Molecular Functions of Protein Disulfide Isomerases in Redox-metabolism and Protein Folding Pathways in Plants

University Of Hawaii, Honolulu

Investigators

Abstract

Intellectual Merit: This project investigates the roles and modes of action of the enzyme protein disulfide isomerase (PDI) in plants. PDI-regulated processes are important for seed formation and endosperm filling, which are central to agricultural yields and grain nutrition. New knowledge by which PDIs transfer electrons between proteins will advance the understanding of basic redox enzyme biochemistry. In Arabidopsis there are 12 genes for members of the PDI family. These 12 PDIs differ in the position and number of thioredoxin domains and in the presence of transmembrane domains. This project has demonstrated some PDIs are located in the endoplasmic reticulum (ER) where they interact with, and correctly fold, substrate polypeptides. In the protein folding process, PDIs use their thioredoxin domains to catalyze the reversible formation and rearrangement of disulfide bonds in substrates. Proper polypeptide folding is necessary for the activity, transport and assembly of the substrates. PDIs have also been shown to chaperone and regulate the activity of substrates outside the ER. PDI5 chaperones and inhibits cysteine proteases during trafficking from the ER to the Golgi apparatus and the vacuoles, where the proteases play essential roles in seed and embryo development. However, little is known about the roles of other members of the PDI family, their biochemical mechanisms of action, interacting partners, and subcellular locations. This research will define the functions and biochemical activities of PDIs, determine where they are located within cells, and identify substrate proteins with which they interact. A variety of molecular, cellular, biochemical, and genetic methods will be used to achieve these goals including yeast two hybrid assays, affinity-tag purification, fluorescence and immunoelectron microscopy, protein refolding activity assays and complementation of protein folding mutants in E. coli and yeast. The physiological, morphological, and cellular effects of over-expressing and eliminating PDIs will also be measured. This basic research forms the underpinnings for understanding fundamental and essential cellular processes common to all plants. Broader Impacts: Methods to manipulate PDI activity as developed in this research will provide solutions for enzyme stability problems for environmental remediation, treating crop diseases and industrial uses. New biochemical knowledge will facilitate the design of novel catalysts and protein folding and stabilizing reagents for agricultural, environmental, food processing and bioenergy uses. Understanding and regulating protein folding is critical for over-expression of valuable proteins in transgenic plants. The graduate student exchange between Hawaii and Florida will promote interdisciplinary training in molecular genetics and cell biology. Central to the mission of this project is to integrate the resulting technologies with learning experiences that will inspire faculty and student development from small colleges lacking research programs. The project will partner with community colleges to continue a successful summer workshop (http://abe.leeward.hawaii.edu/). Faculty members obtain certificates of professional development, while students obtain mentoring, course credit and training in experimental problem-solving. Participants become aware of the fascinating array of activities and career choices in modern life sciences. Hands-on training in molecular biology, genomics, bioinformatics and cellular biological research coupled with synergistic interactions, partnerships and collaboratively developed teaching resources will have far-reaching educational impacts on faculty and students of Pacific Island and Asian decent.