Energized Outer Membrane and Spatial Separation of Metabolic Processes in the Hyperthermophilic Archaeon Ignicoccus Hospitalis

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Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis

Ulf Küpera, Carolin Meyerb, Volker Müllerc, Reinhard Rachelb, and Harald Hubera,1

aInstitute for Microbiology and Archaeal Center, Universität Regensburg, D-93053 Regensburg, Germany; bCenter for Electron Microscopy, Faculty of Natural Sciences III, Universität Regensburg, D-93053 Regensburg, Germany; and cMolecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe Universität Frankfurt, D-60438 Frankfurt am Main, Germany

Edited* by Dieter Söll, Yale University, New Haven, CT, and approved December 18, 2009 (received for review October 9, 2009) ATP synthase catalyzes ATP synthesis at the expense of an electro- To date, in prokaryotes no outer membranes but only cytoplasmic chemical ion gradient across a membrane that can be generated by membranes have been described as harboring ATP synthase com- different exergonic reactions. Sulfur reduction is the main energy- plexes, the key components in cellular bioenergetics (15). These yielding reaction in the hyperthermophilic strictly anaerobic Cren- complexes (bacteria, mitochondria, and chloroplasts: F1FO ATP archaeon Ignicoccus hospitalis. This organism is unusual in having an synthases; Archaea: A1AO ATP synthases) consist of a hydrophilic inner and an outer membrane that are separated by a huge inter- (F1,A1) and a membrane-bound domain (FO,AO) (16). Driven by membrane compartment. Here we show, on the basis of immuno-EM an electrochemical ion gradient (17), the membrane-bound domain analyses of ultrathin sections and immunofluorescence experiments translocates ions (H+;Na+) across the membrane, resulting in ATP with whole I. hospitalis cells, that the ATP synthase and H2:sulfur synthesis by the hydrophilic, catalytic domain. The enzyme is also oxidoreductase complexes of this organism are located in the outer able to reverse this process by hydrolyzing ATP. In contrast to other membrane. These two enzyme complexes are mandatory for the ATP hydrolyzing enzymes, this complex is sensitive to specific generation of an electrochemical gradient and for ATP synthesis. inhibitors. According to the genome annotation of I. hospitalis, Thus, among all prokaryotes possessing two membranes in their cell several ATP hydrolyzing enzymes are present (18); only one set of envelope (including Planctomycetes, Gram-negative bacteria), I. hos- subunits, however, was predicted (A, B, C, D, E, F, a,andc)tobuild MICROBIOLOGY pitalis is a unique organism, with an energized outer membrane and a functional ATP synthase (19) and thought to be located in the ATP synthesis within the periplasmic space. In addition, DAPI stain- inner membrane of I. hospitalis (18). The latter assumption was also ing and EM analyses showed that DNA and ribosomes are localized in based on the fact that primary H+ or Na+ pumps are absent in outer the cytoplasm, leading to the conclusion that in I. hospitalis energy membranes of mitochondria, chloroplasts, and Gram-negative conservation is separated from information processing and protein bacteria (12, 20), so that a gradient sufficient to drive ATP synthesis biosynthesis. This raises questions regarding the function of the two cannot be generated. Therefore, outer membranes are generally membranes, the interaction between these compartments, and the believed to be “non–energy-conserving” (13). To date, neither a general definition of a cytoplasmic membrane. proton motive force across an outer membrane nor ATP synthesis within a periplasmic space has been described. Archaea | ATP synthase | ATPase | immunolabeling | sulfur reductase In this article we show that in I. hospitalis the outer membrane is energized and that ATP synthesis is spatially separated from DNA he hyperthermophilic Crenarchaeon Ignicoccus hospitalis replication, transcription, and protein biosynthesis. These results T TKIN4/I is a strictly anaerobic chemolithoautotrophic sulfur raise questions regarding the function of the two membranes in reducer that grows optimally at 90 °C. It conserves energy by the I. hospitalis, the interaction between the two cell compartments, the reduction of elemental sulfur with molecular hydrogen and uses general definition of a cytoplasmic membrane, and a possible energy CO2 as sole carbon source (1). Together with Nanoarchaeum transfer from I. hospitalis to N. equitans. equitans,itformsan“intimate association,” the only known stable coculture of two Archaea (2, 3). Like all Ignicoccus species (4), I. Results hospitalis cells possess an unusual architecture, with two compart- Purification and Identification of the 440-kDa Subcomplex of ATP ments that can clearly be distinguished in composition and mor- Synthase. To clarify how I. hospitalis conserves energy, we started to phologic appearance. As shown in a number of EM studies (5–7), purify and characterize its A1AO ATP synthase. We solubilized the densely packed cytoplasm is surrounded by two membranes, an membrane proteins of I. hospitalis by addition of n-dodecyl-β-D- “ ” “ ” inner membrane and an outer membrane. These two mem- maltopyranoside (DDM). The solubilisate exhibited a specificATP branes enclose an intermembrane compartment with a variable hydrolysis activity of 1.7 U/mg protein. This activity was completely width from 20 to 500 nm, resulting in a volume exceeding that of the inhibited by the addition of diethylstilbestrol (DES, 1.5 mM) and to cytoplasm (5). Its low electron density suggests that it is devoid of approximately 40% by N′,N′-dicyclohexylcarboiimide (DCCD, 1.5 cellular material like ribosomes or DNA, and it was therefore mM), a property characteristic for a coupled A A ATP synthase “ ” 1 O named periplasm (7). The inner membrane, called the cytoplas- complex. After separating the solubilisate by high-resolution mic membrane, releases numerous vesicles into the periplasmic clear native electrophoresis (hrCNE), the protein complexes were space and also engulfs vesicles into the cytoplasm (7). Both membranes exhibit similar lipid composition, with the exception that the

