SCHULTHEIS POST-DOC/TECHNICIAN SUPPORT

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 P 型 ATP 酶可跨细胞膜运输离子，已分为五个亚家族。 P1-、P2-和 P3-ATP 酶已得到很好的表征，包括重金属 ATP 酶、Na,K-ATP 酶、H,K-ATP 酶、SERCA 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，并且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，除了观察到两种酵母 P5-ATP 酶之一 Cod1p 的缺失会导致糖蛋白加工、内质网相关蛋白降解 (ERAD) 和未折叠蛋白反应 (UPR) 的组成型激活缺陷。因此，我们的目标是获得有关哺乳动物 P5-ATP 酶的分布和离子特异性的基本信息。在初步研究中，我们鉴定了 5 种哺乳动物 P5-ATP 酶，称为 Atp13a1-Atp13a5，确定了它们的序列和 mRNA 组织分布，并开始开发异构体特异性抗体和表达构建体。在目标 1 中，我们将开发异构体特异性抗体和原位杂交探针，并结合组织和亚细胞膜组分的蛋白质印迹分析、免疫细胞化学和原位杂交来确定小鼠 Atp13a1-Atp13a5 的膜位置和细胞类型分布。在目标 2 中，将确定鼠 P5-ATP 酶的离子特异性。将制备来自组氨酸标记版本的 Atp13a1-Atp13a5 过表达的细胞系的膜级分，并进行去污剂溶解。然后，His 标记的转运蛋白将通过镍亲和层析进行纯化，并通过使用 ATP 酶活性和磷酸酶形成的灵敏测定来测量阳离子依赖性 ATP 水解来确定离子特异性。还将使用适当的放射性同位素分析整个细胞或分离的膜囊泡中的离子摄取，以确认离子特异性。这些研究将提供基本信息，对于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至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.