structure, but must be of a more flexible nature consistent with the accommodation of subunit gamma -GFP fusions within functional ATP synthase complexes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

F1F0-ATP synthase uses energy produced from the electrochemical gradient to produce ATP from ADP and Pi. The enzyme complex is described as having two major structural domains, F1 and F0. The globular F1 catalytic sector contains three alpha  and three beta  subunits arranged alternately in a hexamer thereby forming a central shaft space that houses part of the gamma  subunit. Each beta  subunit contains a catalytic site for the synthesis of ATP. These sites are repetitively and sequentially driven through defined conformational states by the rotation of the gamma  subunit, which is in turn linked to the translocation of protons through the membrane bound F0 sector (1). Models of the F1 sector derived from x-ray crystallographic data indicate the presence of an apparent "dimple" in the top of the F1 sector ~15 Å deep that is contiguous with the central shaft space housing the C- and N-terminal portions of subunit gamma  (2).

Despite the considerable advances in determining much of the structure of F1 (2-4), there are many questions that remain unanswered concerning the structure/function relationships of F1F0-ATP synthase components. One important question relates to the structure of F1 in the vicinity of the dimple at the very top of F1. A "cap" at the top of F1 has been visualized in electron microscopic images of ATP synthase isolated from Escherichia coli (5, 6) and bovine mitochondria (7). This cap structure is not seen in models of ATP synthase derived from x-ray crystallographic data of F1. The identity of the proteins that contribute to the cap structure is not clear but may represent, in part, the N-terminal 20-30 amino acids of subunits alpha  and beta  that were disordered and therefore not represented in models derived from the x-ray crystal data, or alternatively, F0 proteins lost upon crystallization of the complex. Using an immuno-electron microscopy approach, bacterial subunit delta , in particular its C-terminal portion, has been localized to the dimple region (8). In earlier studies, proteolysis experiments had indicated that the eukaryotic homologue of subunit delta , OSCP,1 binds to the N-terminal end of the alpha  subunit (9). Subsequent studies have positioned OSCP in association with F1, but sometimes at the top and sometimes at the side (10). Recent evidence obtained using a protein chemistry approach for ATP synthase isolated from rat mitochondria indicates that specific regions at the top of F1 are shielded by F0 components (11). It was suggested that this shielding represents an extension of the stator stalk and that the cap, composed in part by F6 (whose functional homologue in yeast is subunit h (12)) and possibly subunit d, completely covers the N termini of all alpha  and beta  subunits.

It is not clear whether the cap represents a solid structure closing off the surface area at the top of F1 as represented by the mouth of the dimple seen in F1 crystal structures. Under the experimental conditions used by Böttcher and colleagues (6) the cap of the E. coli ATP synthase (EF1F0) was shown to cover much of the area represented by the dimple. Thus, upon binding of the nucleotide analogue AMP-PNP to EF1F0 a cap structure could be detected concomitant with significant shrinkage in the diameter of F1 (6). It was suggested that some of the differences observed on binding AMP-PNP were a result of a significant rearrangement in the N-terminal domains of the alpha  and beta  subunits. When a three-dimensional reconstruction of EF1F0 was viewed from the top looking down the axis of the central stalk a triangular shaped cap appeared to cover the central shaft space. A flexible cap that shifts its position throughout the catalytic synthesis of ATP on F1 would be consistent with the idea that the conformationally flexible N termini of the alpha  and beta  subunits contribute to its structure. The appearance of the cap would alter depending on the state in which the complex was "captured," sometimes completely covering the top of the F1 and sometimes not.

In this study we have investigated the question of the presence of a flexible cap structure in the yeast mitochondrial F1F0-ATP synthase (mtATPase) complex. Although direct evidence for a cap structure has yet to be reported for yeast mtATPase, the high degree of conservation documented between eukaryotic mtATPase complexes would favor the existence of a cap. Thus we have sought to determine whether the putative cap precludes the assembly into mtATPase complexes of subunit gamma , an essential component of the central stalk, fused to a reporter protein, green fluorescent protein (GFP). The N and C termini of subunit gamma  would both lie beneath the cap within the central shaft space formed by the F1 alpha 3beta 3 hexamer, with the C-terminal glycine residue about 15 Å below the position of the N-terminal end of subunits alpha  and beta  (2).

