CHARACTERIZATION OF PUTATIVE MAGNESIUM TRANSPORTING P-TYPE ATPASES

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。P型ATPase包括一个蛋白质超家族，存在于原核生物和真核生物中，可以跨细胞膜运输无机阳离子和其他底物。根据其保守的核心序列，它们被划分为5个亚家族，命名为P1-P5。P1-P3亚家族的成员已经通过生化、分子生物学和遗传学技术得到了很好的描述，包括Na，K-ATPase，Ca-ATPase和H，K-ATPase等。P4-ATPase在所有真核生物中都有表达，包括大约20个具有氨基磷脂转运蛋白功能的哺乳动物泵。最不了解的P型ATPase是P5亚家族的那些，它们只在真核生物中表达。尽管随着进化时间的推移，它们被保留在酵母、蠕虫、鱼和人类等各种生物中，但人们对它们的功能知之甚少。目前提案的总体目标是在小鼠身上进行P5-ATPase的基本表征。具体地说，目的是1)分离每个家族成员的全长cDNA克隆并确定它们的组织分布和膜位置，2)在适当的细胞系中过表达新的P型ATPase以评估它们的离子特异性，以及3)利用RNA干扰(RNAi)和基因打靶技术确定它们在体内的细胞和生理作用。到目前为止，我们已经基本完成了目标1。已经确定了这五个家族成员的全长cDNA克隆及其组织分布。这项工作于2004年10月出版(见出版物)。自从上一次的进展报告以来，我们产生了针对每个转运蛋白的特定异构体多肽的抗体，目前正在通过蛋白质印迹分析来表征这些抗体。我们还建立了稳定的细胞系，表达V5-组氨酸和GFP标记的转运蛋白。这些细胞系正被用于免疫荧光和共定位实验，以确定每个家庭成员的膜位置。这种组氨酸标记的转运蛋白也将通过镍亲和层析进行纯化，并将用于生化研究，以确定转运蛋白的离子专一性。针对两个P5-ATPase Atp13a1和Atp13a2的靶向载体的构建工作也已开始。一项名为“R15”的拨款最近已提交给美国国立卫生研究院。

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 comprise a large superfamily of proteins, present in both prokaryotes and eukaryotes, that transport inorganic cations and other substrates across cell membranes. Based on their conserved core sequences, they have been classified into 5 subfamilies, termed P1-P5. Members of the P1-P3 subfamilies have been well characterized using biochemical, molecular biological, and genetic techniques and include Na,K-ATPases, Ca-ATPases, and H,K-ATPases among others. P4-ATPases are expressed in all eukaryotes and include ~20 mammalian pumps that appear to function as aminophospholipid transporters. The most poorly understood P-type ATPases are those of the P5 subfamily, which are expressed only in eukaryotes. Despite being retained over evolutionary time in organisms as diverse as yeast, worms, fish and humans little is known about their function. The overall goal of the current proposal is to carry out a basic characterization of the P5-ATPases in mice. Specifically, the aims are to 1) isolate full-length cDNA clones for each of the family members and determine their tissue distribution and membrane location, 2) overexpress the novel P-type ATPases in the appropriate cell lines in order to assess their ion specificity, and 3) determine their cellular and physiological roles in vivo using RNA interference (RNAi) and gene targeting technologies. To date, we have almost completed aim 1. Full-length cDNA clones of each of the five family members and their tissue distributions have been determined. This work was published in October of 2004 (see publications). Since the last progress report we generated antibodies to isoform specific peptides of each transporter and these are currently being characterized by Western blot analysis. We have also generated stable cell lines expressing V5-histidine and GFP tagged versions of the transporters. These cell lines are being used in immunofluoresence and colocalization experiments to identify the membrane location of each family member. This histidine tagged transporters will also be purified by nickel affinity chromatography and will be used in biochemical studies to determine the ion specificity of the transporters. Work has also begun on the construction of targeting vectors for two of the P5-ATPases, Atp13a1 and Atp13a2. An R15 grant entitled, " has been submitted to NIH recently.