outer membrane lacks caldarchaeol cores (8). In addition, the latter Author contributions: U.K., V.M., R.R., and H.H. designed research; U.K. and C.M. per- contains multiple copies of a pore-forming complex (9), whereas a formed research; U.K., C.M., V.M., R.R., and H.H. analyzed data; and U.K., V.M., R.R., and surface layer (S-layer), typical for most Crenarchaeota, is lacking H.H. wrote the paper. (10). Therefore, the architecture of the I. hospitalis cell envelope is The authors declare no conﬂict of interest. unique among Archaea. Moreover, owing to its huge intermem- *This Direct Submission article had a prearranged editor. brane compartment and an outer membrane without LPS and 1To whom correspondence should be addressed. E-mail: [email protected]. – porins (11 14), it is fundamentally different from other prokaryotic This article contains supporting information online at www.pnas.org/cgi/content/full/ cell envelopes with two membranes (e.g., Gram-negative bacteria). 0911711107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0911711107 PNAS Early Edition | 1of5 Downloaded by guest on September 29, 2021 checked for their ability to hydrolyze ATP in an in-gel enzymatic assay (Fig. 1A). One prominent active complex with an apparent molecular mass of ≈440 kDa was obtained. This complex was purified by ion exchange chromatography, ultrafiltration, and size exclusion chromatography. After tryptic digestion, MALDI-TOF analysis revealed two subunits, A and B, to be part of the I. hospitalis ATPase (scores 577 and 316, respectively). Using specific antibodies generated against the complex, Western blot analyses after hrCNE gave a single signal at an apparent mass of 440 kDa for the native complex (Fig.1B).After its extraction and separation ofthe subunits under denaturing conditions (SDS/PAGE), Western blot analysis showed that the masses of the major subunits A and B (approximately 65 and 55 kDa; Fig. 1C), correspond to the predicted masses of the annotated A and B subunits of the ATPase (67 and 52 kDa).

