Structural evidence for a constant c 11 ring stoichiometry in the sodium F-ATP synthase Thomas Meier 1 , Jinshu Yu 2 , Thomas Raschle 1 , Fabienne Henzen 1 , Peter Dimroth 1 and Daniel J. Muller 2 1 Institut fu ¨ r Mikrobiologie, Eidgeno ¨ ssische Technische Hochschule, Zu ¨ rich, Switzerland 2 Center of Biotechnology, University of Technology, Dresden, Germany F-type ATP synthases are multisubunit protein com- plexes lo cated i n the membrane of mitochondria, chloro- plasts and bacteria. These enzymes use an electrochemical H + or Na + gradient across the host membrane for the synthesis of ATP, the universal energy currency of living cells [1]. F-ATP synthases are built from two subcomplexes: the water soluble F 1 part, with the composition a 3 b 3 cde, and the membrane-embedded F o part, which in the simplest case has the subunit com- position ab 2 c 10)14 . In the crystal structure of F 1 , a 3 b 3 forms a cylinder around the extended a-helical c sub- unit [2]. Part of the c subunit protrudes from the bottom of the cylinder and forms the central stalk together with the e subunit. A striking feature of the structure is an inherent asymmetry among the catalytic b subunits. In combination with the asymmetric loca- tion of the central c subunit, and in accordance with the binding change mechanism [3], this arrangement suggests a catalytic mechanism in which the c subunit rotates within the a 3 b 3 cylinder. Elegant experiments have subsequently visually verified this rotation [4]. The F o part, a rotary motor by itself, is fueled by the electrochemical H + or Na + gradient and, upon rota- tion, translocates these ions across the membrane. Recent studies of the F o motor have focused on the ion path through the membrane and the coupling Keywords atomic force microscopy; c ring stoichiometry; F-ATP synthase; Ilyobacter tartaricus; Propionigenium modestum Correspondence T. Meier, Institut fu ¨ r Mikrobiologie, Eidgeno ¨ ssische Technische Hochschule Zu ¨ rich (ETH-Ho ¨ nggerberg), Wolfgang-Pauli- Str. 10, CH 8093 Zu ¨ rich, Switzerland Fax: +41 44 6321378 Tel: +41 44 6325523 E-mail: meier@micro.biol.ethz.ch (Received 27 June 2005, accepted 26 August 2005) doi:10.1111/j.1742-4658.2005.04940.x The Na + -dependent F-ATP synthases of Ilyobacter tartaricus and Propioni- genium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corres- ponding gene, extracted from the membrane using detergent and then puri- fied. Subsequent analyses by SDS ⁄ PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the major- ity migrated as monomers. The population of rings consisted mainly of c 11 , but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggre- gates of homogeneous c 11 rings and ⁄ or assemblies of c 11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crys- talline or densely packed assemblies. Most of these rings represented com- pletely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c 11 and c 1 , as observed by SDS ⁄ PAGE. Our results are congruent with a stable c 11 ring stoichiom- etry that seems to not be influenced by the expression level of subunit c in the bacteria. Abbreviations AFM, atomic force microscopy. 5474 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS between ion flux and torque generation [5–7]. Addi- tionally, using various experimental approaches, increasing evidence has accumulated on the overall shape of the F o domain, particularly the ring-shaped c subunit assemblies. The 2.4 A ˚ resolution structure of the Ilyobacter tartaricus c ring, reported recently, imposes important restrictions on proposed models for ion translocation and torque generation [8]. Further- more, a k ring structure from the V-ATPase of Entero- coccus hirae shares features of ion binding with the I. tartaricus c ring, supporting a common ion transloca- tion mechanism in the two types of ATPases [9]. For detailed insights into this mechanism, high-resolution structural data of the F o subunits a and b 2 are required. It has been shown that these subunits flank the c ring on the outside, but details of their structures have not yet been explored [10]. The number of c subunits in the rotor ring is not fixed but varies among species. For example, rings comprising 10, 14 and 11 subunits have been found in yeast [11], chloroplasts [12] and the bac- terium I. tartaricus [13], respectively. Recently, a ring of 15 subunits was found in the alkaliphilic cyanobacte- rium Spirulina platensis, demonstrating that a sym- metry mismatch between the F 1 and F o motor is not an essential feature for function [14]. The