Function follows form: Structural and functional characterization of ABCB transporters in monocots and dicots

Anne Sophie Knoeller, Purdue University

Abstract

ABC transporters perform a multitude of important functions in microorganisms, plants and animals. In microorganisms, many ABC transporters are involved in detoxification processes and uptake of nutrients and hormones. In animals, their functions include maintaining the blood-brain barrier and sterol and lipid transport. In planta, ABC transporters function to excrete of toxins both either out of the cell and or into the vacuole, deposition of lipids that ultimately form the cuticle, are as part a component of the polar auxin transport stream, in the regulaton of stomatal movement, and in the accumulation of defense compounds in specialized organs such as the rhizomes and glandular trichomes. Much progress has been made in elucidating the structure and function of ABC transporters. Partial and complete crystal structures are available-derived from bacterial and animal transporters, coupled with and mutational analyses, have provided deeper mechanistic insights into regarding substrate binding and ATP hydrolysis. However, despite the high degree of sequence similarity, closely related ABC transporters can function in very different processes, and can transport a broad range of structurally unrelated substances. A good example is the reversible auxin importer ABCB4 from Arabidopsis thaliana, which is an ortholog of the importer MDR1 from Coptis japonica, which accumulates that transports the alkaloid defense compound berberine in the rhizome. Therefore, sequence similarity alone is insufficient to draw conclusion about substrate specificity or transport directionality, and a deeper insight understanding of into how small alterations in substitutions in the one-dimensional amino acid sequence translates into a large alterations in the three-dimensional protein structure, and subsequently its function, is needed. This is of particular importance for crop plant breeding, where modification of ABC transporters could improve plant stature, resistance to pests and fungi, and water usage. Here, I present a new phylogenetic analysis of the ABC subfamily B that includes several dicot and monocot species, some of which are important crop plants. I further present a structural model and functional analysis of two closely related members of this family, AtABCB21 from Arabidopsis and OsABCB10 from rice, and propose a possible binding site for substrate acceptance. I identified the regions in the three-dimensional structure that may be responsible for the differences in function. An animated model describing substrate translocation was developed. Transport assays showed that despite very high sequence identity (68% identity, 83% similarity), these transporters do not appear to accept the same substrate. Further, I characterized plants that are deficient in these proteins and drew the conclusion that they are involved in highly specialized functions, which, in the case for OsABCB10, take place in monocot-specific structures that are specific to a grasses.

Degree

Ph.D.

Advisors

Murphy, Purdue University.

Subject Area

Molecular biology|Bioinformatics|Plant sciences

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