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Originally published In Press as doi:10.1074/jbc.M407086200 on August 2, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40670-40676, September 24, 2004
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Building the Stator of the Yeast Vacuolar-ATPase

SPECIFIC INTERACTION BETWEEN SUBUNITS E AND G*

James Féthière{ddagger}§, David Venzke§, Meikel Diepholz¶, Anja Seybert||, Arie Geerlof||, Marc Gentzel**, Matthias Wilm**, and Bettina Böttcher¶{ddagger}{ddagger}

From the Structural and Computational Biology Programme, ||Protein Expression and Purification Core Facility, **Bioanalytical Research Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

Received for publication, June 24, 2004 , and in revised form, July 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F1 and F0 ATP synthases. The V-ATPase is composed of an integral domain (V0) and a peripheral domain (V1) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain controversial. We have expressed subunits E and G in Escherichia coli as maltose binding protein fusion proteins and detected a specific interaction between these two subunits. This interaction was specific for subunits E and G and was confirmed by co-expression of the subunits from a bicistronic vector. The EG complex was characterized using size exclusion chromatography, cross-linking with short length chemical cross-linkers, circular dichroism spectroscopy, and electron microscopy. The results indicate a tight interaction between subunits E and G and revealed interacting helices in the EG complex with a length of about 220 Å. We propose that the V-ATPase EG complex forms one of the peripheral stators similar to the one formed by the two copies of subunit b in F-ATPase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar ATPases (V-ATPases)1 are ATP-dependent proton pumps that play an important role in the pH homeostasis of various intracellular compartments to allow several cellular processes such as endocytosis and intracellular transport to take place (1–3). They are also present at the plasma membrane of various specialized cells where they function to regulate intracellular pH (4–7). Thereby, they participate in a variety of physiological and pathophysiological states such as acid-base balance in the kidney, bone resorption by osteoclasts, and metastatic invasion by tumor cells to name a few (reviewed in Refs. 8 and 9).

V-ATPases are multisubunit protein complexes composed of 13 polypeptide chains. Electron microscopy images as well as analysis of subunit stoichiometry have revealed some analogy in the overall bilobed structure of V-ATPase and F-ATPase (10, 11). It is now well established that the V-ATPase segregates into two main functional domains: V0, which forms the core of the proton translocating machinery associated with the membrane (subunits acc'c''d), is functionally homologous to the F0 domain of the F-ATPases; and V1, which contains the catalytic sites (subunits A-H), is functionally homologous to the F1 domain of F-ATPases. ATP hydrolysis takes place in the A3B3 hexamer of V1, and the energy provided by this reaction is transmitted to V0 via the connecting regions to drive proton translocation. Similarly to the F-type ATPase, a rotational mechanism linking these two functionalities has been proposed (12, 13).

Despite these similarities, V-ATPases differ significantly from F-ATPases in some respect. In particular, electron microscopy has revealed significant protrusions in the connecting elements linking V0 to V1 (14, 15), indicating a more complex structure for this region. In addition, extensive experimental characterization of these connecting elements using different techniques such as chemical cross-linking and low resolution imaging has led to the proposal of several models for their topological arrangement (16–20) confirming the higher complexity of these elements in V-ATPases compared with F-ATPases. Although only limited homology exists between the V- and F-ATPase subunits (21–23), some sequence similarities between the soluble domain of subunit b of the Escherichia coli F-ATPase and subunit G of the V-ATPase have led to the proposal that these proteins are functionally analogous (24–26). Subunit b of F-ATPases serves to fix F1 to F0 and forms a stator through its membrane anchor, thereby preventing rotation of the F1 domain during catalysis (27, 28). In V-ATPases, subunit G lacks the length required to reach the top of the catalytic head and to bridge V1 to V0 in a similar fashion. It also lacks a membrane anchor that would be required for this stator function and is therefore assumed to interact with another subunit to fulfill this presumed role. Sequence analysis has revealed some features that might be important for such interactions (26).