number of binding sites on the c ring determines the ATP to proton ⁄ Na + ratio, and therefore this stoichiometry is an important bioenergetic parameter for the cell. As the number of subunits can vary among species, the question was raised whether this stoichiometry could also vary within one species in order to adapt to speci- fic energetic requirements of the cells [15]. This proposi- tion seemed to be supported by an effect of the carbon source on the expression level of subunit c in Escheri- chia coli. However, structural analyses of rotors from I. tartaricus and from chloroplasts showed that their stoichiometry seems to be constrained by the nearest neighbor interaction between the subunits [16]. This question has also been addressed with subunit c from Escherichia coli [17], where annular shaped particles were detected by electron microscopy after reconstitu- tion from single c subunits. In agreement with the above conclusions, it has been suggested that the primary protein structure determines the ability of subunit c to form rings. Furthermore, it was shown, by gradient gel analysis, that the number of subunits in the oligomer III isolated from the Chlamydomonas reinhardtii chloroplast ATP synthase is not affected by the metabolic state of the cells [18]. However, to date, no structural methods have been applied to clarify whether the stoichiometry of the c rings is influenced by variation of the expression level of subunit c. In the present study we investigated whether the c subunits from the sodium F-ATP synthase of I. tartaricus and of Propionigenium modestum assemble into uniform rings after heterologous overexpression in E. coli. Atomic force microscopy (AFM) topographs showed that complete rings were composed exclusively of 11 subunits and that defective rings exhibited the same diameter as intact ones. Hence, intrinsic features of the I. tartaricus c subunits are responsible for the formation of c 11 oligomers in the fully assembled rings. Results and Discussion Synthesis and assembly of subunit c from I. tartaricus in E. coli To investigate whether heterologously synthesized c subunits from I. tartaricus assemble properly into rings, we used the E. coli strain BL21(DE3) as a host. From wild-type P. modestum or I. tartaricus cells, the c 11 ring is easily purified by extraction from mem- branes using lauroylsarcosine and subsequent ammo- nium sulfate precipitation. As a result of their extreme stability, these rings are easily recognized by SDS ⁄ PAGE. The E. coli BL21(DE3) cells transformed with recombinant plasmids harboring the c subunit genes of I. tartaricus or P. modestum under control of the strong T7 promoter produced large amounts of the appropriate c subunit in the monomeric state, but also sizeable amounts of oligomeric assemblies with a ratio of  9:1 (c 1 :c oligo ). For further analyses, these assemblies were purified by sucrose density gradient centrifugation and subjected to SDS ⁄ PAGE. The results shown in Fig. 1 indicate that the assemblies consisted not only of c 11 , but also of higher aggregates. However, these aggregates are made up exclusively of c subunits because they are converted completely into the monomeric form by treatment with trichloroacetic acid. A similar pattern of bands was observed with the recombinant T67C mutant, with the exception of an additional band corresponding to (c 11 ) 2 and aggregates at the top of the gel. For comparison, the c 11 ring pre- paration of wild-type I. tartaricus cells is shown. Here, the c 11 ring formed the most prominent species, and higher aggregates or the c monomer were less abun- dant than in the preparations from recombinant E. coli cells. Investigation of the aggregation status of c ring preparations by Blue Native PAGE As shown above (Fig. 1) our c ring preparations con- tained various amounts of the c monomer unit and a T. Meier et al. Structural evidence for a constant c 11 ring stoichiometry FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5475 number of aggregates that resisted disassembly into c 11 by SDS. To further investigate the aggregation status of these preparations, we performed Blue Native PAGE in the first dimension followed by SDS ⁄ PAGE in the second dimension. The results (Fig. 2) indicate that the c ring prepared from I. tartaricus wild-type cells in octylglucoside contained not only c 11 and the stable aggregates observed in Fig. 1, but also a number of supercomplexes corresponding to (c 11 ) n , with n ran- ging from two to approximately five. The most abun- dant species was c 11 , and each supercomplex of higher order was present in two- to threefold lower quantities than the previous one. All of these supercomplexes dis- assembled by SDS into the c 11 oligomer (and the SDS- stable aggregates of c 11 , see below) as shown by the equal mobility during SDS ⁄ PAGE (the second dimen- sion in Fig. 2). The aggregation into supercomplexes was prevented if the detergent octylglucoside was replaced by Triton X-100 (Fig. 2A,B). The formation of supercomplexes was also investigated in the recomb- inantly synthesized T67C mutant. Here, in addition to higher aggregates, a (c 11 ) 2 form was observed which Fig. 1. SDS gel electrophoresis of purified c ring preparations. The c rings from Ilyobacter tartaricus and Propionigenium modestum were heterologously expressed in Escherichia coli and purified as described in the Experimental procedures. Two to three micro- grams of each sample was subjected to SDS ⁄ PAGE and the gels were stained with silver. The positions of the monomeric c subunit (c 1 ), the c ring (c 11 ) and the c ring dimer (c 11 ) 2 are marked on the left side. Lanes 1 and 4, purified c rings from P. modestum and I. tartaricus, respectively, isolated from the heterologous expression cultures. Lanes 2 and 5, disintegration of c rings to the c monomer by treatment with trichloroacetic acid. Lane 3, mutant T67C harbouring an SDS-stable c 11 dimer. Lane 6, c ring purified from wild-type I. tartaricus cells. A molecular mass standard is shown. A B C Fig. 2. Supercomplex formation of c 11 rings visualized by Blue Native gel electrophoresis. Five micrograms of c ring in buffer con- taining 10 m M Tris ⁄ HCl, pH 8.0, and 1.5% (w ⁄ v) octylglucoside was loaded on a Blue Native gel (5–17% acrylamide gradient), as described in the Experimental procedures. The samples contained purified c ring from wild-type Ilyobacter tartaricus cells without (A) and with (B) addition of 0.2% (v ⁄ v) Triton X-100. The c ring mutant T67C of Propionigenium modestum was used in (C). After the run in the first dimension, the gel lane was loaded onto a SDS gel for the run in the second dimension. The gels were subsequently stained with silver. Intact c 11 ring and its supercomplexes were marked with c 11 with the indexed numbers (n ¼ 1–4) correspond- ing to the amount of complexed rings (c 11 ) n . The monomeric c sub- unit is marked with c 1 . A molecular mass standard is shown. Structural evidence for a constant c 11 ring stoichiometry T. Meier et al. 5476 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS did not disintegrate into the c 11 oligomer by SDS, indi- cating that a covalent disulfide bond had been formed by the newly introduced cysteine residues. Composition of the SDS-resistant c 11 aggregates As described above, our c 11 ring preparations also con- tained a distinct number of SDS-resistant complexes of higher molecular weight, which might signify rings with different amounts of tightly bound phospholipids. To investigate this possibility, the c ring preparation was incubated with phospholipase C, phospholipase A2 or lipase, and the products were analyzed by SDS ⁄ PAGE. The results (Fig. 3) indicate that none of these enzymes significantly decreased the amount of the stable c 11 aggregates. A similar observation was made after incubating the sample for 5 min at 95 °Cin SDS-containing loading buffer; this result confirms the extreme stability of these aggregates. We conclude, from these results, that the higher molecular weight of the stable aggregates cannot be attributed to strongly bound phospholipids. This conclusion is in agreement with those of previous experiments, which showed that the detergent-purified c ring contained no bound phospholipids and the ones observed on one side of the rings originated from the reconstitution procedure [19]. On SDS ⁄ PAGE, the SDS-resistant aggregates migra- ted between c 11 and (c 11 ) 2 . We therefore reasoned that these complexes might consist of c 11 with one or more c monomers attached. To investigate this possibility, the homogeneous c 11 ring was isolated by electro- elution of the c 11 band excised from the SDS gel (Fig. 4). During storage for at least 1 month, no aggre- gates or monomeric c units were formed from pure c 11 ring preparations. However, after addition of isola- ted c monomers and incubation overnight, the stable aggregates were formed again. This suggests that the c monomer assembled with other c subunits and rings to form a ladder of higher aggregates. To test this hypo- thesis, higher aggregates were specifically electroeluted