Functional
expression of the Oryza sativa NHX1 antiporter in Saccharomyces
cerevisiae.
Olga Kinclova,
Hana Flegelova, Hana Sychrova
Dept. Membrane Transport, Institute of Physiology, Videnska 1083, Prague 4 -
Krc, 142 20, Czech Republic (kinclova@biomed.cas.cz)
Na+/H+
antiporters have been found in a wide variety of organisms. Their main role is
to eliminate high toxic concentrations of Na+ and Li+
from cytosol by pumping the alkali metal cations out of cells or by
sequestration in some compartments, e.g. vacuole. The O. sativa NHX1 gene encodes a Na+/H+
exchanger playing an important role in salt tolerance of rice. The OsNHX1
protein (535 aa) is similar to intracellularly localized antiporters from Arabidopsis
thaliana
(AtNhx1p, 73%) and S. cerevisiae (Nhx1p, 30%). To characterize transport
properties and physiological role of the OsNHX1 antiporter, cDNA of OsNHX1 gene
was expressed in a S. cerevisiae alkali-metal-cations-sensitive strain lacking
its own Na+-transport systems (ena1-4 nha1 nhx1). Overexpression of
OsNHX1 improved significantly the tolerance of S. cerevisiae mutant cells to Na+
and Li+, but not to K+. Microscopy analysis of the
OsNHX1-GFP indicated its localization mainly in ER and vacuolar membranes of S.
cerevisiae
cells. Measurements of Na+/Li+ content and efflux in S.
cerevisiae
cells expressing OsNHX1 proved the antiporter's transport activity and
substrate specificity. Functional expression of a plant antiporter in a yeast
antiporter-less strain confirmed the suitability of yeast cells as a host
system to study individual transport systems of higher eukaryots at the
molecular level. This work was supported by grants from the Grant Agency of the
Czech Republic (204/02/D092) and from the EU (QLK3-2001-00533).