CHARACTERIZATION OF PUTATIVE MAGNESIUM TRANSPORTING P-TYPE ATPASES

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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.