Nitrogen is an essential building block of amino acids and nucleic acids and is crucial for every single form of life on earth. Plants play a key role in converting nitrogen from its inorganic form to an organic form and assimilate more than 90% of the nitrogen through nitrate absorption. In order to efficiently take up nitrate in soils, plants are capable of transporting nitrate across cellular membranes, as well as sensing environmental nitrate concentration changes. A clue to how plants sense soil nitrate changes was revealed by the finding of an unexpected nitrate receptor function in the Arabidopsis nitrate transporter, CHL1. As a member of the Major Facilitator Superfamily, CHL1 is a dual-affinity transporter, displaying a biphasic kinetic property in response to low and high concentrations of nitrate. Phosphorylation of a specific threonine residue switches the transporter between the two affinity modes. Remarkably, independent of its transporter function, CHL1 can also sense and respond to variable nitrate concentrations and control gene expression in a biphasic manner. The same phosphorylation site of CHL1 also regulates its sensor activity. How does CHL1 sense nitrate concentration? What is the molecular mechanism underlying the functions of CHL1 as both a nitrate transporter and sensor? How does phosphorylation regulate CHL1 activities? This project will start to address these questions by revealing the atomic structures of CHL1 in different functional states. Membrane protein X-ray crystallography will be used as the primary approach to achieve the following specific aims: (1) structure determination of CHL1 in the presence of high nitrate concentration; and (2) structure determination of CHL1 in the presence of low nitrate concentration. The results will help unravel the overall molecular architecture of CHL1, the potential nitrate-binding sites that are important for its transporter and receptor functions, and the structural determinants and mechanisms underlying its dual-affinity nitrate transporter and sensor activities.
BROADER IMPACTS This research project will impact science and education in two major areas: (1) Advancing basic research in plant biology in the US with agricultural and environmental implications and (2) Nucleating a regional membrane protein structural biology community. Food, energy, and environment are the three major challenges the world is facing in the 21st century. Solutions to these challenges require a thorough understanding of plant biology at the very fundamental level. This project will promote plant biology research by applying cutting-edge structural biology approaches to plant sciences. It represents an initiative in advancing plant structural biology, with an emphasis on addressing emerging key questions in plant biology and propagating plant sciences in the larger community. The project will also foster further development of regional community of membrane protein structural biologists by offering training in recombinant membrane protein production, crystallization, and structure determination. Our efforts range from hands-on training of undergraduate and graduate students, technicians, and PIs outside the lab, to regular presentations at graduate program meetings, departmental retreats, special interests clubs, and joint university courses.
This project is jointly supported by the Cellular Processes Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.
