SCHULTHEIS POST-DOC/TECHNICIAN SUPPORT

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 P-型ATP酶是一种跨细胞膜转运离子的酶，目前已被分为五个亚家族。P1-、P2-和P3-ATP酶已被充分表征，并且包括重金属ATP酶、Na，K-ATP酶、H，K-ATP酶、SERCA和其它Ca 2 +-ATP酶以及细菌Mg 2 +-ATP酶。P4-ATP酶仅在真核生物中发现，并且与氨基磷脂的转运有关。尽管P5-ATP酶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Cod 1 p的缺失导致糖蛋白加工、内质网相关蛋白降解（ERAD）和未折叠蛋白反应（UPR）的组成性激活缺陷。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，我们已经鉴定了5种哺乳动物P5-ATP酶，命名为Atp 13 a1-Atp 13 a5，确定了它们的序列和mRNA组织分布，并开始开发同种型特异性抗体和表达构建体。在目标1中，我们将开发亚型特异性抗体和原位杂交探针，并确定膜的位置和细胞类型分布的小鼠Atp 13 a1-Atp 13 a5使用相结合的Western印迹分析的组织和亚细胞膜组分，免疫细胞化学，原位杂交。在目标2中，将测定鼠P5-ATP酶的离子特异性。将制备来自过表达组氨酸标记形式的Atp 13 a1-Atp 13 a5的细胞系的膜级分，并进行去污剂溶解。然后通过镍亲和色谱法纯化His标记的转运蛋白，并通过使用ATP酶活性和磷酸酶形成的灵敏测定法测量阳离子依赖性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全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，这将是至关重要的细胞生物学和生理功能的更详细的机制研究，这个最不了解哺乳动物P-型ATP酶的亚家族。

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.