from the gel and subjected to SDS ⁄ PAGE without heat treatment. The results showed that some aggre- gates converted to c 11 and c 1 . It may therefore be con- cluded that c 11 and c 1 form stable aggregates and that these aggregates are in dynamic equilibrium with c 11 and c 1 . The addition of palmitoyl-oleyl-phosphatidyl- choline to pure c 11 did not result in the formation of any stable aggregates, confirming our conclusion that these aggregates do not represent c 11 rings with bound phospholipid molecules. Further experiments showed that the aggregation of c 11 and c 1 was faster at 25 °Cor37°C than at 4 °C Fig. 4. In vitro aggregation of c 11 with c 1 to complexes resistant to SDS. For the preparation of homogeneous c 11 , 1 mg of wild-type c ring from Ilyobacter tartaricus (lane 1) was applied onto a prepara- tive SDS gel. After the run, the c 11 band was cut out with a scalpel and the protein was electroeluted from the gel pieces to obtain pure c ring, as described in the Experimental procedures (lanes 2 and 5). As a control, c-ring bands migrating more slowly were cut out and electroeluted (lane 3). Upon incubation of 2 lg of pure c 11 with 2 lgofc 1 purified in detergent, the slower migrating band reappeared (lanes 4 and 8). Upon incubation of 2 lg of pure c 11 with 2 and 10 lgofc 1 purified in chloroform ⁄ methanol, the slower migrating c ring aggregates did not reappear (lanes 6 and 7). Lane 9, c 1 purified by extraction with chloroform ⁄ methanol. Lane 10, c 1 purified by sucrose density gradient centrifugation with octylgluco- side as the detergent. Lane 11, 2 lg of c ring after incubation with 5 lg of palmitoyl-oleyl-phosphatidylcholine. A molecular mass standard is shown. Fig. 3. Incubation of c ring with phospholipases and lipase. The c ring samples isolated from Ilyobacter tartaricus wild-type cells were incubated with phospholipase C (PLC), phospholipase A2 (PLA2) and lipase (Lip), as described in the Experimental proce- dures, and 4 lg aliquots were loaded onto an SDS gel. The enzymes alone were applied to separate lanes, as indicated. Also shown is the nontreated c ring (–) and the c ring incubated at 95 °C for 5 min. The gel was stained with silver. A molecular mass stand- ard is shown. T. Meier et al. Structural evidence for a constant c 11 ring stoichiometry FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5477 and reached approximately 90% completion after 1 day. Interestingly, the stable aggregates of c 11 and c 1 , or of several c 1 moieties, were formed with c 1 isolated in detergent (by sucrose density centrifugation) but not after the extraction of c 1 with chloroform ⁄ methanol and reconstitution into a water ⁄ detergent mixture. These results suggest different structures for the two different preparations of the c monomer. AFM of c subunit preparations To investigate whether the heterologously expressed c rings exhibited stoichiometries other than the previ- ously observed undecameric composition, all purified samples were reconstituted into lipid bilayers, as des- cribed previously [20], and imaged by AFM. High- resolution AFM topographs of c ring preparations from I. tartaricus (Fig. 5) and P. modestum (Fig. 6) showed surveys of crystalline (A) and densely packed (B) regions of the reconstituted c subunits. The undecameric subunit stoichiometry of the c rings was more clearly visible in the densely packed regions of the unprocessed topographs. Those c rings that were assembled into a 2D crystal exhibited an upside-down orientation, with one oligomer neighbored by three oligomers showing an opposite orientation. In agree- ment with previous results, the more elevated oligo- mers (bright white areas) protruded from the lower and wider c rings by about 1.1 ± 0.2 nm (n ¼ 50) and thus partly prevented the AFM stylus from contouring the wider rings [13]. However, for statistical analyses we performed reference-free single particle analysis of the densely packed c rings. All classes of complete c rings exhibited 11 subunits forming the donut-like o ligo- mer (first image of Figs 5D and 6D). However, some rings were incomplete, missing one or more subunits. Compared with AFM topographs of c rings isolated from wild-type I. tartaricus ATP synthase [13,16], the reconstituted samples investigated in the present study showed more of these structural inconsistencies. The presence of incompletely assembled c rings from I. tartaricus and spinach chloroplast F-ATP synthases was previously observed by AFM [16]. As the dia- meter of the incomplete c rings did not change in any