Carraro Michela, Checchetto Vanessa, Sartori Geppo, Kucharczyk Roza, di Rago Jean-Paul, Minervini Giovanni, Franchin Cinzia, Arrigoni Giorgio, Giorgio Valentina, Petronilli Valeria, Tosatto Silvio C E, Lippe Giovanna, Szabó Ildikó, Bernardi Paolo
Department of Biomedical Sciences, University of Padova, Padova, Italy.
Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Padova, Italy.
Cell Physiol Biochem. 2018;50(5):1840-1855. doi: 10.1159/000494864. Epub 2018 Nov 13.
BACKGROUND/AIMS: The permeability transition pore (PTP) is an unselective, Ca2+-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca2+-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation.
Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers.
Null mutants for the subunits e and g display dimer formation upon Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity.
F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.
背景/目的：通透性转换孔（PTP）是线粒体内膜上一种非选择性的、依赖Ca2+的高电导通道，其分子身份长期以来一直是个谜。最新的假说是孔的形成涉及F-ATP合酶，它持续产生Ca2+激活的通道。现有的结构并未显示出能够容纳通道的明显特征；因此，孔是如何形成的以及其活性是否能完全归因于F-ATP合酶存在争议。在本研究中，我们研究了F-ATP合酶亚基e、g和b在PTP形成中的作用。
构建了亚基e、g和亚基b的第一个跨膜（TM）α螺旋的酵母缺失突变体，并对其线粒体形态（电子显微镜）、膜电位（罗丹明123荧光）和呼吸作用（克拉克电极）进行了评估。通过体外诱变和生物弹道转化产生了亚基a的同质性C23S突变体。通过蓝绿温和聚丙烯酰胺凝胶电泳（BN-PAGE）分析评估F-ATP合酶组装情况。在缺乏亚基e和g的情况下，使用Cu2+处理诱导F-ATP合酶二聚体的形成。在平面脂质双分子层中评估F-ATP合酶的电生理特性。
亚基e和g的缺失突变体在Cu2+处理后显示二聚体形成，并表现出依赖PTP的线粒体Ca2+释放，但无肿胀现象。Cu2+处理导致亚基a的Cys23之间形成二硫键，在缺乏亚基e和g的情况下稳定二聚体，并有利于野生型F-ATP合酶通道的开放状态。缺乏亚基e和g会使F-ATP合酶通道的电导降低约10倍。亚基b的第一个TM缺失，形成了一个与亚基e和g不同的侧向结构域，进一步影响了通道活性。
F-ATP合酶亚基e、g和b在膜内形成一个对高电导通道的产生至关重要的结构域，因此是PTP形成的主要候选者。亚基e和g仅存在于真核生物中，可能已经进化以使F-ATP合酶具有这种新功能。