Recently we have proposed a topology for the assembly of a plant V-ATPase based on a three-dimensional electronic microscopy reconstruction model (15) and published cross-linking data. According to this model, subunits E and G would interact to form a peripheral stalk in analogy to subunits I and II of the chloroplast ATP-synthase (29), subunit b of E. coli F-ATPase (22, 30), and the subunit b of mitochondrial ATPase. Although the number of subunits composing the stalk is known, their arrangement is still a matter of controversy (12, 31, 32). There is currently no clear structural or biochemical data that provide unambiguous solutions to the topological arrangement of the V-ATPase subunits, although understanding this arrangement is essential to determining the enzymatic mechanism and the different modes of metabolic regulation that occur in vivo. The current study attempts to clarify this matter by providing strong biochemical evidence for the tight interaction between subunits E and G. We introduce, for the first time, the use of a co-expression system with differentially tagged subunits for the investigation of their specific interactions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning and Expression—Subunits E and G were cloned by PCR using a Saccaromyces cerevisae genomic DNA library as template. For subunit E, primers were ATG GCC ATG GGC ATG TCC TCC TCC GCT A (forward) and GGG GTA CCT CAA TCA AAG AAC TTT CTT GTC T (reverse). For subunit G they were CAT GCC ATG GGC ATG TCC CAA AAA AAC (forward) and GGG GTA CCT ACA AGG CAT TGA TAT GGA CTT (reverse). PCR products were subcloned into a modified pET expression vector (pETM-41 from G. Stier, European Molecular Biology Laboratory) using the NcoI and KpnI sites (underlined in the above sequences), 3' of a hexa-histidine tag, MBP fusion protein, and TEV cleavage site. Identity of the cloned products was confirmed by sequencing. The resulting vectors were transformed into E. coli strain BL-21 (DE3) (Invitrogen) and plated onto LB-agar supplemented with kanamycin (30 µg/ml). For expression of the subunits, a single colony was used to inoculate 4 ml of LB medium supplemented with 30 µg/ml kanamycin and grown overnight at 37 °C. This culture was diluted 1:1000 into 500 ml of LB medium containing 30 µg/ml kanamycin and grown to an A600 of 0.6 after which induction with 0.2 mM isopropyl-1-thio-{beta}-D-galactopyranoside was started. Expression was performed at 19 °C overnight in Innova shakers at 180 rpm.

For the co-expression experiments, a bicistronic expression vector based on a modified pET24d (Novagen) vector was constructed with a single promoter and a 26-nucleotide linker separating the two open reading frames. An N-terminal histidine tag was kept on subunit G. Subunit E followed in its native form. The resulting bicistronic plasmid was transformed into BL21-star (DE3) cells (Invitrogen) carrying a pRARE plasmid (Novagen) with chloramphenicol selection and colonies selected on kanamycin/chloramphenicol (50 µg/ml, 20 µg/ml) LB-agar plates. For expression of the EG complex, a single colony was picked and grown in 500 ml of terrific broth medium (supplemented with antibiotics) to A600 of 0.8 after which expression was induced with 0.2 mM isopropyl-1-thio-{beta}-D-galactopyranoside. Expression was carried out at room temperature for 20 h.