Our strategy has been to express in yeast cells subunit gamma  fused at its C terminus to GFP via a polypeptide linker. Correctly folded GFP forms a very rigid and stable 11-stranded beta -barrel structure 24 Å in diameter and 48 Å in length threaded by an alpha -helix running up the axis of the barrel (13) that is remarkably stable to the action of a range of denaturing agents and proteases (14). The fluorescence properties of GFP have been utilized widely as a convenient tag to report the presence of a protein in various subcellular locations. We have used these properties of GFP to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and mtATPase subunit gamma . Functional incorporation of subunit gamma -GFP fusions into mtATPase would suggest that the putative cap structure in vivo is not at any time a "solid" inflexible structure that completely occludes the dimple in the top of the F1, but must be of a more flexible nature consistent with the incorporation of subunit gamma -GFP fusions within functional ATP synthase complexes. In such complexes it is presumed that that the polypeptide linker connecting subunit gamma  with GFP would not be obstructed in exiting the central shaft space at the top of F1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Dodecyl beta -maltoside and complete protease inhibitors were purchased from Roche Molecular Biochemicals (Sydney, Australia). Vistra ECF substrate was purchased from Amersham Biosciences (Sydney, Australia).

Construction of Yeast Expression Vectors-- A DNA cassette encoding a yeast enhanced variant of GFP, referred to here as YEGFP3L, was retrieved by PCR using the primer pair YGFP3UP/YGFP3DO (YGFP3UP, 5'-GGATCCATCGCCACCATGTCTAAAGGTGAAGAATTATTCACTGG-3'; YGFP3DO, 5'-CAGCTGTTATTTGTACAATTCATCCATACCATGG-3') with the vector pYGFP3 (15) as template. The PCR product was cloned into the BamHI/NotI site of the multicopy yeast expression vector pAS1NB to produce pAS1NB::YEGFP3L from which the DNA cassette encoding wild-type GFP had been removed. pASN1B is a derivative of pAS1N (16) in which a BamHI restriction site has been removed from the PGK promoter region. This vector allows the expression of GFP not fused to a partner protein.

The DNA cassettes encompassing segments of the yeast ATP3 gene for subunit gamma  were recovered by PCR from YRD15 genomic DNA (17). The first, ATP3PO, encompasses 729 bp of sequence upstream of the initiation codon flanked by HindIII and nested BamHI/BglII/NotI restriction sites at the 5' and 3' ends, respectively. The second, ATP3T, encompasses a transcription terminator cassette representing the terminator region of the ATP3 gene flanked at the 5' and 3' ends by restriction sites for NotI and SacII, respectively. The oligonucleotide pairs ATP3POUP/ATP3PODO and ATP3TUP/ATP3TDO, respectively, were used for these amplifications (ATP3POUP, 5'-TGAAAGCTTGAATGTACAGGTTATGACAACC-3'; ATP3PODO, 5'-AACGATGCGGCCGCAGATCTGAGGATCCCAAAGAGGAAGCACCAG-3' and ATP3TDO, 5'-ATCGTTGCGGCCGCGCATTGCCTCTTTATTTGACG-3'; ATP3TDO, 5'-TACTCCGCGGTCTGGGCATACGCTTGGTAAAAAACCAAATC-3'; in each case the underline indicates the position of relevant restriction sites). The ATP3PO and ATP3T DNA cassettes were cloned sequentially into the yeast expression vector pRS306 (18) as HindIII/NotI and NotI/SacII fragments, respectively, to produce the expression vector denoted pRS306::ATP3. A BglII/NotI DNA fragment encoding YEGFP3L was excised from pAS1NB::YEGFP3L and then cloned into the BglII/NotI site of pRS306::ATP3 to produce a vector (pRS306::ATP3-YEGFP3L27) encoding subunit gamma  fused to YEGFP3 with a polypeptide linker of 27 amino acids (gamma -27-GFP; Fig. 1). A vector (pRS306::ATP3-YEGFP3L4) encoding subunit gamma  fused to YEGFP3 with a polypeptide linker of 4 amino acids (gamma -4-GFP) was derived from pRS306::ATP3-YEGFP3L27 by removal of a 69-bp fragment flanked by BamHI sites (Fig. 1).