Localization of ATP synthase in I. hospitalis. By EM of ultrathin sections of I. hospitalis cells, we investigated the subcellular localization of ATP synthase. Surprisingly, immunolabeling with the antibody raised against the purified 440-kDa ATPase complex showed a highly predominant labeling of the outer membrane of I. hospitalis cells (Fig. 2A). The same result was obtained with antibodies raised against the heterologous membrane-bound subunit a (Fig. 2B)and against the catalytic subunit A of Methanocaldococcus jannaschii (Fig. S1). In all cases, less than 10% of the signals could be detected within the cytoplasm, the inner membrane, and the periplasmic space (including the membrane-coated vesicles). This clearly indicates that thebyfarmajorpartofATPsynthasemoleculesofI. hospitalis is located in the outer membrane. To confirm this result, we carried out fluorescence light microscopy experiments (Fig. 3 A–E)usingDNA staining with DAPI (Fig. 3B) and concomitantly the antibody against the 440-kDa ATPase complex (Fig. 3C). According to these images the DNA is, as expected, exclusively located inside the inner mem- Fig. 1. Detection of ATP synthase by ATP hydrolysis in-gel assay and brane (i.e., in the cytoplasm), whereas we observed the fluorescence Western blotting. (A) ATP hydrolysis activity test (90 min at 80 °C) after of the antibodies bound to the A1AO ATP synthase to a great extent native protein complex separation (hrCNE) of the I. hospitalis solubilisate. (B) in the outer membrane, as illustrated in the merge of the two fluo- Immunoblot of an hrCNE gel using the 440 kDa complex antibody of I. rescence images (Fig. 3D). A similar observation was made for hospitalis (dilution 1:5,000). (C) Immunoblot of an SDS/PAGE gel using both dividing I. hospitalis cells, with two daughter cytoplasms existing antibodies raised against the A and B subunits of M. jannaschii (dilution within the outer membrane (7) (Fig. 3 E and F). 1:10,000). Immunolabeling was visualized by HRP-conjugated secondary On the basis of these results, we conclude that the outer mem- antibodies (Sigma-Aldrich; dilution, 1:5,000). brane of I. hospitalis is energized and that energy conservation takes place across this membrane. As a consequence, it is necessary to the cytoplasmic membrane is energized but that energy can be assume that the ion potential-generating enzyme is also located transmitted to the outer membrane via, for example, the TonB here. As an obligate chemolithoautotroph, I. hospitalis conserves system (12, 20, 22). A reverse transport has never been observed, energy exclusively by reduction of elemental sulfur using molecular with the exception of the uptake of “host derived-ATP” by members hydrogen as electron donor (1). Therefore, we aimed at localizing of the genera Rickettsia or Chlamydia (23). Other bacteria that have, H :sulfur oxidoreductase complex, which generates a proton motive 2 at a first glance, some structural similarity to Ignicoccus, are the force used for ATP generation by A A ATP synthase. 1 O Planctomycetes: they lack murein but possess two membranes, I. hospitalis named intracytoplasmic and cytoplasmic (24). Hence, two com- Localization of H2:Sulfur Oxidoreductase in . Labeling of “ ” ultrathin sections with specific antibodies against H :sulfur oxidor- partments are found: the inner riboplasm, containing DNA and 2 “ ” eductase complex from Pyrodictium abyssi strain TAG11 (21) (Fig. ribosomes, and the outer paryphoplasm (24). The latter was 4A) demonstrated that this complex was also located in the outer postulated on the basis of histochemical experiments to contain membrane of I. hospitalis. A similar result was obtained by immu- RNA, but its nature was not further investigated. In fact, the dis- nofluorescence light microscopy (Fig. 4 B and C). In a parallel tribution of key metabolic enzymes in Planctomycetes cells is not yet experiment, the solubilisate was analyzed by Western blotting after known, and therefore no data are available regarding which of the separation under native conditions (hrCNE). A strong signal in an two membranes is energized. in-gel hydrogenase assay and in Western blot analyses with specific Among the Archaea, the cells of all Ignicoccus species are distinct antibodies gave direct evidence for the presence of an active H2: and unique in possessing a two-membrane system (1, 4, 5, 7), sulfur oxidoreductase complex (Fig. S2 A and B). In contrast, no defining two structurally distinguishable compartments: the cyto- signals were detected when both experiments were carried out with plasm and the huge periplasm. The outer membrane of I. hospitalis purified ATP synthase complex. These data prove that H2:sulfur was suggested to only contain lipids and one major protein (9), thus oxidoreductase complex (primary proton pump) as well as ATP surrounding a more or less empty intermembrane compartment like synthase are located in the outer membrane of I. hospitalis. an inert blanket. This model for the outer membrane was consistent with the observation that the inner membrane compartment divides Discussion independently from the outer membrane (7). Other Archaea, like To date, the existence of an energized outer membrane in prokar- members of Sulfolobales, Desulfurococcales (with the exception of yotes harboring more than one membrane system has not been all Ignicoccus species), and Methanococcales, possess a single previously described. For Gram-negative bacteria, it is known that membrane only and a “quasi-periplasmic space” between this

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.0911711107 Küper et al. Downloaded by guest on September 29, 2021 Fig. 2. Localization of A1AO ATP synthase on I. hospitalis cells by EM of ultrathin sections. (A) Labeling with antibodies speciﬁcally raised against the puriﬁed 440-kDa ATPase complex, (B) Labeling with antibodies raised against the membrane-bound subunit a. For both images the secondary antibody with ultrasmall gold particles is visualized by silver enhancement (dilution, 1:200). Dilution of the primary antibodies, 1:200. C, cytoplasm; IM, inner membrane; V, vesicles in the periplasm; OM, outer membrane. (Scale bars, 1 μm.)

