9604246 Rea Re-establishment of the resting state after stimulus-coupled elevations of cytosolic free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. The investigator in collaboration with Drs. Hirschi and Fink, Whitehead Institute for Biomedical Research, has recently isolated two cDNAs, CAXl and CAX2, from Arabidopsis thaliana. These cDNA's suppress a mutation in a Saccharomyces cerevisiae strain defective in vacuolar Ca2+ accumulation (Hirschi et al., 1996). Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAXl or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAXl gene product indicate that it is the high capacity transporter responsible for contributing to the maintenance of low cytosolic free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation. Having cloned CAXl and CAX2 and broadly defined the functional characteristics of their translation products in yeast, the investigator aims now to extend his studies of one of these, CAXlp, the putative vacuolar H+/Ca2+ antiporter, to better defining its transport capabilities and membrane localization in the intact plant. The main objectives of the research program are be four-fold: (1) Determination of the plant membrane fraction(s) with which CAXlp is associated. The investigator's data strongly indicate that CAXlp corresponds to the vacuolar H+/Ca2+ antiporter but this remains to be tested directly. (2) The development of procedures for the purification and reconstitution of CAXlp. This is necessary for studies of the sufficiency of CAXlp for H+/Ca2+ antiport, and possibly other antiport functions, and the provision of material for future investigations of transport stoichiometry and structure-function relations. (3) Elucidation of the electrogenicity, and thence transport stoichiometry, of CAXlp-mediated H+/Ca2+ exchange. It is critical to know the numbe r of H+ ions exchanged per Ca2+ since this determines the maximum accumulation ratio that can be achieved by CAXlp under the conditions prevailing in vivo. This, in turn, will provide indications of the physiological function of CAXlp. (4) Examination of the capacity of CAXlp for the transport of Na+ as well as Ca2+ or Na+ as well as H+. The investigator has shown that CAXlp catalyzes Ca2+ transport in preference to other divalent cations but it is not known if Na+ can substitute for H+ as driver ion or whether CAXlp is competent in Na+/H+ antiport as well as H+/Ca2+ antiport, as is the case for some non-plant H+/Ca2+ and Na+/Ca2+ antiporters. Knowledge of its membrane localization, sufficiency, likely physiological poise and ion-selectivity will provide a firm foundation for future investigations, by both the investigator and others, of the regulation, structure-function characteristics and physiological impact of CAXlp on general ion homeostasis and Ca2+-dependent stimulus-response coupling in plants. The coordination of many plant cell responses to stimuli, such as light, gravity, touch and cold, is mediated by changes in cytoplasmic calcium ion ( Ca2+) concentration. A major intracellular reservoir for some of the Ca2+ involved in these processes is the vacuole. Since the vacuolar concentration of Ca2+ is often 10,000 times higher than that of the cytoplasm, and the maintenance of low cytoplasmic concentrations is critical for normal cell function, efficient systems for concentrating Ca2+ in the vacuole are required. One of these is a transporter, a calcium-proton antiporter, located in the membrane surrounding the vacuole. This membrane protein harnesses the electrochemical energy contained in the hydrogen ion (H+) concentration gradient across this membrane to drive the transport of Ca2+ from the cytoplasm into the vacuole by exchanging vacuolar H+ for cytoplasmic Ca2+. This research is concerned with understanding the molecular mechanism of this transporter.