Construction of Yeast Strains-- pRS306::ATP3-YEGFP3L27 was linearized by digestion at the unique XbaI site 363 bp upstream of the initiation codon for subunit gamma  and used to transform YRD15 cells by the method of Schiestl and Gietz (19). Transformants were selected by plating onto solid minimal medium supplemented with histidine and leucine. Loss of plasmid sequences leaving only sequences encoding a gamma -GFP fusion at the chromosomal ATP3 gene locus was induced as follows. Individual ura+ transformant colonies were isolated, cultured in non-selective glucose-containing medium, and then subjected to positive selection for the loss of the URA3 marker (ura-) by plating onto medium containing 5'-fluoro-orotic acid (20). One isolate was designated strain MPgamma -27 and expresses gamma -27-GFP.

A PCR product was generated with the primer pair ATP3POUP/ATP3TDO using pRS306::ATP3-YEGFP3L4 as a template and Dynazyme EXT thermostable polymerase (Finnzymes, Espoo, Finland). The product was digested with XbaI and used to transform YRD15 cells. Transformants (ura+) and then ATP3 gene replacement events were selected as described above. Strain MPgamma -4 expresses gamma -4-GFP. A PCR product encoding YEGFP3 linked, without any intervening amino acids, to the C terminus of subunit gamma  (gamma -0-GFP), the yeast ADH1 terminator, and the Kluyveromyces lactis URA3-selectable marker and bearing regions of homology sufficient for recombination with the ATP3 gene was prepared using a template to be described elsewhere. The PCR product was used to transform YRD15 cells. Following selection of ura+ transformants and then ATP3 gene replacement events (as described above), one isolate was designated strain MPgamma -0 (expresses gamma -0-GFP).

Expression vector pAS1NB::YEGFP3L was introduced into strain YRD15 to produce the strain MPGFPc. Cells of this strain express GFP not fused to another protein, and therefore GFP is localized to the cytoplasm.

Subcellular Fractionation-- Mitochondria were prepared from spheroplasts (21) and stored in aliquots at -70 °C until required. For the isolation of cytoplasmically expressed YEGFP3, MPGFPc cells were cultured in a glucose-containing medium. Cells were harvested, resuspended in 50 mM Tris/HCl, pH 7.5, 1 mM EDTA containing Complete Inhibitors (Roche Molecular Biochemicals) and broken open by vortexing with glass beads at 4 °C for 1 min. Soluble proteins were retrieved by centrifugation (20,000 × g for 20 min) and stored in aliquots at -20 °C.

Assays of ATP Hydrolysis Activity-- Assays of mitochondrial lysates were performed as described previously (22). ATPase activity of mtATPase separated on clear native gels was determined in situ by incubating the gel slices in a solution of 5 mM ATP, pH 8.6, in 50 mM glycine/NaOH, pH 8.6, 0.05% lead acetate, 1 mM magnesium acetate with gentle agitation at room temperature (23). A white precipitate in the gel was indicative of ATPase activity. Images of gel sections made for detection of ATPase activity were recorded against a black background using a flat bed document scanner.

Electrophoresis-- Clear native gel electrophoresis was performed essentially as described previously (24). 200 µg of mitochondrial protein was pelleted for 10 min at 100,000 × g. Each pellet was solubilized in 20 µl of extraction buffer (50 mM NaCl, 2 mM 6-aminocaproic acid, 1 mM EDTA, 50 mM imidazole-HCl, pH 7.0, 5 mM phenylmethylsulfonyl fluoride, and 3% (w/v) dodecyl beta -maltoside), incubated on ice for 20 min, and centrifuged 100,000 × g for 10 min. Supernatants (20 µl) were loaded into wells of 4-16% gradient acrylamide gels (13 × 10 × 0.075 cm). Gels were run for 1 h at 100 V, then 2-3 h at 500 V with current limited to 5 mA/gel. Gels, while still between the glass plates, were then imaged for GFP fluorescence using a Wallac multi-wavelength imager in "edge-illumination mode" equipped with filters for excitation (480 ± 20 nm) and emission (535 ± 20 nm). After imaging, gels were separated from gel plates and sliced into sections for further individual analysis. Protein was stained with Coomassie Brilliant Blue G-250 (24). Proteins extracted from cells (25) were subjected to SDS-PAGE on 4-20% gradient acrylamide gels according to standard procedures (26) and using a Bio-Rad mini-gel apparatus.

