Poster: Membrane Transport

Abs # 1224: Topology and structure-function analysis of the Arabidopsis vacuolar Na⁺/H⁺ antiporter

Presenter:	Yamaguchi, Toshio , tyamaguchi@ucdavis.edu
Authors	Yamaguchi, Toshio  (A)   Shi, Huazhong  (A)   Blumwald, Eduardo  (A)
Affiliations:	(A): University of California, Department of Pomology, Davis, CA 95616

Cation/H⁺ antiporters play a major role in maintaining cellular pH and ion homeostasis of cells throughout the biological kingdoms. AtNHX1 is the most abundant of the vacuolar Na⁺/H⁺ antiporters in Arabidopsis and plays an important role in maintaining ion homeostasis. Additionally, AtNHX1 is needed for normal leaf development and its overexpression has been shown to confer salt tolerance in several plants by increasing vacuolar Na⁺ sequestration in the leaves. Although the structural and functional analysis of mammalian and bacterial plasma membrane-bound Na⁺/H⁺ antiporters is emerging, little is known about the topology, structure and mode(s) of action of plant vacuolar Na⁺/H⁺ antiporters. AtNHX1 has 10-12 predicted membrane spanning regions according to hydropathy analysis, however the membrane topology of the protein has not been determined experimentally. We made 13 constructs of AtNHX1 with a hemagglutinin tag (HA-tag) at different hydrophilic regions of the protein. These fusion proteins were expressed in yeast and isolated vacuoles were used for subsequent protease protection assays to determine the membrane topology of AtNHX1. Furthermore, N- and C- terminal truncations and site-directed mutagenesis constructs were used to determine amino acid residues and domains critical to AtNHX1 function and ion selectivity.