Purification and Complex Formation—Cells were harvested by centrifugation and lysed in PBS supplemented with one tablet/100 ml of complete EDTA-free protease inhibitors (Roche Applied Science) using a cell emulsifier (Emulsiflex-C5, Avestin). Lysed cells were centrifuged at 200,000 x g for 45 min, and recombinant protein in the supernatant was purified by metal chelating affinity chromatography using a Talon cobalt column (Clontech) pre-equilibrated with PBS. After an extensive wash of the resin with PBS, the protein was eluted with a 0–0.5 M imidazole gradient, and fractions were analyzed by gel electrophoresis. The fractions containing the protein were pooled, diluted 2-fold with Tris buffer, pH 8.0, and the buffer was exchanged for 50 mM Tris-HCl, pH 8.0, by anion exchange chromatography. The elution product was then used directly for cleavage from the fusion partner using the TEV protease. Digestion was allowed to proceed overnight at 4 °C with 10 µg/ml TEV. Initially, both subunits were purified separately for independent characterization of their properties. After cleavage, the MBP fusion partner, which retained the histidine tag, was separated from the protein of interest using the cobalt affinity column. The recombinant subunits in the flow-through were further purified by ion exchange chromatography (HiTrapQ-Sepharose, Amersham Biosciences) and gel filtration (Superose 12 column, Amersham Biosciences) to yield the final pure proteins. Samples were concentrated by ultrafiltration on centriprep YM30-membranes (Millipore), and protein concentration was determined with the BCA assay (Pierce). Purity was assessed by SDS-PAGE. To characterize the specific interaction between subunits E and G, purified MBP-E and MBP-G were mixed and cleaved with TEV protease (10 µg/ml). The cleaved product was separated from the MBP fusion partner with a second cobalt affinity step. The flow-through of this step consisted essentially of the EG complex and uncomplexed monomers. The complex was further purified by ion exchange and gel filtration. Recombinant EG complex co-expressed from the bicistronic vector was purified by metal chelating affinity chromatography followed by anion exchange and gel filtration.

CD Measurement—The purified samples were diluted to 200 µg/ml and dialyzed in PBS, pH 7.5. Spectra were acquired in a 0.1-cm path length cell on a Jasco J-710 spectrometer. Far UV scans were collected between 190 nm and 240 nm in 0.1-nm steps at 22 °C. The raw data were converted to molar mean residual ellipticity ({Theta}) in a standard manner. Secondary structural content was estimated from the CD measurements with the K2D package (33).

Cross-linking—The co-expressed EG complex from the bicistronic vector was used for the cross-linking experiments. The protein at a concentration of 0.8 mg/ml in PBS, pH 7.5, was incubated with 0.1 mM 1,5-difluoro-2,4-dinitrobenzene or Bis-(sulfosuccinimidyl) suberate and 5mM EDC/N-hydroxysulfosuccinimide for 30 min at room temperature. After incubation, 10 µl of 1 M Tris-HCl, pH 8.0, was added to stop the reaction. Samples were then analyzed by SDS-PAGE. To analyze the content of the cross-linked species, in-gel proteolytic digestion was performed with trypsin (modified sequencing grade, Roche Applied Sciences), and the proteolytic fragments were analyzed by MALDI mass spectrometry (Bruker Ultraflex, Bruker Daltonik, Bremen, Germany).

Electron Microscopy Analysis of the EG Complex—Prior to sample analysis, large debris and aggregates were removed by centrifugation at 16,000 x g for 5 min. The supernatant was diluted 5- to 10-fold with a solution containing 100 mM NaCl and 35% sucrose. The grids were prepared as described in Ref. 34. In brief, the thin carbon film was floated from a small piece of mica onto the surface of the sample. Then the carbon film was picked up again with the mica and floated on the surface of staining solution (2% uranyl formate). A second carbon film was floated on another aliquot of staining solution. Both films were picked up subsequently with a copper grid so that the sample and stain were sandwiched between the two carbon films. After drying, the grids were examined in a CM-120 Biotwin (FEI Company) microscope operated at 100 kV. Images were recorded at a nominal magnification of x52,000 on a charge-coupled device or on Kodak S0163 films. Some micrographs were scanned with a Zeiss Scai scanner using a step size of 14 µm for further analysis. Particle images were selected and boxed using the MRC package (35). Basic image processing (like adjustment of gray values, alignment, and averaging) was done with the IMAGIC V package (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of the Recombinant Subunits— Both subunits E and G were expressed as soluble MBP fusion proteins in E. coli using low temperature for expression. Expression at 19 °C in BL21 (DE3) E. coli cells made it possible to obtain good yield of soluble fusion protein (25–50 mg/liter) in contrast to studies in which subunit E could be expressed only in inclusion bodies (37) or with the use of a special osmoregulated system (31). As seen in Fig. 1A, immediately after expression (lanes 3 and 5), the fusion proteins are detectable in the crude supernatant of the cell lysate. The subunits purify readily using a combination of metal-chelating affinity, ion exchange, and gel filtration chromatography. The purity of the overexpressed subunits is verified by SDS-PAGE (Fig. 1B). As can be seen on this figure, subunits E and G have apparent molecular masses of 28 kDa and 14 kDa, respectively.