Western Blotting-- Proteins were transferred to polyvinylidene difluoride membranes (Gelman Laboratory, Pall Corporation) after SDS-PAGE by standard procedures. Membranes were probed with rabbit polyclonal antisera against subunit gamma  (diluted 1:1000). Secondary antibodies were alkaline phosphatase-conjugated anti-rabbit. Signals were generated using chemifluorescent Vistra substrate and incubating for 10 mins at room temperature. Chemifluorescence was detected using a Amersham Biosciences Storm PhosporImager (27). Image data were analyzed using ImageQuant software (Amersham Biosciences). Integrated volumes for each of the relevant bands were determined and expressed as a percent of the relevant intact fusion protein after background correction.

Modeling of Fusion Proteins-- The x-ray crystal structures of bovine mitochondrial F1-ATPase (Ref. 2; Protein Data Bank identifier 1BMF) and GFP (Ref. 28; Protein Data Bank identifier 1EMB) were obtained from the Protein Data Bank (www.rcsb.org; Ref. 29). Using the tools available within the program Quanta (Accelrys Inc., San Diego, CA), and to simplify the modeling procedure, a "template" structure was created containing subunit gamma  plus all residues within a 40-Å radius of its C terminus. Thus, the template contained the dimple and surrounding regions located at the top of F1.

To build the model for the zero length linker, the GFP structure was positioned "end on" with its N terminus of GFP proximal to the C terminus of subunit gamma . Manual positioning of the GFP structure was carried out so that steric clashes were minimized (using the contacts package within Quanta to highlight close contacts) in the merging of the two termini such that the GFP moiety became a C-terminal extension of subunit gamma . The entire F1-derived portion of the model was then constrained (i.e. so that only the GFP portion of the model was allowed to move) and the model subjected to CHARMm minimization. Further minimization was performed using dihedral constraints applied to residues in the N-terminal region of GFP. Minimization was performed to convergence, and upon completion of the modeling procedure all residues in the GFP molecule were in allowed conformations. We predict that in order for the GFP molecule to correctly link to subunit gamma , the N-terminal helix must partially unwind. To create the full model of F1 containing subunit gamma  fused to GFP, the coordinates of the fusion were copied back into 1BMF (since the F1-derived portion of the template was constrained during minimization the subunit gamma  chain of 1BMF could be simply replaced with that of the template). Models containing longer linkers were created similarly, the linkers being added to the C terminus of subunit gamma  using the "edit protein" facility available within Quanta. Linkers were modeled as extended regions so as to create the maximum possible distance between the C terminus of subunit gamma  and the GFP moiety.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In YRD15 yeast cells lacking endogenous subunit gamma , we expressed individually the fusions gamma -27-GFP, gamma -4-GFP, and gamma -0-GFP (Fig. 1) and tested their ability to act as a functional replacement for the native subunit. Yeast cells lacking expression of subunit gamma  are unable to grow on non-fermentable substrates because of the absence of a functional ATP synthase (30). Thus, strains MPgamma -27, MPgamma -4, and MPgamma -0 were assessed for growth in liquid SaccE medium containing ethanol as carbon source (31). All three strains grew on ethanol-containing medium, enabling comparison of their growth rates at 28 °C with that of the parent strain YRD15 (Table I). There was no significant difference2 (p > 0.05) in generation time for yeast MPgamma -27 (10.9 h) and the control strain YRD15 (10.4 h). The generation times for MPgamma -4 (14.7 h) and MPgamma -0 (23.9 h), however, were significantly longer than that for the control strain (p > 0.05). Collectively these results indicate that functional mtATPase complexes are assembled in each of the strains MPgamma -27, MPgamma -4, and MPgamma -0 at levels sufficient to support growth on a non-fermentable substrate.


