Mechanisms of Ion Selection in P-type ATPases

许多重要的细胞和生理事件，包括营养摄取、信号转导和细胞周期进程都是由跨膜离子梯度介导的。阳离子泵的一个广泛的多基因家族，P-ATP酶，已经进化为运输各种不同的离子（仅举几例，Ca 2+、Na+、K+、H+、Mg 2+、Cu 2+）。 与它们的基本作用一致，P-ATP酶是药物干预疾病（如充血性心力衰竭和胃溃疡）的靶标，并且在各种遗传性疾病（Menkes、Wilson、Brody和Hailey-Hailey病）中是有缺陷的。 尽管该家族在序列、结构和机制上具有相似性，但单个成员在离子选择性上存在显著差异。离子泵中选择性的分子基础仍然是膜生物能学领域中未回答的基本问题之一。 为了解决这个问题，我们将：专注于高尔基体Ca 2+，Mn 2 +-ATP酶，Pmrl，在遗传上易处理的生物酵母，应用简单而强大的表型筛选，将识别功能丧失或选择性突变，开发严格的生化工具来分析有缺陷的泵。在目的1中，我们将鉴定酵母Pmrl中二价阳离子选择性的分子决定因素，Pmrl是新定义的高尔基体/分泌途径Ca 2 +-ATP酶亚组的创始成员。具体而言，我们将重点关注Mn 2+与Ca 2+离子的选择性。 在一种方法中，我们将使用定向和随机诱变技术结合生物测定的Ca 2+螯合剂和Mn 2+毒性，以确定突变，改变离子选择性。 在第二种方法中，我们将使用酵母Pmrl的同源性建模，基于已知的SERCA泵的晶体结构，以设计用于诱变的合理靶标。靶残基将包括预测为离子传导途径的衬里、稳定相邻膜螺旋或形成结构域界面的那些。 在目标2中，将进一步诱变具有感兴趣的特性（例如离子选择性改变或ATP酶水解与离子转运解偶联）的功能丧失突变体，并对其进行表型选择以鉴定基因内抑制突变。 这些将提供独特的洞察力域之间的关键相互作用，和膜螺旋内或之间，这将补充离子泵的结构信息。 在目标3中，将从发酵罐生长的巴斯德毕赤酵母培养物中大规模纯化Pmrl，用于阳离子结合和伴随的构象变化的结构研究。 总之，这些目标构成了一个强大的方法，对破译的选择性和离子泵中的运输的分子基础。

Many important cellular and physiological events, including nutrient uptake, signal transduction and cell cycle progression are mediated by transmembrane ion gradients. An extensive, multigene family of cation pumps, the P-ATPases, have evolved to transport a wide variety of different ions (Ca2+, Na+, K+, H+, Mg2+, Cu2+, to name a few). In keeping with their essential roles, the P-ATPases are a target for pharmacological intervention in disease (such as congestive heart failure and stomach ulcers), and are defective in various inherited disorders (Menkes, Wilson, Brody and Hailey-Hailey disease). Despite the similarities in sequence, structure and mechanism within this family, individual members differ strikingly in ion selectivity. The molecular basis of selectivity in ion pumps remains one of the fundamental unanswered problems in the field of membrane bioenergetics. To approach this problem, we will: focus on the Golgi Ca2+, Mn2+-ATPase, Pmrl, in the genetically tractable organism yeast, apply simple and powerful phenotypic screens that will identify loss of function or selectivity mutations, develop rigorous biochemical tools to analyze the defective pumps. In Aim 1, we will identify the molecular determinants of divalent cation selectivity in yeast Pmrl, a founding member of the newly- defined subgroup of Golgi/secretory pathway Ca2+-ATPases. Specifically, we will focus on selectivity for Mn2+ versus Ca2+ ions. In one approach, we will use directed and random mutagenesis techniques in conjunction with biological assays for Ca2+ chelator and Mn2+ toxicity to identify mutations that alter ion selectivity. In a second approach, we will use homology modeling of yeast Pmrl, based on the known crystal structure of the SERCA pump, to design rational targets for mutagenesis. Target residues will include those predicted to line the ion conducting pathway, stabilize adjacent membrane helices or form domain interfaces. In Aim 2, loss-of-function mutants with interesting properties such as alterations in ion selectivity or uncoupling of ATPase hydrolysis from ion transport, will be further mutagenized and subjected to phenotypic selection in order to identify intragenic suppressor mutations. These will provide unique insight on critical interactions between domains, and within or between membrane helices, that will complement structural information on ion pumps. In Aim 3, large-scale purification of Pmrl from fermentor-grown Pichia pastoris cultures will be undertaken for structural studies on cation binding and the concomitant conformational changes. Taken together, these aims constitute a powerful approach toward deciphering the molecular basis of selectivity and transport in ion pumps.