Defining the structural organization of the polysaccharide-synthesizing multiprotein complexes localized in Golgi

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This interdisciplinary project will uncover the structures of biosynthetic machines involved in polysaccharide biosynthesis in plant Golgi and unravel exactly how they function. Xyloglucan is the major hemicellulose component of primary cell walls in most plants and provides integral functions within plant systems for growth, development, signaling, and stress responses. The work should be applicable to a broad variety of plant species and hence contribute to both fundamental research and the development of more desirable cell wall structures for robust and productive crops. Overall, the findings could lay foundation for biotechnological strategies towards improving crop quality and yield increasing food and energy security and independence. The project will provide research training across a broad range of cutting-edge research methodologies in modern cell and structural biology and biochemistry for junior scientists at the high school, undergraduate, and graduate level. Graduate students will develop mentoring and teaching skills supervising high school and undergraduate students through the framework of this project. The overarching goal of this project to generate knowledge of mechanisms that control polysaccharide pattern assembly through the accomplishment of three specific aims: 1) Investigate xylosyltransferase - substrate interactions to confirm the acceptor substrate binding mode, 2) Investigate formation of xylosyltransferase T heterodimers or hetero-tetramers and xylosyltransferase - CSLC4 complexes in Golgi to elucidate the structural organization of the xyloglucan synthesizing complex, and 3) Structurally characterize the complexes and their dynamics using single particle EM and computational modeling. The research proposed will be carried out through a collaboration in applications across structural, biochemical, and computational biology through live cell imaging, protein immunoprecipitation and enzymatic assays, X-ray crystallography, single particle EM and computational modeling applied to multi-scale systems, such as recombinant proteins, plant and yeast cells and a whole plant. This project is jointly funded by the Cellular Dynamics and Function and Molecular Biophysics Clusters in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.