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Fig. 1.   The structure of the linker joining subunit gamma  and GFP in fusion proteins. The position of the BglII and BamHI sites, within the polylinker into which the DNA encoding the subunit gamma  precursor was cloned, is underlined. The length of the polypeptide linker for each fusion protein is defined by the number of amino acids between the C-terminal amino acid of subunit gamma  (G in shaded box at left) and the initiating methionine of GFP (M in open box at right). An arrow indicates for each of the fusion proteins, gamma -27-GFP, gamma -4-GFP, and gamma -0-GFP, the position at which the C-terminal amino acid of subunit gamma  was fused to the polypeptide linker. The number below each arrow indicates the length of the linker in amino acids.

                              
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Table I
Generation times of strains and oligomycin-sensitive ATPase activities of isolated mitochondria
Mitochondria were isolated from cells grown at 28 °C with 2% ethanol as the carbon source. Assays were performed in the presence of 100 µg of oligomycin/mg of protein, where indicated. Numbers represent the mean ± S.E. (n = 3).

Individual cells of strains MPgamma -27, MPgamma -4, and MPgamma -0 were observed by fluorescence microscopy. For cells of each strain fluorescence due to GFP was distributed in a filamentous manner characteristic of a mitochondrial location (data not shown). This result indicated that GFP had been correctly targeted to the mitochondrion and that the correct folding of the protein and maturation of the chromophore had occurred.

The integrity of the gamma -GFP fusion proteins was further investigated as follows. Proteins were extracted from cells of yeast strains MPgamma -27, MPgamma -4, MPgamma -0, and YRD15 and subjected to SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride membrane. Polypeptides of Mr ~63,800 (Fig. 2, lane 2) and Mr ~61,600 (Fig. 2, lanes 3 and 4) were detected when a blot was probed with polyclonal antibodies against subunit gamma . The polypeptides migrated with mobility corresponding to a size slightly larger than that predicted for the corresponding fusion proteins (Mr 59,997, 57,891, and 57,495 respectively, for gamma -27-GFP, gamma -4-GFP, and gamma -0-GFP). Native subunit gamma  (not fused to GFP) from YRD15 mitochondria (Fig. 2, lane 1) was also found to migrate more slowly, Mr 32,400, than its predicted size (Mr 30,661). Probing an equivalent blot with antibodies against GFP confirmed that each of these polypeptides contained GFP (data not shown). An additional band (Mr ~38,000) not present in YRD15 and representing only minor amounts (1.9%, MP-27; 0.47%, MP-4; and 2.8%, MP-0) of intact fusion protein was observed when blots were probed with antisera against subunit gamma  (Fig. 2). If it is assumed this polypeptide was assembled into functional mtATPase complexes, the amounts of this polypeptide relative to the intact fusion protein cannot explain the levels of oligomycin sensitive ATPase assayed in isolated MPgamma -27, MPgamma -4, and MPgamma -0 mitochondria (see Table I and below). Thus, compared with YRD15 reductions in ATPase activity of greater than 97% would be expected instead of the 40% observed for MP-0 (Table I). These findings confirm the identity of the fusion proteins and indicate that only complete fusion proteins are present within the vast majority of ATP synthase complexes. In other experiments in which a hexahistidine tag was added to the C terminus of the fluorescent protein moiety, it was possible using nickel-nitrilotriacetic acid chromatography, to recover assembled mtATPase complexes that contain only intact gamma -fusion proteins. Under conditions where mtATPase complexes from YRD15 mitochondria did not bind to nickel-nitrilotriacetic acid resin, complexes containing gamma -4-GFP or gamma -0-GFP were recovered from lysates of mitochondria that when assayed for ATPase were found to be oligomycin sensitive (data not shown). Thus, it can be concluded that gamma -27-GFP, gamma -4-GFP, and gamma -0-GFP are able to functionally replace native subunit gamma  in ATP synthase complexes. This conclusion is consistent with the results of other studies from our laboratory that show different subunits of the yeast mtATPase complex can be functionally replaced by the cognate GFP fusion protein (16, 32, 33).3


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Fig. 2.   Immunochemical detection of subunit gamma -GFP fusion proteins. Proteins extracted from cells of strains YRD15, MPgamma -27, MPgamma -4, and MPgamma -0 were subjected to reducing SDS-PAGE. Blots were probed with polyclonal antisera against subunit gamma . Blots were developed with Vistra ECF substrate and bands visualized by scanning for chemifluorescence using a Wallac phosphorimager. The mobilities of protein size standards (RainbowTM Markers, Amersham Biosciences) are indicated at the left.