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  		FIG. 1.
A, overexpression of recombinant subunits E and G in E. coli. Lanes 2–4 represent the post-lysis supernatant of BL21 E. coli cells carrying the pETM-41-E or -G subunits, uninduced (lanes 2 and 4) and induced with isopropyl-1-thio-{beta}-D-galactopyranoside (lanes 3 and 5 for subunits E and G, respectively). B, pure subunits E (right panel) and G (left panel) after the different chromatographic purification steps and fusion protein cleavage. Proteins were separated by SDS-PAGE on a 15% acrylamide gel subsequently stained with Coomassie Blue G-250.

 
EG Complex Formation—In an analogy to subunit b of the F0F1-ATP synthase, subunits E and G are believed to form part of the peripheral stalk of the V-ATPase. Several reports have already indicated that these subunits might interact (16, 17, 38). However, very few data exist on the isolated subunits. Using our system, we have been able to verify a direct physical interaction between E and G. The EG complex was initially formed by cleaving MBP-E and MBP-G from the MBP fusion partner simultaneously, using an excess of MBP-G fusion protein. The cleaved MBP was washed away by a second Co2+-Talon chromatographic step. Further purification of the complex by ion exchange took advantage of the basic pI of subunit G. At pH 8.0 free subunit G is not retained on an anion exchanger and can be separated from the EG complex. The EG complex together with some free E is then eluted from the column with a salt gradient. Monomeric E elutes very close to the EG-complex from the anion exchanger at 120 mM NaCl, and an extra gel filtration step is necessary to separate them. However, complete separation of the monomer from the complex is not easily achieved. The chromatogram shown in Fig. 2A indicates that subunit E elutes as a shoulder on the EG complex elution peak.



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  		FIG. 2.
Gel filtration chromatogram of subunits E and G and the EG complex in PBS. A, gel filtration analysis was conducted on subunits G and E and the EG complex on a Superose 12 column (Amersham Biosciences) using PBS as a buffer. Subunits E and G and the EG complex were run separately on the Superose 12 column, and then the chromatograms were overlaid into one graph. B, using the Low Molecular Weight Calibration kit (Amersham Biosciences) with the Superose 12 column to make the standard curve of Kav versus molecular weight, the Kav and molecular weights of subunits G and E and the EG complex were determined from this standard curve.

 
Considering the inability of uncomplexed subunit G to bind to the anion exchanger under the conditions used, its co-elution with E from the ion exchange column is a clear indication of their interaction. One additional piece of evidence indicating the favorable interaction between subunits E and G is the increased stability of both subunits in the complex. Fig. 3 shows significantly reduced proteolytic degradation of subunits E and G after prolonged storage in the EG complex compared with either subunit alone.



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  		FIG. 3.
Stabilization of the subunits E and G in the EG complex after prolonged storage. Lanes 1, 3, and 5 show the respective yeast subunits loaded on SDS-PAGE (12%) right after the last gel filtration step. Lanes 2, 4, and 6 represents the same subunits after an overnight incubation at room temperature.

 
To confirm this interaction and obtain pure EG complex, we co-expressed subunits E and G using a bicistronic vector. This strategy achieves selective purification of the complex with elimination of uncomplexed single subunits. Both subunits were overexpressed in this system, and we obtained over 10 mg/liter of pure EG complex. With subunit G carrying the histidine tag, both subunits E and G co-purified from the metal affinity column, indicating their specific interaction. Extensive high salt washes of the bound EG complex did not cause significant release of subunit E, confirming the strength of the interaction. Upon elution with an imidazole gradient, the EG complex as well as uncomplexed subunit G excess was recovered. As for the previous MBP fusion protein isolation, this excess subunit G was cleared on an anion exchange column, and the complex was further purified by gel filtration. In this system, in which the complex forms within the cell, no subunit E was detected in the flow-through of the metal column. We have verified that the interaction between subunits E and G was not caused by some nonspecific effect mediated by the histidine tag by cleaving the latter with TEV protease. The product was recovered in the unbound supernatant of the nickel-nitrilotriacetic acid beads and contained the EG complex (Fig. 4). In addition, gel filtration analysis after cleavage of the histidine tag showed a single elution peak containing both subunits, indicating the integrity of the complex in the absence of the tag (data not shown). The EG complex isolated from this co-expression experiment had properties similar to those of the complex obtained from separate expression of the subunits.



