Cation pumps belonging to the family of P-ATPases generate the transmembrane ion gradients underlying many important cellular and physiological events such as nutrient uptake, signal transduction, and cell cycle progression. Although clearly related to one another in structure and mechanism, members of this family are functionally diverse, having evolved to transport different cations (H+, Na+, K+, Ca2+, Mg2+, Cd2+, Cu2+, Hg2+, Zn2+) and thus fulfill a broad array of biological needs. With cloned genes and working models of the ion pumps in hand, we will focus on elucidating the molecular determinants of ion specificity and transport within this multigene family. Yeast was chosen as a model system because it is readily amenable to both biochemical and genetic approaches. We have developed novel strategies in yeast that will, 1. maximize expression levels of ion pumps, 2. amplify intact membrane compartments in which the pumps reside, and 3. allow phenotypic selection of recombinant pumps with altered specificities. We begin our analysis with a set of three yeast pumps having different ion specificities: (i) PMA1, a plasma membrane H+ pump, (ii) PMR1, a Ca2+ pump in Golgi membranes, and (iii) PMR2, a plasma membrane Na+ pump. Our first goal will be to define the selectivity profile for each ion pump, using a series of alternative cations. This will set the stage for our second goal, which will be to map the molecular determinants of ion selectivity using a variety of convergent approaches. One approach will involve interchange of functional domains between the three pumps to generate chimeras with altered ion specificities. A second strategy will be to identify 'gain of function mutations' that confer novel specificities by phenotypic selection of randomly mutagenized pumps. We will also use directed mutagenesis to examine individual residues suspected of altering ion selectivity. Our final goal is to identify residues essential for ion transport. Building on the information gained thus far, we will subject individual transmembrane segments to saturation mutagenesis and screen for loss of function mutations. Useful information on interactions between transmembrane segments (such as salt bridges) or long range conformational coupling with cytoplasmic domains will be obtained from the identification of suppressor mutations. Critical residues identified by these studies will be substituted to determine the effect of charge, hydrophobicity and size on the kinetics of transport. Ultimately, we hope to use this powerful molecular genetic approach to describe the location, size and shape of the transport pathway in ion transporting ATPases.
