CAREER: Functional Analysis of the Tomato Ca-ATPase Mechanism

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The goal of this project is to understand the functional mechanism of plant Calcium-ATPases. Ca-ATPases belong to the ubiquitous protein family of P-type ATPases, which exist in all taxonomic phyla. Although investigations of these transporters began in the late 1950's, studies of plant members have only recently begun to appear. This project aims to identify key structural features involved in nucleotide selectivity and membrane trafficking of the ER-type Ca-ATPase from Lycopersicon esculentum (LCA, i.e. the tomato plant). Interestingly, although animal Ca pumps are obligatory for ATP, it appears that plant Ca pumps have a rather relaxed requirement for nucleoside triphosphates. Since phosphorus has limited availability in soil (thus the need for adding P-containing fertilizers to crops), cytoplasmic ATP levels can fluctuate dramatically. Thus, it is possible that plant pumps have evolved mechanisms to utilize many high-energy phosphate compounds to combat phosphate starvation. This research will compare structure-function differences between tomato LCA and animal sarco/endoplasmic reticulum Ca pump (SERCA) to identify critical contact points in the catalytic center that provide relaxed nucleotide selection without altering cation coupling. An important goal of this project is to determine the structure of the LCA nucleotide-binding domain using X-ray crystallography. This atomic resolution structure would allow direct comparisons to the recent animal SERCA structure. Also, the cellular distribution of plant Ca-pumps remains unresolved. For example, calmodulin-stimulated Ca pumps are not only limited to the plasma membrane and "ER-type" Ca pumps are not restricted to organelles. Thus, an additional goal is to identify the membrane pool for LCA and whether its delivery is regulated via phosphorylation. The goals of this investigation will be achieved by 1) expressing full-length wild-type and mutant LCA constructs in yeast, 2) overexpressing and purifying intracellular domains of LCA from E. coli, and 3) engineering transgenic tomato plants expressing green fluorescent protein-tagged versions of LCA. Broader Impact. This project is designed to actively involve undergraduates from underrepresented groups (principally African-American and Hispanics, but including Native Americans and Pacific Islanders) to make significant contributions to the understanding of plant P-type ATPases. This goal includes enhancing the undergraduate curriculum by developing a two credit hour Introduction to Undergraduate Research course that will actively recruit undergraduate minority students from two core sophomore-level courses (i.e. Genetics and Cell Biology). This laboratory-based course will provide opportunities for critical thinking and scientific discovery. Student participation will be directed by the Principle Investigator in conjunction with doctoral students in the laboratory (also enrolled in the scholar-educator program). In addition to receiving academic credit, this course will be a stepping-stone towards entering a thesis-driven undergraduate research project. To enhance participation, funds are budgeted to support interested students during the summer. Lastly, the undergraduate participants will synergistically join a current outreach effort in the department to bring current scientific investigation to local middle- and high-schools. These activities will enhance Illinois State University retention of underrepresented students and encourage diversity for future applicants.