The human Na+ /H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity.

• Keywords
• N-terminal auto-inhibition, Na+/H+ antiporter, human NHA2, phloretin, yeast,
• MeSH
• Humans MeSH
• Sodium-Hydrogen Exchangers * chemistry   genetics   MeSH
• Protons * MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Sodium-Hydrogen Exchangers * MeSH
• Protons * MeSH
• SLC9B2 protein, human MeSH Browser

The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.

• MeSH
• Biosensing Techniques instrumentation   MeSH
• Chemical Engineering MeSH
• Golgi Apparatus chemistry   MeSH
• Isoenzymes chemistry   MeSH
• Hydrogen-Ion Concentration MeSH
• Saccharomyces cerevisiae Proteins chemistry   MeSH
• Saccharomyces cerevisiae chemistry   enzymology   MeSH
• Vacuolar Proton-Translocating ATPases chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Isoenzymes MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Vacuolar Proton-Translocating ATPases MeSH

The Saccharomyces cerevisiae genome contains three genes encoding alkali metal cation/H+ antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Systematic genome sequencing of other yeast species revealed highly conserved homologous ORFs in all of them. We compared the yeast sequences both at DNA and protein levels. The subfamily of yeast endosomal/prevacuolar Nhx1 antiporters is closely related to mammalian plasma membrane NHE proteins and to both plasma membrane and vacuolar plant antiporters. The high sequence conservation within this subfamily of yeast antiporters suggests that Nhx1p is of great importance in cell physiology. Yeast Kha1 proteins probably belong to the same subfamily as bacterial antiporters, whereas Nhal proteins form a distinct subfamily.

• MeSH
• DNA, Fungal analysis   MeSH
• Potassium-Hydrogen Antiporters chemistry   classification   genetics   MeSH
• Phylogeny MeSH
• Membrane Proteins chemistry   classification   genetics   MeSH
• Molecular Sequence Data MeSH
• Sodium-Hydrogen Exchangers chemistry   classification   genetics   MeSH
• Cation Transport Proteins chemistry   classification   genetics   MeSH
• Saccharomyces cerevisiae Proteins chemistry   classification   genetics   MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Homology, Amino Acid MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Fungal MeSH
• Potassium-Hydrogen Antiporters MeSH
• KHA1 protein, S cerevisiae MeSH Browser
• Membrane Proteins MeSH
• Sodium-Hydrogen Exchangers MeSH
• NHA1 protein, S cerevisiae MeSH Browser
• NHX1 protein, S cerevisiae MeSH Browser
• Cation Transport Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH