This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.
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