Fig. 5. Atomic force microscopy (AFM) topographs of c subunit oligomers from Ilyobacter tartaricus F-ATP synthase overexpressed in Escherichia coli. The undecameric oligomers were reconstituted into the lipid bilayer and imaged in buffer solution. (A) A survey of oligomers assembled into a 2D crystal. The donut-shaped oligomers were inserted into the membrane exposing either one of their surfaces to the AFM stylus. (B) A survey of densely assembled oligomers. Arrows point out oligomers either missing one subunit or showing additional subunits inside their central cavities. (C) A gallery of c rings observed from the densely packed arrangement. The first topograph represents a reference-free single particle average obtained from more than 300 c rings. Most of the examples selected exhibit additional central pro- trusions. (D) A gallery of c rings observed from the crystalline arrangement. Examples selected exhibit additional central protrusions. The dashed circles with a diameter of 5.7 nm demonstrate that the outer diameters of the c rings are very consistent with each other. Topo- graphs exhibit a gray scale corresponding to a vertical height of 3 nm. Structural evidence for a constant c 11 ring stoichiometry T. Meier et al. 5478 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS preparation investigated, it was concluded that the diameter of the c rings may be determined by the struc- ture of the c monomer and not by the number of assem- bled subunits. This finding is in agreement with our present analysis of the defective rings overexpressed in E. coli, which exhibited the same outer diameter 5.7 ± 0.3 nm (n ¼ 300) as the complete rings 5.6 ± 0.3 nm (n ¼ 280) within an experimental error of 0.1 nm. This structural agreement did not depend on the number of subunits missing to complete the c ring. The incompletely assembled c rings prepared from chloroplasts and bacteria represented less than 5% of all rings imaged [16]. In contrast, c rings from I. tar- taricus (Fig. 5) or P. modestum (Fig. 6) synthesized recombinantly in E. coli showed an increased amount of incomplete c rings, exhibiting a total content of  8% (n ¼ 2000). Among these, c 10 ,c 9 and c 8 assem- blies represented the most abundant species. These defective rings could probably not be observed on the SDS gel because the detergent dissociates the less stable c 2 to c 10 assemblies into monomeric units. Therefore, we assume that upon insertion of the last, 11th, c subunit, the assembly becomes resistant to SDS or heat treatment. The observed accumulation of the incomplete c 10 complex in the recombinant c ring preparations suggests that the insertion of the last c subunit forms the limiting step in the assembly process of a functional oligomer. Upon closer inspection, the occurrence of additional protrusions in the cavity, and sometimes at the side of some oligomers, became apparent (galleys of Figs 5 and 6). It may be assumed that these protrusions rep- resent one or more c subunits attached to the ring- shaped oligomer. Such a finding is in agreement with the observation presented in Fig. 4, in which the com- plete c subunit oligomers, hosting additional c sub- units, migrate at higher molecular weights in the SDS gel electrophoresis. Furthermore, it also corresponds to the recent observation that the analogous rotor from chloroplast F-ATP synthase may accommodate small transmembrane proteins within its central cavity [21]. Fig. 6. Atomic force microscopy (AFM) topographs of c subunit oligomers from Propionigenium modestum F-ATP synthase overexpressed in Escherichia coli. The oligomers were reconstituted into the lipid bilayer and imaged in buffer solution. (A) Survey of undecameric oligo- mers assembled into a 2D crystal. The donut-shaped oligomers were inserted into the membrane exposing either one of their surfaces to the AFM stylus. (B) A survey of densely assembled oligomers. Arrows point out oligomers either missing one subunit or showing additional subunits inside their central cavities. (C) A gallery of c rings observed from the densely packed arrangement. The first topograph represents a reference-free single particle average obtained from more than 200 c rings. Most of the examples selected exhibit additional central protru- sions. (D) A gallery of c rings observed from the crystalline arrangement. Examples selected exhibit additional central protrusions. The dashed circles with a diameter of 5.7 nm demonstrate that the outer diameter of the c rings is very consistent with each other. Topographs exhibit a gray scale