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  		FIG. 4.
TEV cleavage of His-E/native-G complex expressed from the bicistronic vector. First lane, markers in kDa; second lane, His-E/native subunit G before cleavage; third lane, supernatant of nickel-nitrilotriacetic acid beads incubated with the cleavage reaction (purified EG complex).

 
Mass Characterization of the Recombinant Subunits—Gel filtration analysis of the retention times for the different protein species in comparison with globular protein markers is presented in Fig. 2B. It is observed that, using these markers, the estimated molecular weights do not agree with those calculated from the respective sequences. Both subunits E and G eluted earlier than expected, indicating high molecular weight oligomeric species of these subunits (Fig. 2B). The EG complex eluted before the respective monomers, confirming formation of the complex. Because the elution volume theoretically relates to the Stokes radius and not to the molecular size, one can hypothesize that the apparent size obtained from the retention times of the respective monomers is partly related to a difference in shape of the subunits rather than a true size difference. In accordance with this, both subunits E and G have been predicted to form long helices (20, 25, 39), a shape that will make them behave quite differently from globular proteins on a gel filtration column.

Cross-linking—Additional confirmation of the close interaction between subunits E and G came from cross-linking experiments. We have used three different cross-linkers differing in their arm length to generate covalent association between subunits E and G in the EG complex and have an idea of the proximity of the subunits. EDC in combination with N-hydroxysulfosuccinimide is a zero length cross-linker reactive against amino and carboxylic groups. 1,5-Difluoro-2,4-dinitrobenzene is a bifunctional hydrophobic compound with a 3-Å spacer arm, which preferentially reacts with amino groups, and Bis-(sulfosuccinimidyl) suberate is bifunctional agent with an 11-Å arm reactive against the {epsilon}-amine of lysine. The results presented in Fig. 5 reveal the formation of a higher molecular weight common cross-linked product for all three cross-linkers. The appearance of this covalent species coincides with reduced staining of subunits E and G, and its mass corresponds to that of an expected EG complex. The identity of both subunits E and G on the gel was confirmed by MALDI analysis, and the same analysis of the isolated cross-linked complex indicated the presence of both subunits confirming the identification of the latter as an EG complex.



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  		FIG. 5.
Chemical cross-linking of the EG complex expressed from the bicistronic vector. Lane 1, molecular mass markers in kDa; lane 2, control (not cross-linked); lane 3, EDC/N-hydroxysulfosuccinimide-treated; lane 4, 1,5-difluoro-2,4-dinitrobenzene-treated; lane 5, Bis-(sulfosuccinimidyl) suberate-treated.

 
CD of Subunits and EG Complex—The secondary structure of the EG complex and the respective monomers were characterized by far-UV CD spectroscopy. The results presented in Fig. 6 show strong minima at 208 and 222 nm for subunit E, indicating a high helical content. However, the ratio of the molar ellipticities at 222 and 208 nm ([{Theta}]222/[{Theta}]208) is calculated to be significantly lower than 1 (0.84), which characterizes non-interacting helices (40, 41) and argues for the absence of self-interacting monomers. The spectrum of subunit G shows a broad minimum around 200 nm with a small peak at 222 nm ([{Theta}]222/[{Theta}]208 of 0.63). This spectrum is most likely that of a misfolded protein commensurate with the lack of stability of this subunit as shown by the rapid proteolysis. Upon formation of the EG complex, the spectrum obtained shows a shift in the [{Theta}]222 and [{Theta}]208 minima with a ratio of 0.99. This characteristic shift is often observed when two helical elements interact to form higher order structures such as coiled coils (40, 41).