cytoplasmic membrane and the S-layer (10). In these organisms, obvious candidates for ATP-dependent ion pumps or other immunolabeling data show that A1AO ATPase is, as expected, means to energize the membrane are apparent (18). located in the cytoplasmic membrane (e.g., Sulfolobus solfataricus; Consequently, neither the inner nor the outer membrane of Fig. S3). Therefore, it was unexpected and surprising to find ATP I. hospitalis satisfies all criteria of a cytoplasmic membrane. synthesis within the periplasm of I. hospitalis. It demonstrates that Although the outer membrane is energized by a primary proton several assumptions (18) about the function and localization of the pump and contains ATP synthase, the inner membrane surrounds different cell envelope components of I. hospitalis were wrong. First, the compartment containing the machinery of information pro- it is not the inner but rather the outer membrane that is energized cessing and biosynthesis reactions. This extraordinary cellular MICROBIOLOGY architecture, whereby features of a classical cytoplasmic membrane (H2:sulfur oxidoreductase) and the site of energy conservation (A A ATP synthase). Second, at least a certain amount of energy- are distributed to two membranes, raises the fundamental question 1 O fi consuming cellular processes must take place in the periplasmic of how to de ne a cytoplasmic membrane in general and in partic- space. Because a specific biophysical separation of the inner and ular in I. hospitalis. outer compartment is not yet possible, we can only speculate about Our results may also help shed light on the interaction of I. hospitalis with N. equitans (3) and a central question encountered such metabolic reactions: although DNA (Fig. 3 B and D)and with N. equitans: how is energy supply ensured in an organism ribosomes are found in the cytoplasm, they are absent in the peri- that possesses no primary proton pumps? N. equitans only con- plasm, and therefore it can be excluded that transcription, trans- tains (according to the genome data and annotation) a rudi- lation, and DNA replication take place here. In contrast, reactions fi fi mentary, incomplete ATP synthase (30) which was supposed to like the rst steps of the CO2 xation pathway (25), or uptake and be unable to synthesize ATP on its own (15), although this has to activation of external molecules like acetate or succinate (26), have be proven experimentally. The ATP formation in the periplasm to be considered to be located in the periplasm, because high of I. hospitalis facilitates the ATP uptake for N. equitans: it avoids amounts of energy are needed for these reactions (25). the otherwise complex import from the cytoplasm of I. hospitalis According to our results, I. hospitalis possesses a very complex across three membranes (7), into the cytoplasm of N. equitans. and unique ultrastructure, with two independent functional Finally, our results can even lead to an extreme hypothesis: it partitions inside a prokaryotic cell: the inner membrane sepa- is assumed that the eukaryotic cell may have originated from an rates energy conservation in the periplasm from information archaeal ancestor, which incorporated bacterial organisms (31). processing and protein biosynthesis in the cytoplasm. The latter If this is correct (which is controversially discussed), would not processes are highly dependent on a constant energy supply, but an organism like I. hospitalis, with its huge and energized peri- there is hardly any energy-conserving ATP synthase in the sur- plasmic space, be an ideal candidate for such an ancestor, which rounding inner membrane. Therefore, ATP has to be channeled provides ATP and further metabolites but without an interaction from the periplasm to the cytoplasm, for example, by diffusion as between the cytoplasms of symbiont and host? in the case of the outer membrane of mitochondria, or by a potential-driven antiport of ADP and ATP, as known for the Materials and Methods inner membrane of mitochondria (27). The resurvey of the I. Cultivation Conditions. I. hospitalis KIN4/IT cells were cultivated in 1/2 SME hospitalis genome (18) and analysis of major membrane proteins (synthetic sea water) Ignicoccus medium at 90 °C as previously described (1, 2), with elemental sulfur as electron donor and a gas phase consisting of H / (28) did not give any hint for known ATP transport systems. 2 CO2 (250 kPa; 80:20 vol/vol). Mass cultivation was carried out in a 300-L However, such transporters are most likely hard to detect on the enamel-protected fermentor with the following minor modifications: stir- gene level because the similarity among the known ADP/ATP ring was decreased from 150 to 100 rpm, and the gassing rate was increased