corresponding to a vertical height of 3 nm. T. Meier et al. Structural evidence for a constant c 11 ring stoichiometry FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5479 Conclusion Recently, the crystal structure of the rotor ring from the I. tartaricus F-ATP synthase has been solved and provides striking details concerning the mechanism of the F o motor of ATP synthase [8]. Of particular interest in this structure is the architecture of the Na + -binding site, which closely resembles that of the k ring from the E. hirae V-ATPase [9]. As a result of its extreme stability [22], the c 11 rotor ring from the Na + -translocating F-ATP synthase from I. tartaricus seems to be particularly suitable for structural investi- gations. For a more detailed characterization of this system, and to increase experimental options, we have now investigated the aggregation behavior of the c 11 ring isolated from wild-type I. tartaricus cells, and we have explored the subunit c assembly of the protein expressed heterologously in E. coli. Under all investi- gated conditions, these assemblies were found to con- sist exclusively of rings of uniform size, allowing tight packaging of 11 monomeric units. In accordance with previous observations, some of these rings had gaps indicative of the absence of, in most cases, one c sub- unit [16]. As these rings had the same diameter as the c 11 rings, they were regarded as incompletely assembled. In preparations derived from recombinant E. coli cells, the incomplete assemblies were more abundant than in preparations derived from wild-type I. tartaricus cells. In both wild-type and recombinant preparations, the majority of the incomplete rings lacked only one monomer. It can therefore be conclu- ded that the insertion of the last monomer is the lim- iting step in the assembly of the ring. Whether this step, which is particularly demanding, requires a spe- cific assembly factor, is completely unknown. A can- didate for such a factor is the membrane insertion protein, YidC, which was recently shown to be required for in vitro assembly of the c ring from E. coli [23]. Irrespective of the assembly mechanism, our results clearly show that the size of the ring is not changed by massive overexpression of subunit c in the E. coli host cells, indicating that intrinsic fea- tures of the monomeric unit determine the number of subunits that can be packed into the ring. These data are fully compatible with the recent finding that the stoichiometry of the subunit III cylinder within the ATP synthase of the green algae, C. reinhardtii,is not affected by the metabolic state of the cells [18]. However, such findings are difficult to reconcile with the proposed variation of c ring stoichiometries in E. coli, which are dependent on the expression level or the nutritional status of the cells [15]. Knowledge on the aggregation behavior of the c ring has been of considerable value in exploring suitable crystallization conditions for structure determination. Two types of aggregates had to be taken into account. The first were supercomplexes of the (c 11 ) n type. The formation of these supercomplexes is dependent on the detergent because they are formed in octylglucoside, but not in Triton X-100. These supercomplexes dis- aggregate completely into the c 11 rings in the presence of SDS. The second type of aggregate appears as a ladder above the original c 11 band on SDS ⁄ PAGE and consists of c 11 rings hosting varying amounts of the c monomer. Aggregates are particularly abundant in c ring preparations from E. coli expression clones where the c monomer is present in high amounts. Once these aggregates were formed they remained stable and were minimally influenced by additives such as deter- gents, organic solvents, salts or lipids (like 1-palmitoyl- 2-oleyl-sn-glycero-3-phosphocholine). AFM topographs of these samples showed an exclusively undecameric stoichiometry in the completely assembled rings. This is also observed in the noncrystalline areas of the reconstituted vesicles, demonstrating that it is not an artifact from the 2D crystallization. Slower migrating bands of c rings, as observed on SDS gels, suggest that a certain fraction of c rings may host additional subunits. The AFM topographs indi- cate that these additional subunits may be hosted at the outer sides and within the central cavities of the rings. That these bands are composed exclusively of c sub- units has been proven by biochemical analyses and, in addition, the formation of these aggregates from pure c 11 and c 1 has been