Characterization of the P5 Subfamily of P-type Transport ATPases in Mice

描述（由申请人提供）：p型atp酶，通过细胞膜运输离子，被分为五个亚家族。P1-， P2-和p3 - atp酶已被很好地表征，包括重金属atp酶，Na， k - atp酶，H， k - atp酶，SERCAs和其他Ca2+ atp酶，以及细菌Mg2+ atp酶。p4 - atp酶仅存在于真核生物中，并与氨基磷脂的转运有关。尽管它们存在于所有真核生物中，但人们对p5 - atp酶知之甚少，除了观察到酵母中两种p5 - atp酶之一Cod1p的缺失会导致糖蛋白加工缺陷、内质网相关蛋白降解（ERAD）和未折叠蛋白反应（UPR）的组成性激活。因此，我们的目标是获得关于哺乳动物p5 - atp酶的分布和离子特异性的基本信息。在初步研究中，我们已经鉴定了5种哺乳动物P5-ATPases，命名为Atp13a1-Atp13a5，确定了它们的序列和mRNA的组织分布，并开始开发同型特异性抗体和表达结构。在Aim 1中，我们将开发异构体特异性抗体和原位杂交探针，并使用组织和亚细胞膜组分的Western blot分析，免疫细胞化学和原位杂交相结合，确定小鼠Atp13a1-Atp13a5的膜位置和细胞类型分布。在Aim 2中，将确定小鼠P5-ATPases的离子特异性。将制备组氨酸标记Atp13a1-Atp13a5过表达的细胞系的膜组分，并进行洗涤剂溶解。然后通过镍亲和层析纯化his标记的转运体，并通过使用ATP酶活性和磷酸酶形成的敏感测定来测量阳离子依赖性ATP水解来确定离子特异性。离子摄取在整个细胞或分离的膜囊泡也将分析使用适当的放射性同位素，以确认离子特异性。这些研究将为更详细地研究哺乳动物p型atp酶亚家族的细胞生物学和生理功能提供重要的基础信息。本研究的主要目的是获得哺乳动物p5 - atp酶的分布和离子特异性的基本信息，以便更好地了解其细胞生物学和生理功能。考虑到p5 - atp酶在酵母和人类等多种生物体中的存在，以及相关转运体的突变导致达里尔病、海利病和威尔逊病等疾病，它们很可能也会被发现在人类健康和疾病中发挥关键作用。

DESCRIPTION (provided by applicant): P-type ATPases, which transport ions across cell membranes, have been grouped into five subfamilies. The P1-, P2-, and P3-ATPases have been well characterized and include heavy metal ATPases, Na,K-ATPases, H,K-ATPases, SERCAs and other Ca2+ATPases, and bacterial Mg2+ATPases. The P4-ATPases are found only in eukaryotes, and have been implicated in the transport of aminophospholipids. Although they are present in all eukaryotes, little is known about the P5-ATPases, other than the observations that loss of Cod1p, one of the two yeast P5-ATPases, leads to defects in glycoprotein processing, endoplasmic reticulum associated protein degradation (ERAD), and constitutive activation of the unfolded protein response (UPR). Thus, our objective is to obtain basic information regarding the distribution and ion specificity of the mammalian P5-ATPases. In preliminary studies we have identified 5 mammalian P5-ATPases, termed Atp13a1-Atp13a5, determined their sequences and mRNA tissue distribution, and begun development of isoform-specific antibodies and expression constructs. In Aim 1 we will develop isoform-specific antibodies and in situ hybridization probes, and determine the membrane location and cell-type distribution of mouse Atp13a1-Atp13a5 using a combination of Western blot analysis of tissues and subcellular membrane fractions, immunocytochemistry, and in situ hybridization. In Aim 2 the ion specificity of the murine P5-ATPases will be determined. Membrane fractions from cell lines in which histidine-tagged versions of Atp13a1-Atp13a5 are over-expressed will be prepared and subjected to detergent solubilization. The His-tagged transporters will then be purified by nickel affinity chromatography and ion specificities will be determined by measuring cation-dependent ATP hydrolysis using sensitive assays of ATPase activity and phosphoenzyme formation. Ion uptake in whole cells or isolated membrane vesicles will also be analyzed using the appropriate radioisotope to confirm ion specificity. These studies will provide basic information that will be critical for more detailed mechanistic studies of the cell biological and physiological functions of this most poorly understood subfamily of mammalian P-type ATPases. The primary goal of the proposed study is to obtain basic information regarding the distribution and ion specificity of the mammalian P5-ATPases in order to better understand their cell biological and physiological functions. Given the presence of P5-ATPases in organisms as diverse as yeast and humans and that mutations in related transporters result in Darier's, Hailey-Hailey, and Wilson's disease among others, it is likely that they will also be found to play a critical role in human health and disease.