The E1E2- or P-ATPases are a large and physiologically important family of cation pumps, ranging from the K+, Ca2+, Cd2+, and Cu2+-ATPases of bacteria to the H+, K+-, Na+, K+, and Ca2+-ATPases of animal cells. In recent years, structural genes have been cloned for 15 members of the family, and there is now considerable interest in defining structure- function relationships by means of site-directed mutagenesis. A serious limitation to such research is the small amount of material available from standard expression systems. In this FIRCA project, an alternative approach will be explored for the sarcoplasmic reticulum Ca2+-ATPase, using a new expression strategy developed under the auspices of the parent grant (GM15761). The strategy makes use of a temperature- sensitive sec6 strain of yeast, in which the mutant ATPase to be analyzed is targeted to secretory vesicles that accumulate when the cells are shifted to 37o. The vesicles are readily isolated, and because they are oriented inside-out relative to the plasma membrane, can be used for measurements of ATP-dependent cation uptake. Preliminary experiments have established that the Ca2+-ATPase from rabbit fast-twitch muscle is synthesized in yeast. The next step will be to determine whether the ATPase is fully functional and to compare this kinetic and transport properties with those of enzyme isolated from sarcoplasmic reticulum. Once the characterization of wild-type enzyme has been completed, site- directed mutants will be assayed for Ca2+ binding and Ca2+-induced changes in the intrinsic fluorescence to pinpoint residues that contribute to the transport pathway. The results should provide useful insights into the fundamental mechanism of ATP-coupled cation pumping across cell membranes.
