Supramolecular organization of ATP synthase and protonic energy coupling

线粒体三磷酸腺苷合成酶是线粒体和细胞生理学的关键酶。来自氧化代谢的能量储存在细胞器内膜的质子动力(PMF)中。ATP合成酶(CV)利用PMF产生ATP，但也有助于维持PMF处于不利条件下。人三磷酸腺苷合成酶是由29个亚基组成的复合体(包括调节蛋白IF1)。该旋转机由膜部(F0)和膜-外部部(F1)组成，膜部由中央和外围的柄连接。线粒体F1FO-ATP合成酶是以二聚体的形式组织起来的，这些二聚体沿着眉骨的边缘形成一排排。这种特殊的超分子结构也有助于形成和稳定特定的冠状结构，紧随其后的是与冠状连接相关的蛋白质复合体OPA1和MICOS复合体。分别报道了三磷酸腺苷合成酶和OPA1、MICOS化合物之间的功能和物理相互作用，这表明形成冠状突的蛋白质之间存在串扰。E和g亚基促进了ATP合成酶的二聚化，而DAPIT/USMG5亚基可能参与了二聚体-二聚体的稳定。同样，抑制因子IF1除了具有调节三磷酸腺苷合成酶活性的功能外，还促进二聚体-二聚体的相互作用。我们最近还发现，IF1还决定了ATP合成酶的时空组织和它在鸡冠膜上的迁移率。在即将到来的资助期，我们的目标是更好地了解ATP合成酶的超分子组织与其酶活性之间的关系。为此，我们将通过改变DAPIT-和IF1亚基的浓度来操纵ATP合成酶的寡聚。使用pH、m和ATP值的荧光生物传感器将在活细胞中测量CV寡聚减少对酶活性的影响。我们推测，寡聚体结构的破坏会导致酶的不稳定和向ATP水解酶的转变，这可能也涉及到冠状突超微结构的变化。这可以通过上调IF1来抵消。与P02合作，我们将研究DAPIT/ATP合成酶与MICOS复合体，特别是Mic10亚单位的串扰。我们的细胞系将用于该联盟内的进一步研究(P05，P06，P07)，以进一步研究超分子ATP组织和MICOS之间的串扰。

Mitochondrial ATP synthase is an enzyme pivotal for mitochondrial and cellular physiology. Energy derived from oxidative metabolism is stored in a proton-motive force (PMF) across the inner membrane of the organelle. The ATP synthase (CV) harness the PMF to generate ATP but also contributes to maintain the PMF under unfavourable conditions. Human ATP synthase is a complex of 29 subunits (including the regulatory protein IF1). The rotary machine consists of a membrane part (F0) and membrane-extrinsic (F1) part linked by central and peripheral stalks. Mitochondrial F1FO ATP synthase is organized in dimers that form rows along the rims of cristae. This specific supramolecular structure also contributes to the formation and stabilization of the specific cristae architecture, next to the cristae-junction related protein complexes OPA1 and the MICOS complex. Functional and physical interaction between ATP synthase and OPA1, respectively MICOS compounds were reported, suggesting a crosstalk between the cristae-shaping proteins. The dimerization of ATP synthase is promoted by subunits e and g, while the subunit DAPIT/USMG5 is likely involved in dimer-dimer stabilization. Likewise, the inhibitory factor IF1 also promotes dimer-dimer interaction in addition to its ATP synthase activity-regulating function. We recently showed that also the spatiotemporal organization of ATP synthase and its mobility in cristae membranes is determined by IF1. In the upcoming funding period, we aim to better understand the relationship between the supramolecular organization of ATP synthase and its enzyme activity. To this end, we will manipulate the oligomerization of ATP synthase via changing concentrations of the DAPIT- and IF1 subunits. The effects of decreased CV oligomerization on enzyme activity will be measured in living cells using fluorescent biosensors for pH, m, and ATP. We hypothesize that disruption of oligomeric structure leads to enzyme instability and a shift toward ATP hydrolysis, likely also involving ultrastructural changes of cristae. This could be counterbalanced by upregulation of IF1. In collaboration with P02, we will study the crosstalk of DAPIT/ATP synthase with the MICOS complex, specifically the Mic10 subunit. Our cell lines will be made available for additional studies within the consortium (P05, P06, P07) to further investigate the crosstalk between supramolecular ATP organization and MICOS.