Determinants of amino acid transporter oligomerization in membranes

摘要 氨基酸-多胺-有机阳离子(APC)超家族膜转运蛋白是 最大的溶质载体家族。它们参与营养和氨基酸在所有王国的运输。 生活。在人类中，它们对肾脏的氨基酸运输、突触的神经递质重新摄取、 物质跨越血脑屏障的运动，以及许多其他生理作用。虽然它们的功能 是不同的，它们采用了一种称为“Leut-折叠”的通用拓扑结构，包括一种倒置的 5+5个跨膜螺旋的拓扑重复，在C末端另外有2+个螺旋参与 某些异构体的齐聚作用。近年来，许多Leut-折叠同系物的结构已被解决 几乎在运输周期沿线的每一个州。在检查这些结构时，存在一个主要问题 Out-为什么观察到Leut-折叠转运蛋白以不同的寡聚体形式(即单体、二聚体和三聚体) 尽管拥有相同的亚基折叠？我们假设寡聚在APC转运蛋白中起作用。 所有的Leut-折叠转运蛋白都具有先天的二聚化潜能，这是由于 保守的亚基结构。然而，观察到的低聚物的多样性是由于不同的稳定性造成的 由于蛋白质和脂肪接触面的不同。为了研究这一点，我们将研究一对APC 运输商为这一问题的严格调查提供了一个模型系统。这些是 精氨酸/胍丁胺逆向转运蛋白ADIC和谷氨酸/GABA逆向转运蛋白GADC。这两个传送器是 在进化上与同一亚家族相关，并且都参与了大肠杆菌的极端耐酸性，然而， ADIC只作为二聚体被观察到，而GADC是单体。此外，这些蛋白质很容易 提纯并可在重组系统中进行功能研究，提供严格的模型系统进行研究 亮氨酸转运蛋白的二聚化决定因素。在这次调查中，我们将使用一种组合 跨学科的研究，使我们能够检查其他膜蛋白的二聚化稳定性 例如ClC Cl-/H+逆向转运体和双拓扑氟离子通道。计算研究将 画出蛋白质配对，与水或脂的相互作用，以及膜结构 表格。将进行单分子光漂白分析实验，以测量平衡 膜上亚基的二聚化亲和力，同时测试关键蛋白质和脂类变量 计算分析。在目标1中，我们将列出ADIC二聚化的定义，并在目标2中确定 定义使GADC成为单体的特征，并进行最终测试，将GADC设计为功能二聚体。这 对两种型号的Leut-Fold运输机进行严格的比较/对比研究将提供一个基本的 了解与所有APC转运体相关的寡聚机制，包括那些重要的转运体 在人类健康和疾病方面。

ABSTRACT The Amino Acid-Polyamine-organoCation (APC) superfamily of membrane transport proteins is one of the largest families of solute carriers. They are involved in nutrient and amino acid transport across all kingdoms of life. In humans, they are essential for amino acid transport in kidneys, neurotransmitter re-uptake in synapses, movement of substances across the blood-brain barrier, and many other physiological roles. While their functions are diverse, they adopt a common topological arrangement known as the "LeuT-fold", involving an inverted topology repeat of 5 + 5 transmembrane helices, with an additional 2+ helices at the C-terminus that engage in oligomerization in some isoforms. In recent years, structures of many LeuT-fold homologues have been solved and in nearly every state along the transport cycle. When examining these structures, a major question stands out - why are LeuT-fold transporters observed in diverse oligomeric forms (i.e. monomers, dimers and trimers) despite possessing the same subunit fold? We hypothesize that oligomerization plays a role in APC transporter regulation, and that all LeuT-fold transporters possess an innate potential to dimerize in membranes due to the conserved subunit structure. However, the diversity of observed oligomers arises because of different stabilities due to differences in protein and lipid contact surfaces. To investigate this, we will study a pair of APC transporters that provide a model system for the rigorous investigation of this question. These are the arginine/agmatine antiporter AdiC and glutamate/GABA antiporter GadC. These two transporters are evolutionarily related within the same sub-family, and are both involved in extreme acid resistance in E. coli, yet, AdiC has only ever been observed as a dimer, while GadC is monomeric. In addition, these proteins are easy to purify and can be studied functionally in reconstituted systems, providing rigorous model systems to study dimerization determinants of LeuT-fold amino-acid transporters. In this investigation, we will use a combination of interdisciplinary studies that have allowed us to examine dimerization stability of other membrane protein systems, such as the CLC Cl-/H+ antiporter and the dual-topology Fluc F- ion channel. Computational studies will map out protein partners, interactions with water or lipids, and membrane structure in associated and dissociated forms. Single-molecule photobleaching analysis experiments will be carried out to measure the equilibrium dimerization affinity of subunits in membranes while testing key protein and lipid variables identified in the computational analysis. In Aim 1, we will map out what defines AdiC dimerization, and in Aim 2, identify the defining features that make GadC monomeric, with a final test to engineer GadC as a functional dimer. This rigorous compare/contrast study between two model LeuT-fold transporters will provide a fundamental understanding of oligomerization mechanisms relevant to all APC transporters, including those that are important in human health and disease.