Assembly and structure of native PMCA-Neuroplastin/Basigin complexes

细胞内钙离子浓度的瞬时变化，通常被称为钙信号，是控制从递质释放、酶活性调节到激发-转录偶联、细胞运动和轴突生长等一系列细胞过程和反应途径的普遍机制。所有这些过程都是通过钙离子进入胞浆而启动的，而它们又被位于肌浆/内质网(SERCA)或质膜(PMCA)中的各种钙离子转运体，最重要的是钙-ATPase(或泵)的协同作用而关闭。在最近的蛋白质组学分析中，我们发现任何类型的细胞中的PMCA都是ATPase亚基(PMCAs1-4)和单跨膜蛋白神经纤溶素(NPTN)或basigin(BASI)的异构体复合体。后者与PMCA蛋白的共同组装对于复合体的稳定和运输以及有效的底物运输都是必不可少的。事实上，NPTN或BASI的结合增加了钙离子的转运速度，从而在几十毫秒内促进了钙的清除，而不是之前假设的几秒钟。PMCA与NPTN和BASI组装的结构基础目前尚不清楚，PMCA-NPTN/BASI核心复合体在不同组织中的相互作用伙伴也是如此。这项建议旨在全面了解PMCA-NPTN/BasI复合体的组装和结构及其在自然组织中的分子形态(S)。为此，我们将追求以下密切相关的目标和实验方法/步骤：(1)用冷冻EM(Aim1A)阐明PMCA2-NPTN和PMCA4-BASI复合体的三维结构，以及PMCA2处于辅助亚基自由‘apo’状态的PMCA2(Aim1A)；(2)通过一种新的基于MS的活性分析(Aim1B)测量纯化的PMCA2-NPTN/BASI复合体的ATP消耗(Aim1B)；(3)解开天然PMCA复合体(AIM2)的相互作用组。

Transient changes in the intracellular calcium (Ca2+) concentration, generally known as Ca2+ signaling, represents a prevalent mechanism to control a multitude of cellular processes and reaction pathways from transmitter release and regulation of enzymatic activities to excitation-transcription coupling, cell motility and neurite outgrowth. All these processes are switched on by influx of Ca2+ into the cytosol, and they are switched off by the concerted action of various Ca2+ transporters, most importantly Ca2+-ATPases (or pumps) that are located in the sarco/endoplasmic reticulum (SERCA) or the plasma membrane (PMCA). In recent proteomic analyses, we identified PMCAs in any type of cell as heteromeric complexes of ATPase subunits (PMCAs1-4) and the single-span membrane proteins Neuroplastin (NPTN) or Basigin (BASI). Co-assembly of the latter with the PMCA proteins is obligatory for both stabilization and trafficking of the complexes, as well as for effective substrate transport. In fact, binding of NPTN or BASI increases the rates of Ca2+-transport thus promoting Ca2+-clearing within tens of milliseconds rather than seconds, as previously assumed. The structural basis behind the assembly PMCAs with NPTN and BASI are currently unknown, as are the interaction partners of the PMCA- NPTN/ BASI core complexes in distinct tissues. This proposal aims at a comprehensive understanding of the assembly and structure of PMCA-NPTN/BASI complexes and their molecular appearance in native tissue(s). For this purpose we will pursue the following closely interrelated aims and experimental approaches/steps: (1) Elucidate 3D-structures of PMCA2-NPTN and PMCA4-BASI complexes, and of PMCA2 in auxiliary subunit-free ‘apo’ state by cryo-EM (Aim1A), (2) Measure ATP-consumption of purified PMCA2-NPTN/BASI complexes by a novel MS-based activity assay (Aim1B, (3) Unravel the interactome of native PMCA complexes (Aim2).