The integrity and function of isolated ATP synthase complexes containing gamma -GFP fusion proteins were next investigated. Lysates of mitochondria isolated from cells of yeast strains MPgamma -27, MPgamma -4, MPgamma -0, and YRD15 were subjected to clear native gel electrophoresis. This gradient gel electrophoresis technique (23) is capable of separating proteins and protein complexes over a wide size range. After electrophoresis, gels were sliced longitudinally into several sections. Each section was subjected to one of the following: staining for protein, fluorescence imaging, or an in situ assay for ATPase activity (Fig. 3). When stained for protein (Fig. 3A), a band having mobility corresponding to that of assembled monomer ATP synthase, purified by the method of Rott and Nelson (34), was observed for each of the mitochondrial lysates isolated from YRD15, MPgamma -27, MPgamma -4, and MPgamma -0 cells. Since proteins were extracted from mitochondria using the detergent dodecyl beta -maltoside, the species of ATP synthase observed in this study corresponds, as expected, to the monomeric form. The Coomassie Blue staining and fluorescence profiles for monomeric mtATPase complexes from MPgamma -0 cells shown in Fig. 3 (A and B) were of reduced intensity and presumably result from a decreased extractability of complexes. However, similar amounts of each of the gamma -GFP fusion proteins were present in whole cell lysates (Fig. 2). ATP synthase complexes in a dimer form can be isolated if digitonin is used to extract the ATP synthase (35). In a separate experiment fluorescent bands of lower mobility corresponding to ATP synthase dimers were observed on clear native gels when samples of MPgamma -27, MPgamma -4, and MPgamma -0 mitochondria were extracted using digitonin (data not shown).


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Fig. 3.   Analysis of ATP synthase complexes containing gamma -GFP fusion proteins by clear native gel electrophoresis. Lysates of mitochondria from strains YRD15, MPgamma -27, MPgamma -4, and MPgamma -0 were subjected to clear native gel electrophoresis. Gels were stained for protein with Coomassie Blue (A), imaged for fluorescence due to GFP (B), or subjected to an in situ gel assay for ATPase activity (C). Also imaged at the right of B is GFP not fused to another protein, wtGFPL.

One gel slice was imaged for fluorescence due to GFP (excitation 480 ± 20 nm, emission 535 ± 20 nm). A single fluorescent species with mobility similar to that observed for mtATPase stained with Coomassie Blue (Fig. 3A, lanes 1-4) was observed for mitochondria lysates isolated from the three strains expressing a subunit gamma -GFP fusion protein (Fig. 3B, lanes 2-4). As expected, fluorescence was not detectable for the mitochondrial lysate isolated from YRD15 cells (Fig. 3B, lane 1). A cytosolic extract prepared from cells of strain MPGFPc (Fig. 3B, lane 5) was used to indicate the mobility of GFP not fused to another protein. Collectively, these results indicate that mtATPase complexes isolated from MPgamma -27, MPgamma -4, and MPgamma -0 cells are stably assembled and contain fluorescent GFP. In other experiments regions of clear native gels corresponding to mtATPase complexes extracted from mitochondrial isolates of MPgamma -27, MPgamma -4, and MPgamma -0 cells were excised and subjected to a second dimension of reducing SDS-PAGE. Proteins were then transferred to polyvinylidene difluoride membrane and probed with antibodies against subunit gamma . Polypeptides with mobilities corresponding to each of the fusion proteins were detected (data not shown). These findings provide additional confirmation that complete fusion proteins are present in assembled and functional mtATPase complexes.

The ATP hydrolytic activity of mtATPase complexes containing gamma -GFP fusions was investigated for osmotically lysed mitochondria using a spectrophotometric assay. ATPase activity was measured in the absence and presence of oligomycin, an inhibitor of the F0 proton channel. Such an assay gives an indication of the degree of functional coupling between the F1 and F0 sectors of the complex, since ATPase activity is only sensitive to inhibition by oligomycin if the two sectors are functionally coupled. Inhibition by oligomycin (Table I) was found to be in the range of 64-83%, within the range observed for ATPase isolated from wild-type yeast cells. These findings indicate that the fusion of GFP to the C terminus of subunit gamma  does not compromise the ability of the gamma  subunit to assemble into active mtATPase complexes.

