ATP synthase from human p 0 (rho zero) cells was almost fully assembled in spite of the absence of subunits a and A6L using clear native electrophoresis (CNE or CN-PAGE). From this we conclude that subunits a and A6L are the last subunits to complete the ATP synthase assembly. Under the CNE conditions small amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was compar- able to the reduced amounts of dimeric and tetrameric ATP synthases from yeast subunit e and g null mutants detected by CNE. The dimer/ oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF 1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer whereas nonprocessed pre-IF 1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in p 0 cells and not to the dimeric form. This supports previous suggestions that IF 1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function. doi:10.1016/j.bbabio.2010.04.135 2P.39 Resolving stepping rotation of V-ATPase with an essentially drag-free probe Shou Furuike 1,2 , Masahiro Nakano 3,4 , Kengo Adachi 1 , Hiroyuki Noji 4 , Kazuhiko Kinosita Jr. 1 , Ken Yokoyama 3,5 1 Department of Physics, Faculty of Science and Engineering, Waseda University, Japan 2 Department of physics, Osaka medical college, Japan 3 Chemical Resources Laboratory, Tokyo Institute of Technology, Japan 4 Institute of Scientific and Industrial Research, Osaka University, Japan 5 ICORP, ATP Synthesis Regulation Project, Japan Science and Technology Agency (JST), Japan E-mail: yokoyama.k.ab@m.titech.ac.jp Vacuole-type ATPases (V- or VoV1-ATPases), together with F O F 1 ATP synthases, constitute a superfamily of rotary molecular machines that couple ATP hydrolysis/synthesis in the soluble V 1 /F 1 portion with proton (or Na + ) flow in the membrane-embedded V O /F O portion through rotation of a common central shaft. Here we have observed at submillisecond resolutions the ATP-driven rotation of isolated V 1 and of the whole V O V 1 from Thermus thermophilus, by attaching a 40-nm gold bead for which viscous drag is almost negligible. At saturating ATP of 4 mM, V 1 rotated at about 60 revolu- tions/s, with about 5 ms dwells every 120°. Dwell time analyses indicated that at least two events other than ATP binding, one likely ATP hydrolysis, occur in each dwell, as in F 1 . Unlike F 1 , however, the dwells were at ATP-waiting positions that were resolved at μM ATP. V O V 1 rotated an order of magnitude slower, and exhibited dwells separated by about 30°. The twelve positions, though not always fully populated, match the twelve-fold symmetry of the V O rotor in T. thermophilus, indicating that the ATP-driven rotation must go through stator–rotor interactions in V O . doi:10.1016/j.bbabio.2010.04.136 2P.40 Heterologous expression of the peripheral stalk Aquifex aeolicus F 1 F O ATP synthase in Escherichia coli Chunli Zhang, Guohong Peng, Hartmut Michel Max Planck Institute of Biophysics, Department of Molecular Membrane Biology, Frankfurt am Main, Germany E-mail: Hartmut.Michel@biophys.mpg.de The hyperthermophilic bacterium Aquifex aeolicus possesses a nine- subunit F 1 F O ATP synthase [1]. A part of the complex, called the peripheral stalk, provides the connection between the membrane embedded F O part and the soluble F 1 part, acting as a stator to counteract the rotation of the catalytic F 1 part during ATP synthesis. Structural information is available to date for the peripheral stalk subunits of the bovine mitochondrial F 1 F O ATP synthase and the Thermus thermophilus A 1 A O ATP synthase, respectively [2–5]. However, further structural characterization is necessary because the peripheral stalk is the least conserved component of the complex, differing substantially in composition and stoichiometry among ATP synthase subtypes [5]. In particular, in A. aeolicus, the peripheral stalk is exceptional because it is hetero- and not homodimeric and so it differs from that of all other currently known F 1 F O ATP synthases of non-photosynthetic organisms [1]. It mainly contains subunits b 1 and b 2 , encoded by genes aq_1586 and aq_1587, which overlap by 1 bp in the genome. We have cloned the two genes and expressed the b 1 /b 2 subunits heterologously in Escherichia coli. They localize both in E. coli membranes and inclusion bodies. Two- dimensional Blue native (2-D BN)/SDS-PAGE, together with peptide mass fingerprint mass spectrometry (PMF-MS) shows that they form a complex in E. coli membranes. The b 1 /b 2 complex can be isolated from the membranes to a high level of purity in a single chromatographic step. Further studies are in progress to optimize the expression level and to characterize the folding and stability of the b 1 /b 2 complex by size exclusion chromatography, circular dichroism and differential scanning calorimetry. The final aim of the project is the determination of the structure of the b 1 /b 2 complex by 3-D crystallography. References [1] Peng G et al. (2006) FEBS Lett. 42: 5934–5940. [2] Dickson VK et al. (2006) EMBO J. 25: 2911–2918. [3] Carbajo RJ, et al. (2007) JMB 368: 310–318. [4] Rees DM et al. (2009) Proc. Natl. Acad. Sci. 106: 21597–21601. [5] Lee LK et al. (2010) Nat. Struct. Mol. Biol. 17: 373–378. doi:10.1016/j.bbabio.2010.04.137 Abstracts 40