Hydrogen ion translocating ATPases (H+ -ATPases) are the primary ion pumps in the plasma membrane and tonoplast (vacuolar membrane) of plant cells. Together, these enzymes play a central role in 1) ion and metabolite transport, 2) cellular bioenergetics, 3) plant responses to hormones and 4) plant responses to environmental stimuli. Because of the central importance of these enzymes in plant cell function, they are likely targets in molecular genetic manipulations of plant cell function. This grant continues studies of the molecular basis of H+ transport at both the plasma membrane and tonoplast of plant cells. The information gained will be essential in designing and implementing strategies to modify the regulation and function of these enzymes. Progress to date includes 1) raising and characterizing antibodies to the plasma membrane H+ -ATPase catalytic subunit and to two subunits of the tonoplast H+ -ATPase, 2) identifying and characterizing cDNA clones from tomato encoding these H+ - ATPase subunits, and 3) using site-specific reagents to identify functional domains of the H+ -ATPases. Now, efforts will focus on sequencing full length cDNA or genomic clones encoding the H+ -ATPase subunits. The nucleotide and deduced amino acid sequence will used to: 1) assess structural homologies between related H+ -ATPases, 2) analyze topology of the membrane-associated H+ - ATPase subunits using antibodies to synthetic peptides, and 3) in conjunction with site-specific labeling reagents, identify functional domains of the H+ -ATPases. In addition antibodies and cloned DNA sequences will be used to examine regulation of expression of the three H+ -ATPase subunit genes. The coordination of expression of H+ -ATPase mRNAs and protein will be analyzed in relation to normal development and in response to environmental stimuli. This basic information is critical in assessing the extent that regulation of H+ -ATPase genes contributes to the regulation of membrane function.//