carriers (mainly found in endosymbionts and mitochondria) (23, from 15 L/min to 60 L/min N2/H2/CO2 (65:15:20, vol/vol/vol) after reaching a 27, 29) is quite low, and to our knowledge no such system has cell density of ≈5 × 106 cells/mL. been identified to date for any Archaeon. Besides an import of Cells of M. jannaschii JAL-1T and S. solfataricus DSM1616T were grown as ATP, transport of molecules across the inner membrane requires previously reported (16, 32). an electrochemical gradient sufficient to drive these processes. Electron Microscopy. For EM analysis, fresh cells were cultivated in cellulose Because the amount of H2:sulfur oxidoreductase and ATP syn- capillaries and cryo-immobilized by high-pressure freezing as previously thase is negligible, other (as yet unknown) processes generating described (33). After freeze-substitution fixation in 95% ethanol/0.5% glu- an electrochemical gradient must be present in the inner mem- taraldehyde/1% formaldehyde/0.5% uranyl acetate and 5% water, samples brane of I. hospitalis. From the genome sequence, however, no were embedded in Epon; for immunolabeling on ultrathin sections, the

Küper et al. PNAS Early Edition | 3of5 Downloaded by guest on September 29, 2021 Fig. 4. Localization of H2:sulfur oxidoreductase complex on I. hospitalis cells with antibodies specifically raised against the purified complex of P. abyssi strain Fig. 3. Epifluorescence microphotographs of I. hospitalis cells. (A) Phase TAG11. (A) EM of ultrathin sections: the immunolabeling was performed similar contrast image. (B) Merged image of phase contrast and DAPI-stained to Fig. 2; C, cytoplasm; IM, inner membrane; V, vesicles in the periplasm; OM, cytoplasm (blue). (C) Localization of ATP synthase immunolabeled with the outer membrane. (Scale bar, 1 μm.) (B) Merged image of phase contrast and fi speci c 440-kDa ATPase complex antibody; secondary antibody coupled to DAPI-stained cytoplasm (blue). (C) Colocalization of H2:sulfur oxidoreductase Alexa Fluor 488 (green). (D) Merge of Fig. 2 B and C.(E) Merge of a phase complex and cytoplasm visualized by immunofluorescence (secondary antibody contrast image and an immunofluorescence image of a dividing I. hospitalis coupledtoAlexaFluor 488, green) and DAPI staining. (Scalebars in B and C,2μm.) cell. (F) Scheme of the dividing cell in Fig. 2E. (Scale bars, 2 μm.)

primary antibodies for ATP synthase subunits A, B, and a of M. jannaschii and

primary antibody was detected using secondary antibodies with ultrasmall for H2:sulfur oxidoreductase complex from P. abyssi were generated as gold and silver enhancement (7). Electron micrographs were digitally previously described (16, 21, 34). Antibodies against the 440-kDa sub- recorded using a slow-scan CCD camera (TVIPS) attached to a CM12 trans- complex of I. hospitalis were raised in rabbits (Davids Biotechnology) from mission electron microscope (FEI). the puriﬁed native complex.

Light and Fluorescence Microscopy. Phase contrast and fluorescence micro- Membrane Preparation. Frozen or freshly cultivated and centrifuged I. hos- scopy were carried out with an Olympus BX 60 fluorescence microscope (BV pitalis cells were resuspended in sterile lysis buffer (25 mM Tris/HCl, 10 mM fi and NB lter) as previously described (2). Micrographs were recorded digi- MgCl2, and 0.1 mM PMSF, pH 7.5) at 4 °C. Cells were disrupted by two French tally using a PixelFly CCD camera (1,024 × 1,024 pixels; PCO). Digital images press passages (3.5 MPa; Aminco), and the cell debris were removed by were edited with Photoshop CS3 (Adobe). Phase contrast and fluorescence centrifugation (Beckman Avanti J-25, JA25.50 rotor, 3,000 × g, 15 min, 4 °C). images were merged with ImageJ (open source). Membranes were sedimented from the extract by ultracentrifugation, DNA of I. hospitalis cells was stained with freshly prepared DAPI staining washed, and resuspended in 25 mM 2-(N-morpholine)-ethane sulphonic acid

solution, containing (for 100 μL): 30 μL 2 M Na-acetate (pH 4.7), 50 μL 100 mM (Mes)/NaOH, 10 mM MgCl2, 0.1 mM PMSF, and 10% glycerol (vol/vol), pH 6.0, Na2-EDTA, 10 μL DAPI (0.2 mg/mL), and 10 μL 1% SDS solution. For optimal as previously described (17). All protein concentrations were determined staining, 1 μL of this solution was added to 8 μLofanI. hospitalis culture. with a BCA Protein Assay Kit (Pierce Biotechnology).

