Mutants of the plant cation/H+ antiporter AtNHX1 that confer greater halotolerance were
generated by random mutagenesis and selected in yeast by phenotypic complementation.
The amino acid substitutions that were selected were conservative and occurred in the
second half of the membrane-associated N terminus. AtNHX1 complemented the lack of
endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on
hygromycin B and vanadate media agreed with a generally improved endosomal …

MATERIALS AND METHODS Strains and Culture—S. cerevisiae strains AXT3 (ena1-4::
HIS3, nha1:: LEU2, nhx1:: TRP1), WX1 (nhx1:: TRP1), and OC02 (nhx1:: HIS3, vnx1::
KanMX4) have been described previously (4, 5). For cation tolerance testing, saturated
cultures were diluted to 2 106 cell/ml, and 5-l aliquots of 10-fold serial dilutions from
saturated yeast cultures were spotted onto AP plates (8 mM phosphoric acid, 10 mM L-
arginine, 2 mM MgSO4, 0.2 mM CaCl2, 2% glucose plus vitamins and trace elements) …