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  		FIG. 6.
CD analysis of subunits E and G and the EG complex. The wavelength dependencies of the ellipticities of subunit G (solid line), subunit E (dashed line) and the EG complex (thick line) are shown.

 
Electron Microscopy Analysis of the EG Complex—The expected mass of a binary EG complex is 40 kDa, which is generally considered too small to give valuable structural information by electron microscopy (42). However, specific features such as an exceptionally extended shape should still be recognizable. We have imaged the negatively stained EG complex by electron microscopy. Despite the inherently low resolution of negatively stained samples, an elongated shape could be recognized in the class average (Fig. 7). From the projection images, we have obtained a rough estimate of the dimensions of this structure. It is rod shaped with an estimated smallest diameter of 23 ± 4 Å and a maximal length of 220 ± 20 Å.



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  		FIG. 7.
Gallery of electron micrographs of the negatively stained EG complex. The right picture in the bottom row shows an average of the preceding particle images. The length of the scale bar equals 100 Å.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To establish a definitive and accurate structural and topological model of large macromolecular complexes, one needs to identify specific interactions occurring between the different subunits of these complexes. We have applied a divide and conquer strategy to the investigation of the subunit interactions of yeast V-ATPase with the goal of more accurately interpreting low resolution density maps obtained by electron microscopy for a functional understanding of the complex. We have looked at the interaction between isolated subunits E and G forming the stator using a combination of different techniques. Based on secondary structure predictions and sequence alignment, a similar role of subunit G as a stator linking the catalytic V1 domain to the proton translocating machinery V0 (like the subunit b in E. coli F-type ATPase) has been proposed (25, 26). Moreover, the alignment of the homologous regions (in particular the N terminus) indicates that these functional homologs might fulfill their role in a similar manner, i.e. by interacting with other conserved regions of the enzyme. Based on the alignment of sequences of subunits G from several species, a region mapping to a single face of a predicted helix (26, 43, 44) has been proposed to be involved in protein-protein interaction. Cross-linking studies and native complex isolation also indicated that subunits E and G could form part of the peripheral stator (16, 19). The data presented in this study clearly show that subunit E is a partner for subunit G. The fact that these subunits spontaneously associate after cleavage from their respective fusion partner and purify as a single entity when co-expressed is a clear indication of a tight and specific interaction. In addition, we have observed by CD analysis that upon EG complex formation there is an increase in the total molar ellipticity with a [{Theta}]222/[{Theta}]208 ratio approaching 1. This increase in helical content and change in ratio are indicative of helix interaction. Comparison with spectra obtained for either subunit alone in which this ratio was significantly smaller is consistent with the formation of a coiled coil quaternary fold upon EG complex formation similar to the subunit b of F-ATPases. In addition, it is observed that this association is mutually beneficial to the stabilization of both subunits as shown from the reduced proteolysis of both subunits E and G. This is consistent with the in vivo stabilization of subunit E upon complex formation with subunit G (16). Considering that subunit G alone is misfolded, this increased stabilization can be associated with a more structured polypeptide chain, implying that the interaction with subunit E stabilizes a folded conformation of subunit G. This unfolded state of the isolated subunit G has been reported previously (37, 45, 46), and similar behavior in terms of aggregation and stability was observed for some mammalian orthologs of subunit G that share high sequence identity, suggesting that this assisted folding might be a general feature of the subunit G.