Labeling of Whole I. hospitalis Cells for Immunofluorescence Analyses. For Solubilization and Purification of the 440-kDa Protein Complex. Proteins from immunofluorescence, fresh log-phase cells were fixed by addition of form- the resuspended membranes were solubilized with 1.5 mg DDM (Anatrace) per aldehyde (30% in PBS), washed with PBSG (PBS + 50 mM glycine) and PBS. milligram protein and a final concentration of 2.5% (wt/vol) DDM. After the Subsequently, PBS was replaced by PBSBT (PBS + 2% BSA + 0.02% Tween 20), addition of DDM, the membranes were incubated for 2 h at 40 °C and stored and the primary antibody was added (1:250). After incubation for 2 h, cells overnight atroomtemperature.Solubilizedmembraneproteins were separated were washed with PBS, incubated with the secondary antibody (Alexa Fluor from residual membrane particles by ultracentrifugation (Beckman Optima LE- 488 goat anti-rabbit IgG; Invitrogen, 1:500 in PBS) for 2 h, and finally washed 80K, 70.1 Ti rotor, 120,000 × g, 90 min, 4 °C). The 440-kDa complex was further twice in PBS. All procedures were carried out at room temperature. The enriched by negative purification via cation exchange chromatography [HiTrap

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.0911711107 Küper et al. Downloaded by guest on September 29, 2021 SP HPcolumn,GEHealthcare; CXC buffer A:25mMMes/NaOH,10mMMgCl2,0.1 phosphate, released by hydrolysis of ATP, with reactive lead ions from the mM PMSF, 10% glycerol (vol/vol), and 0.05% DDM (wt/vol), pH 6.0; CXC buffer B: test buffer (lead nitrate). This results in opaque precipitations at the corre- CXC buffer A + 1 M NaCl]. The flow-through, containing the active 440-kDa sponding positions of ATP hydrolyzing enzymes in the gel. The test was complex, was diluted 1:10 with AXC buffer A [25 mM Tris/HCl, 10 mM MgCl2,0.1 carried out with the following modifications: hrCNE gels (20 × 20 cm; 5–13% mM PMSF, 10% glycerol (vol/vol), and 0.05% DDM (wt/vol), pH 8.0; AXC buffer B: acrylamide) were run with 5 mA/gel for 16 h at 4 °C, and the assays were AXC buffer A + 1 M NaCl] and then applied onto an anion exchange chroma- performed at 80 °C for 1 h in the dark. The hydrogenase activity was tography column (MonoQ 5/50 G; GE Healthcare). Proteins were eluted by a detected after hrCNE by incubation of the gel with methylene blue staining linear gradient from 0 to 40% AXC buffer B (20 column volumes). The fractions solution (200 mM Tris, pH 8.5; 2.5 mM methylene blue) for 40 min under showing ATP hydrolysis activity were pooled and concentrated in ultrafiltration anoxic conditions. The gel was washed twice with H2-saturated Tris/HCl spin column devices (Vivaspin, 100,000 molecular weight cut-off; Sartorius). A buffer (100 mM, pH 8.5) and incubated for 20 min at 80 °C. Active enzymes final purification of the 440-kDa complex was carried out by size exclusion were detected by a bleaching of the blue color at the corresponding gel chromatography (HiLoad 16/60 Superdex 200 pg; SEC buffer: AXC buffer A + 150 positions. Tricine SDS/PAGE and Western blot analyses were carried out as mM NaCl) of the concentrated AXC eluate. previously described (36, 37).

ATPase Activity and Inhibition Assays. ATPase activity tests and inhibitor ACKNOWLEDGMENTS. We thank K. Y. Pisa, U. Friedrich, and R. Wirth for studies, using DES and DCCD, were performed as previously described (17). stimulating discussions; M. Thomm for ongoing support; R. Dirmeier and K. Y. Pisa for providing antibodies; and T. Heimerl, A. Röhl, N. Wasserburger, Electrophoresis, In-Gel Enzyme Activity Assays, and Immunodetection. Protein C. Neuner, G. Leichtl, T. Hader, and K. Eichinger for technical support. This complexes were separated by hrCNE and tested for ATP hydrolysis by an work was supported by Deutsche Forschungsgemeinschaft Grants HU 703/2- ATPase in-gel assay (35). The assay is based on the precipitation of inorganic 1 (to H.H. and R.R.), SFB807 (to V.M.), and SFB699 (to R.R.).

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