![]() ![]() In yeasts and mammals, subunit a is mitochondrially-encoded (known as ATP6 in human mitochondria), whereas in Chlamydomonas reinhardtii and, presumably, its close relative Polytomella, it is nuclear-encoded ( Funes et al., 2002). Sequences of functionally important subunit a regions are highly conserved ( Figure 1-figure supplement 3) and likely to have similar structures. The remaining eight ASA subunits in the peripheral stalks, the central stalks and catalytic F 1 heads are shown in transparent cyan. Showing the two c-rings (yellow), subunits a (blue) and ASA 6 (brick). distortion corr.)ģD map classification and refinement software Like the a-subunit helix hairpins themselves, the channels appear to be conserved in all rotary ATPases ( Kühlbrandt and Davies, 2016). Two proton channels were proposed to provide access to the c-ring protonation sites ( Vik and Antonio, 1994) and first observed in the 6.2 Å Polytomella structure ( Allegretti et al., 2015). ![]() Subsequent structures of F-type ( Guo et al., 2017 Hahn et al., 2016 Morales-Rios et al., 2015) and V-type ATPases ( Mazhab-Jafari et al., 2016) at 3.7 to 7 Å resolution have shown that the long membrane-intrinsic helix hairpins are a conserved and apparently essential feature of all rotary ATPases ( Kühlbrandt and Davies, 2016), but the reason for this was not understood until now. The longest helix bends around the c-ring, positioning the strictly conserved aArg239 and other key subunit a residues next to the c-subunit protonation site. The helix hairpins run roughly at right angles to the c-ring helices. The previous 6.2 Å cryo-EM map of the Polytomella ATP synthase dimer indicated two long, membrane-intrinsic helix hairpins in subunit a ( Allegretti et al., 2015), but did not resolve sidechains. A conserved glutamate ( cGlu111 in Polytomella) serves as the c-subunit proton-binding site ( Meier et al., 2005 Pogoryelov et al., 2009). Mammalian mitochondria have a c 8-ring, while yeasts ( Hahn et al., 2016 Stock et al., 1999) and Polytomella ( Allegretti et al., 2015) have 10 c-ring subunits, which we refer to as cA to cJ. Rotor rings of F-type ATP synthases consist of 8 ( Watt et al., 2010 Zhou et al., 2015) to 15 ( Pogoryelov et al., 2009) identical c-subunits that each form a hydrophobic helix hairpin. Protonation and deprotonation of these glutamates drives ring rotation and ATP synthesis. Our structure reveals two prominent aqueous channels, each spanning one half of the membrane, that conduct protons to and from the conserved glutamates in the rotor ring. We have determined the structure of the complete mitochondrial ATP synthase dimer from the unicellular green alga Polytomella sp. The recent cryo-EM structure of the F o subcomplex dimer isolated from yeast mitochondria ( Guo et al., 2017) indicated the positions of key residues in the proton pathway. Until now, no high-resolution structure of an intact, functionally competent mitochondrial ATP synthase has been reported. ![]() Understanding how this fundamental process generates rotary force requires an atomic model of the proton pathway. Rotation is driven by protons flowing down the membrane gradient through the F o subcomplex. ![]() ATP synthases consist of the catalytic F 1 head and the F o subcomplex in the membrane ( von Ballmoos et al., 2009). Mitochondrial ATP synthase uses the energy of the electrochemical proton gradient across the inner mitochondrial membrane to produce ATP from ADP and phosphate by rotary catalysis ( Abrahams et al., 1994 Gresser et al., 1982). ![]()
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