Characterization of the ion transport activity of the budding yeast Na+/H+ antiporter, Nha1p, using isolated secretory vesicles.
The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.
Ohgaki
Ryuichi
R
Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Nakamura
Norihiro
N
Mitsui
Keiji
K
Kanazawa
Hiroshi
H
D000609:Aminoacridines / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry / Q000502:physiology*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007477:Ions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D012492:Salts / Q000494:pharmacology; D012964:Sodium / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000502:physiology*; D013379:Substrate Specificity; D013696:Temperature
D000609:Aminoacridines; C484692:COS3 protein, S cerevisiae; D027682:Cation Transport Proteins; D002412:Cations; D007477:Ions; D008565:Membrane Proteins; C000631624:NHA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D017923:Sodium-Hydrogen Exchangers; D049452:Green Fluorescent Proteins; C010847:9-amino-6-chloro-2-methoxyacridine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D012964:Sodium