In a separate experiment the ATP hydrolytic activity of mtATPase complexes containing gamma -GFP fusions and separated on clear native gel electrophoresis was confirmed using an in situ gel ATPase assay. In the presence of lead acetate, free phosphate liberated by hydrolysis of ATP forms a white precipitate indicating the site of ATPase action within the gel (23). Enzyme activity (as indicated by the presence of a white precipitate) was observed for mitochondrial lysates (Fig. 3C) isolated from the control strain YRD15 and the three strains expressing a subunit gamma -GFP fusion at a position in the gel corresponding to the Coomassie-stained or fluorescent species detected in the corresponding lanes of A and B. This result confirms that mtATPase complexes migrating with the expected mobility of monomers and containing GFP retain ATPase activity.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we have shown that fusion proteins in which GFP is linked to the C terminus of mtATPase subunit gamma  with a polypeptide linker of 0, 4, or 27 amino acids are able to assemble into complexes that are functional in vivo. Furthermore, ATP synthase complexes isolated from these yeast cells expressing each of these fusion proteins are correctly assembled, fluorescent, and functionally coupled.

There are little structural data available concerning the nature of the cap structure. The results in this study indicate that any putative cap structure in yeast mtATPase complexes containing the gamma -GFP fusion proteins cannot entirely cover the dimple located at the top of F1. There must be a route passing through, or around, the cap structure sufficient to accommodate the polypeptide chain linking the C terminus of subunit gamma  to GFP. Furthermore, such a route accommodating the linker must be present throughout all states of the catalytic cycle of the mtATPase. It would be expected that ATP synthase complexes prevented from undergoing the full range of co-operative subunit interactions required for multisite catalysis would be severely compromised in their ability to synthesize ATP. Such complexes would not be capable of providing sufficient ATP synthetic capacity to support growth on respiratory substrates such as ethanol. Cells in which subunit gamma  was replaced by each one of the fusion proteins were capable of growth on ethanol and exhibited specific activities for oligomycin-sensitive ATPase similar to that of the control. Unless the linker of the gamma -GFP fusion causes some particular displacement of the cap compatible with continued mtATPase function, it can be concluded that complexes containing native subunit gamma  would also possess potential polypeptide "threading" routes through the cap structure. A significant displacement of the cap might be expected to result in some uncoupling of the F1 and F0 sectors of the complex because of an adverse influence on interactions between OSCP and alpha /beta pairs. OSCP is known to support important structural interactions between F1 and F0 and also modulates proton channel function at a distance (36, 37). However, instability of mtATPase arising from uncoupled F1-F0 was not apparent according to the results presented here.

The generation time for yeast expressing gamma -27-GFP is not significantly different from that of the cells expressing gamma  subunit not fused to GFP. The x-ray crystal structure coordinates for F1 and GFP were used to model the position of GFP in relation to the top of the F1 alpha 3beta 3 hexamer (Fig. 4). Given the distance of the GFP moiety in gamma -27-GFP from the top of F1 (estimated to be some 40 Å), an effect on growth would not be expected. However, expression of the shorter linker length fusions, gamma -4-GFP and gamma -0-GFP, resulted in longer generation times. Since growth under these experimental conditions is dependent upon ATP production by mtATPase, increased generation times can be taken to indicate that the net rate of ATP production by the complex is compromised when GFP is tethered closer to the top of the F1 catalytic sector. The model of F1 incorporating gamma -4-GFP (Fig. 4) indicates the distances between the loops extending from the end of the beta -barrel of GFP and the proximal loops of the alpha  and beta  subunits are of the order of 10 Å such that some interference may occur. Much more extensive contacts between GFP and the top of F1 are predicted for complexes containing gamma -0-GFP (Fig. 4). The bottom of the beta -barrel of GFP in these complexes may be expected to make significant contacts with the surface of the alpha  and beta  subunits at the top of F1 (Fig. 4). If the putative cap structure includes those N-terminal amino acids of alpha  and beta  not resolved in the x-ray crystal structure then these segments must be placed to one side to prevent interference with the beta -barrel of GFP. For complexes containing gamma -0-GFP the N-terminal domains of the alpha  and beta  subunits of F1 may be moved apart to accommodate the GFP like a "cork in a bottle."