demonstrated in the present study. Experimental procedures Materials Chemicals were purchased from Fluka (Buchs, Switzerland) including lipase from Aspergillus oryzae. N-Lauroylsarcosine sodium salt and n-octyl-beta-d-glucopyranoside were pur- chased from Sigma (Buchs, Switzerland) and Glycon Biochemicals (Luckenwalde, Germany), respectively. Primers were custom synthesized by Microsynth (Balgach, Switzer- land). Phospholipase A2 from hog pancreas, and phospho- lipase C from Bacillus cereus, were purchased from Sigma (St Louis, MO, USA). Construction of plasmid pt7cIT The atpE gene from I. tartaricus [24] was amplified with Pfu polymerase and the following two primers: Structural evidence for a constant c 11 ring stoichiometry T. Meier et al. 5480 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5¢-GGAGGAAATAAGCATATGGATATG-3¢ (forward), containing an NdeI site, and 5¢-CCTTTCAGGAAGCT TCCTCC-3¢ (reverse), containing a HindIII site. The PCR product and plasmid pt7-7 [25] were digested with these two restriction enzymes and ligated before transformation into E. coli DH5a. Plasmid pt7c [26] was mutagenized with the Quick Change Site Directed Mutagenesis Kit (Strata- gene, La Jolla, CA, USA) to yield the single mutation, T67C, in the P. modestum subunit c (plasmid pt7cT67C). Synthesis and purification of c oligomers from strain BL21(DE3) transformed with various plasmids E. coli BL21(DE3) (Novagen, Madison, WI, USA) was transformed with plasmids pt7c, pt7cIT and pt7cT67C, as described above. The transformed E. coli cells were grown in 2 L of Luria–Bertani (LB) medium to reach an attenua- nce (D) of 0.6 at 37 °C in the presence of 200 lgÆmL )1 ampicillin. After cooling on ice for 5 min, the expression was induced with 0.7 mm isopropyl thio-b-d-galactoside and allowed to continue for 6 h at 30 °C, to yield typically 2.5 g of cells per L of medium. The cells (1 g wet weight) were suspended in 8 mL of 50 mm potassium phosphate buffer, pH 8.0, containing 1 mm 1,4-dithio-dl-threitol, 0.1 mm diisopropylfluorophosphate and a spatula tip of DNaseI. Preparation of membranes was performed at 4 °C. The cell suspension was passed twice through a French pressure cell at 12 000 psi (8.3 · 10 4 kPa). After the removal of cell debris by centrifugation at 15 000 g for 20 min, ultracentrifugation was performed at 200 000 g for 60 min. The membrane pellet was washed once with 4 mL of 20 mm Tris, 5 mm EDTA, and then adjusted to pH 8.0 with HCl. Solubilization of the membranes was accom- plished with 2 mL of 20 mm Tris ⁄ HCl, pH 8.0, containing 5mm EDTA and 1% (w ⁄ v) N-lauroylsarcosine for 10 min at 65 °C. After ultracentrifugation at room temperature, the pellet was discarded and contaminating membrane pro- teins were precipitated with (NH 4 ) 2 SO 4 at 65% (w ⁄ v) sat- uration. After 20 min of incubation at 20 °C, the sample was centrifuged for 20 min at 39 000 g. The filtrated super- natant containing the c oligomer was dialysed against 5 L of 10 mm Tris buffer, which was adjusted to pH 8.0 with HCl using a dialysis membrane with a molecular cut-off of 6000 Da. The protein sample was concentrated by ultrafiltration with Centricon tubes YM-10 (Millipore, Billerica, MA, USA) to a concentration of 1 mgÆmL )1 and applied to the top of a density gradient (5 mL) of 5–30% sucrose con- taining 20 mm Tris ⁄ HCl, pH 8.0, 10 lm 1,4-dithio-dl-thre- itol and 1% (w ⁄ v) octylglucoside. After ultracentrifugation (4 °C, 16 h, 150 000 g) in a Beckman SW55-Ti rotor (Beckman, Coulter, Inc., Fullerton, CA, USA), fractions of 0.5 mL were collected from the top and analysed by SDS ⁄ PAGE [27]. The c ring-containing samples were pooled and concentrated by ultracentrifugation (18 h, 200 000 g,4°C). The final protein concentration was typ- ically between 1.5 and 3 mgÆmL )1 . Fractions containing the monomeric c subunit were also collected and used to study the association with c 11 rings to stable c 11 (c 1 ) n aggregates. Reconstitution of densely packed and 2D crystalline c ring samples The c rings purified from these expression cultures were crystallized in two dimensions by mixing octylglucoside-sol- ubilized protein with 1 mgÆmL )1 1-palmitoyl-2-oleyl-sn-gly- cero-3-phosphocholine at a lipid : protein ratio of 0.8 (w ⁄ w) in a total volume of 50 lL, followed by dialysis for 24 h at 25 ° C against 200 mL of buffer (10 mm Tris ⁄ HCl, pH 7.5, containing 200 mm NaCl and 0.02% NaN 3 ), then for another 24 h at 37 °C. The crystals were stored at 4 °C for further analysis. Subunit c monomers solubilized in chloroform ⁄ methanol were purified according