SCHULTHEIS POST-DOC/TECHNICIAN SUPPORT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 P型ATPase是一种跨细胞膜转运离子的酶，分为五个亚家族。P1-、P2-和P3-ATPase已经得到了很好的研究，包括重金属ATPase、Na，K-ATPase、H，K-ATPase、SERCAs和其他Ca2-ATPase，以及细菌的Mg2-ATPase。P4-ATPase仅在真核生物中发现，并与氨基磷脂的运输有关。虽然它们存在于所有真核生物中，但对P5-ATPase知之甚少，除了观察到两种酵母P5-ATPase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)的结构性激活。因此，我们的目标是获得有关哺乳动物P5-ATPase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个哺乳动物P5-ATPase，命名为Atp13a1-Atp13a5，确定了它们的序列和mRNA的组织分布，并开始开发异构体特异性抗体和表达载体。在目标1中，我们将开发针对异构体的抗体和原位杂交探针，并结合组织和亚细胞膜组分的Western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，将确定小鼠P5-ATPase的离子特异性。来自组氨酸标记的Atp13a1-Atp13a5过表达的细胞系的膜组分将被制备并经受洗涤剂的增溶。组氨酸标记的转运体随后将通过镍亲和层析纯化，离子特异性将通过使用敏感的ATPase活性和磷酸酶形成分析来测量阳离子依赖的ATP水解度来确定。也将使用适当的放射性同位素来分析整个细胞或分离的膜泡的离子摄取，以确认离子的特异性。这些研究将为更详细地研究这个哺乳动物P型ATPase亚家族的细胞、生物学和生理功能提供关键的基本信息。

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.