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Fig. 4.   Modeling of the F1 alpha 3beta 3 hexamer with the gamma -GFP fusion proteins. The x-ray crystal structure coordinates for F1 and GFP were used to model the position of GFP in relation to the top of the F1 alpha 3beta 3 hexamer. The models show a side view of F1 and the position of the bottom end face of the beta -barrel of GFP relative to the N termini of the F1 alpha  and beta  subunits in mtATPase complexes containing gamma -4-GFP (left of panel) and gamma -0-GFP (right of panel).

To maintain chemical equivalency of the three alpha /beta pairs during catalysis two models of the stator stalk have been proposed recently by Pedersen and colleagues (38). In these models it is envisaged that the top of the stator stalk makes contact with the top of F1 via a cap consisting of the delta  subunit in E. coli or F6, OSCP, and possibly subunit d in the complex isolated from rat liver mitochondria (11, 38). The cap covers the dimple region in the "swinging" stator model or alternatively dips into the dimple in the "stepping" model. In both cases the cap is anticipated to exchange contacts between the three alpha /beta pairs as either the cap undergoes rotation (swinging model) or the alpha 3beta 3 hexamer rotates (stepping model). The position of GFP in the model of a complex containing the fusion protein gamma -0-GFP suggests that contact between subunits of the stator stalk and alpha /beta subunit pairs must occur close to the edge of the rim of the dimple formed by several beta  sheets at the N termini of alpha  and beta  and that contact regions cannot extend downwards into the central shaft space housing subunit gamma . It is noteworthy that the exact location of the OSCP (or subunit delta ) component of the cap remains uncertain and variously has been positioned at the top of F1 or alternatively on the outer face of F1 extending from the nucleotide binding sites toward the top of F1 (7, 39).

Does GFP rotate during catalysis? It is now accepted that that ATP synthase is a rotatory motor and that subunit gamma  together with subunit epsilon  (delta  in mitochondria) and the subunit c (nine in mitochondria) ring forms the rotor of the motor (40-42). Rotation of the gamma  subunit appears to be an absolute requirement for the synthesis or hydrolysis of ATP. However, ATP synthase is capable of synthesizing ATP through uni-site catalysis where the subunit gamma  is not required to rotate (43). In this case ATP synthesis occurs at a greatly reduced rate compared with complexes in which the subunit gamma  is allowed to rotate without interference. No evidence is yet available concerning rotation of GFP in the strains investigated here, but rotation of subunit gamma  is assumed on the basis of restoration of mtATPase function in host cells lacking native subunit and the results of in situ ATPase assays for purified mtATPase complexes. However, it is conceivable that sufficient freedom exists in the polypeptide linker, joining the C-terminal region of subunit gamma  to GFP, to allow an uncoupling of rotation between subunit gamma  and the relatively bulky GFP. Evidence suggesting such a possibility is feasible is provided by the observation of forced full rotation of subunit gamma  in EF1F0 complexes when the penultimate residue of subunit gamma  was cross-linked to subunit alpha  (44). Further experiments are now under way in an attempt to obtain evidence for the rotation of GFP in these complexes. As we have already shown that other subunits of yeast ATP synthase can be functionally replaced with GFP fusions proteins (16, 32, 33),3 complexes containing binary combinations of appropriate fusion proteins may provide the means for monitoring catalytic activity by ATP synthase in live cells.

    FOOTNOTES

* This work was supported in part 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 These two authors contributed equally to this study.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, P. O. Box 13D, Monash University, Clayton Campus, Victoria 3800, Australia. Tel.: 61-3-9905-3782; Fax: 61-3-9905-4699; E-mail: Rodney.Devenish@med.monash.edu.au.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M204556200

2 Significance levels of difference between generation times were determined using Student's t test, comparing control strain YRD15 and cells expressing subunit gamma -GFP fusions.

3 M. Prescott, P. Gavin, K. McKee, S. Kashyap, and R. J. Devenish, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: OSCP, oligomycin sensitivity-conferring protein; AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate; mt, mitochondrial; GFP, green fluorescent protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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