The role of membrane scaffolds for the spatial organization and heterogeneity of the mitochondrial inner membrane

线粒体内膜是一种富含蛋白质的复合膜结构，是细胞内重要的代谢屏障，具有多种代谢过程。不同的功能在形态定义的区域中执行，例如蛋白质在内边界膜上的转位或通过线粒体内的OXPHOS复合体产生ATP。此外，越来越多的证据表明，内膜在纳米尺度上存在巨大的功能异质性。SPFH家族的多聚膜支架蛋白--禁止素(PHB1，PHB2)和类口腔蛋白SLP2在内膜形成大的环状复合体，被认为是脂类和蛋白质类支架。这些支架蛋白与AAA蛋白酶在相对的膜表面组装，构成了内膜的蛋白分解中心。PHB膜结构域的缺失严重损害了线粒体的功能，并与神经退行性变、肥胖和肾衰竭有关。在上一次资助期间，我们已经确定TMBIM5(GHITM，MICS1)是PHB膜结构域的一个新成分。我们的功能分析表明，TMBIM5在内膜既是钙/H+交换者，也是m-AAA蛋白酶的抑制者。因此，TMBIM5将线粒体质子梯度与蛋白质周转率结合起来，重塑线粒体蛋白质组并调节细胞新陈代谢。这些发现表明，空间受限的、局部的蛋白分解有助于内膜的功能和结构的异质性。因此，我们将研究膜支架和TMBIM5依赖的质子梯度在内膜蛋白分解的空间调节中的作用。我们将应用各种先进的蛋白质组学方法来监测PHB膜结构域的局部蛋白降解，定义这些结构域中的蛋白质拓扑结构，并研究TMBIM5和质子基序作用力对内膜蛋白稳定性的影响。总之，我们的实验将进一步定义将蛋白质周转与线粒体的能量状态相耦合的调节电路，并揭示由膜支架调节的局部蛋白质降解如何有助于内膜的功能异质性。

The mitochondrial inner membrane is a protein-rich, complex membrane structure, which serves as an important metabolic barrier in cells and houses numerous metabolic processes. Distinct functions are carried out in morphologically defined regions, such as protein translocation in the inner boundary membrane or the production of ATP via OXPHOS complexes in mitochondrial cristae. Moreover, increasing evidence suggests large functional heterogeneity at the nanoscale within the inner membrane. Multimeric membrane scaffold proteins of the SPFH-family, prohibitins (PHB1, PHB2) and the stomatin-like protein SLP2, form large ring complexes in the inner membrane, which are proposed to serve as lipid and protein scaffolds. These scaffold proteins assemble with AAA proteases at opposite membrane surfaces constituting proteolytic hubs in the inner membrane. Loss of PHB membrane domains severely impairs mitochondrial functions and is associated with neurodegeneration, obesity and kidney failure. During the last funding period, we have identified TMBIM5 (GHITM, MICS1) as a novel constituent of PHB membrane domains. Our functional analysis revealed that TMBIM5 acts both as a Ca2+/H+ exchanger and inhibitor of the m-AAA protease in the inner membrane. TMBIM5 thus couples the mitochondrial proton gradient with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism. These findings suggest that spatially restricted, localized proteolysis contributes to the functional and structural heterogeneity of the inner membrane. We will therefore examine the role of membrane scaffolds and the TMBIM5-dependent proton gradient for the spatial regulation of proteolysis in the inner membrane. We will apply various advanced proteomic approaches to monitor localized proteolysis at PHB membrane domains, define the protein topology within these domains and examine the effect of TMBIM5 and the proton motif force on the stability of inner membrane proteins. Together, our experiments will further define regulatory circuits coupling protein turnover to the energetic status of mitochondria and unravel how localized proteolysis regulated by membrane scaffolds contributes to the functional heterogeneity of the inner membrane.