to the proce- dure described previously [26]. Purification of pure c 11 without supercomplexes and attached monomers The c ring was isolated from wild-type I. tartaricus cells as previously described [20]. One milligram of the protein was loaded onto a preparative SDS-containing gel, according to Scha ¨ gger et al. [27], together with a prestained marker. After the run, the c 11 ring was visible, without staining, as a result of the high local protein concentration. The band was excised from the gel with a scalpel and subjected to electroelution (at 25 mA) for 6 h at 4 °C. Phospholipase A 2 , phospholipase C and lipase digestions Eighty micrograms of purified subunit c 11 ring from I. tar- taricus [20] was incubated for 14 h at 37 °C with 10 U of phospholipase A2, 2 U of phospholipase C or 5 U of lipase, in the presence of 50 mm Tris ⁄ HCl buffer with a pH adjusted to 8.0, 7.2 or 8.0, respectively, and 1.5% (w ⁄ v) octylglucoside. Blue Native PAGE Blue Native PAGE was carried out as described previously [28]. Separation gels with a linear gradient of 5–17% acryl- amide were prepared and overlayed with 4% sample gels. Samples of 2–5 lg protein each were mixed with sample buffer [50 mm Tris ⁄ HCl, pH 6.8, containing 12% (v ⁄ v) gly- cerol and 0.01% (w ⁄ v) Serva blue G]. After running for 1 h at 100 V with cathode buffer (50 mm Tricine, 15 mm Bis- Tris ⁄ HCl, pH 7.0) containing 0.02% (w ⁄ v) Serva blue G, T. Meier et al. Structural evidence for a constant c 11 ring stoichiometry FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5481 the cathode buffer was replaced with buffer containing only 0.002% (w ⁄ v) Serva blue G and the run continued at 400 V. Native protein complexes were then analyzed by SDS ⁄ PAGE, as described previously [27], with the lanes from the Blue Native PAGE embedded into a 4% stacking gel. Atomic force microscopy The samples were diluted to a concentration of 10 lgÆmL )1 in 200 mm NaCl, 10 mm Tris ⁄ HCl, pH 7.5. To allow adsorption of the membranes, a drop of 30 lL was placed onto freshly cleaved mica. After an adsorption time of 15 min, the sample was gently washed using the above buffer solution containing no membrane proteins to remove weakly attached material from the mica surface. Contact mode AFM topographs were then recorded in the same buffer, at room temperature, at forces of < 100 pN applied to the AFM stylus, and at scanning line frequencies of typically 4–6 Hz. The AFM used was a Nanoscope E (Digital Instruments, Santa Barbara, CA, USA) equipped with a 120 lm piezo scanner and a fluid cell. Cantilevers (Olympus, Tokyo, Japan) had oxide-sharpened Si 3 N 4 tips and a spring constant of 0.09 NÆm )1 . No differences between topographs recorded simultaneously in trace and in the retrace direction were observed, indicating that the scanning process did not influence the appearance of the biological sample. AFM data analysis and image processing Individual particles of the AFM topographs were selected manually and subjected to reference-free alignment and averaging using the SPIDER image processing system (Wadsworth Laboratories, New York, NY, USA). Refer- ence-free averages generated by translational and rotational alignment of single particles enhanced common structural features among the c oligomers (Figs 5 and 6). To assess the rotor symmetry, the rotational power spectrum of the averaged image was calculated using the semper image processing system (Synoptics Ltd, Cambridge, UK). Alter- natively, the rotational power spectrum of each individual particle was calculated and then averaged (data not shown). It appeared that all averaged classes showed a stoi- chiometry of 11 subunits except for those of defect parti- cles. The diameter of intact and defective c rings was determined as described previously [16]. Other methods Gels were stained with silver [29]. The protein concentra- tion of samples was determined according to the bicinchon- inic acid method [30] with bovine serum albumin as the standard. Acknowledgements The authors thank Marijke Koppenol for critically reading the manuscript. 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Structural evidence for a constant c 11 ring stoichiometry FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5483 . the amount of the stable c 11 aggregates. A similar observation was made after incubating the sample for 5 min at 95 °Cin SDS-containing loading buffer;. the retrace direction were observed, indicating that the scanning process did not in uence the appearance of the biological sample. AFM data analysis and