We believe that this coiled coil formed by the interaction between subunits E and G is an important factor in the stabilization and formation of the stator similar to the b dimer forming the stator of the F-type ATPases. The association of subunit G with subunit E could compensate for the short length of subunit G to link V0 to the top of V1 in a similar way to the subunit b of F-ATPases, which reaches all the way to the tip of F1 to bind to the {delta} subunit. Thus, this interaction with subunit E could be a prerequisite for its participation in the formation of a peripheral stator in the yeast V-ATPase. A dimer of subunit G has been shown to form an elongated helix with a length of 80 Å (39), not sufficient to bridge V0 to V1. Our gel filtration results as well as electron microscopy data on the EG complex also indicate the existence of an elongated shape for this complex, and together with the aforementioned report provide additional evidence for a similar structural role of subunit G (in association with subunit E) of V-ATPase and the subunit b of F-ATPase. The dimension we derived from the electron microscopy images for the EG complex does not correlate with the reported SAXS data for a G-dimer. Although there is an agreement on the overall elongated shape with a diameter in the order of 20–30 Å, the length of both complexes differ substantially with the EG complex being almost 3-fold longer than the G-dimer. This shows that the interaction between subunits E and G is necessary to elongate subunit G in order for it to act as a stator. The positioning of the EG complex at the periphery of the V0-V1 complex and its extended character are in accordance with cross-linking data of cysteine mutants that have shown that subunits E and G are in close contact to the outer surface of subunit-B of V1, reaching almost to the top of the V0-V1 complex (19).

An additional handicap for this complex to serve as a genuine stator is the absence of a membrane anchor. This may be linked to the in vivo regulation of V-ATPase by reversible dissociation of the complex and may be an additional argument supporting subunit interaction for anchoring to the membrane. In this respect, it has been shown that subunit E could be cross-linked to subunit a (17) and thus provide a membrane anchor for the peripheral stator. This provides additional support for the stator role of the EG complex because it confirms the functional significance inferred from the homology between subunit G and the subunit b of the F-ATPase. Through the interaction with other subunits such as the N terminus of subunit a, the EG complex fulfills its stator role. We are currently verifying such interactions using a similar approach to the one described in this study.

Both the sequences of subunits E and G display regions predicted to have left-handed coiled coil structure based on the criteria established by Lupas (47). In the case of subunit G, a clear 28-residue coiled coil with a probability of 1 is identified at the N terminus, and subunit E displays a shorter sequence with equally high probability for coiled coil formation. As predicted for coiled coils, a high frequency of hydrophobic residues is found at the a and d positions of a heptad repeat in the respective sequences. The importance of this motif for the formation of the EG complex is supported by conservation of these residues in all sequences of subunit G and subunit E. In addition, mutagenesis studies of the N terminus of subunit G have demonstrated the importance of this region for stabilization of the complex (44). When compared with subunit b of the F-type ATPases, these conserved residues align with the hydrophobic residues of the latter, forming the coiled coil interface.

A basic topology that is common to all ATPases is a central stalk lying in the catalytic core and a more or less complex peripheral connection playing a stator role. Early data obtained from yeast two- and three-hybrid experiments and native subcomplex isolation in subunit deficient mutant strains, as well as co-immunoprecipitation, have helped identify important interactions between the different subunits of yeast V-ATPase (16, 38, 48, 49). In addition, neighboring subunits have been identified by chemical cross-linking experiments (17, 19) in native complexes. These data paved the way to the establishment of a consensus on the topology of the ATPase, especially of the peripheral subunits. The data we present further support and extend these hypotheses. We have used for the first time a co-expression system enabling the in vivo formation of a native complex. A tight interaction between subunits E and G most likely forming a complex analogous to the subunit b dimer of F0F1 is demonstrated. This complex forms an elongated structure with an estimated length sufficient to bridge V0 to the top of V1 to form a stator at the periphery of the complex. In addition, our data suggest that this interaction is required to stabilize both subunits as well as promote folding of subunit G.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

{ddagger} To whom reprint requests may be addressed. E-mail: fethiere{at}embl.de. {ddagger}{ddagger} To whom correspondence and reprint requests may be addressed. Tel.: 49-6221-387304; Fax: 49-6221-387265; E-mail: boettcher{at}embl.de.

1 The abbreviations used are: V-ATPase, vacuolar ATPase; MBP, maltose binding protein; PBS, phosphate-buffered saline; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; MALDI, matrix-assisted laser desorption ionization; CD, circular dichroism; TEV, tobacco etch virus. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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