Targetting of the S. cerevisiae Na +/H + antiporter Nha1 to the plasma membrane depends on the final step in ergosterol biosynthesis.
Katerina Malinska, Hana Sychrova
Dept. Membrane Transport, Institute of Physiology CAS, Videnska 1083, Prague 4, 142 20, Czech Republic
Nha1p is a plasma-membrane secondary active transport system that mediates efflux of alkali metal cations (Na, Li, K, Rb). Its physiological role in cells includes, besides elimination of toxic cations, participation in regulation of intracellular potassium homeostasis, pH and cell volume. The localization of Nha1p in living cells was studied using GFP tagging. Its localization was not homogenous within the plasma membrane. Clusters of Nha1-GFPp observed even in the individual transversal confocal section formed diffuse patchy-like structures on the cell surface. When the plasma membranes were isolated and solubilized using the cold Triton X-100, Nha1p was found in insoluble fractions suggesting its association with lipid rafts. Targetting of the Nha1p into the plasma membrane was strongly affected in strains with diminished amount of one of the raft components, sphingolipids ( lcb1-100 ). When the trafficking of Nha1p was followed in mutants blocked in different steps of ergosterol biosynthesis, the mutations in earlier steps of synthesis ( erg6 and erg 24 ) had no effect on proper Nha1p targeting to the plasma membrane. Only strains impaired in last step of ergosterol biosynthesis ( erg4, lacking the C-24(28) sterol reductase) showed a severe defect in Nha1p localization, and the overexpression of Nha1p in erg4 cells had a lethal phenotype. This work was supported by Czech Grant Agencies (GA AV CR A5011407, AVOZ 50110509).