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 alteration of the fine-tuned balance of phospho/dephosphorylation reactions in the cell often results in functional disturbance. In the yeast Saccharomyces cerevisiae, the overexpression of Ser/Thr phosphatase Ppz1 drastically blocks cell proliferation, with a profound change in the transcriptomic and phosphoproteomic profiles. While the deleterious effect on growth likely derives from the alteration of multiple targets, the precise mechanisms are still obscure. Ppz1 is a negative effector of potassium influx. However, we show that the toxic effect of Ppz1 overexpression is unrelated to the Trk1/2 high-affinity potassium importers. Cells overexpressing Ppz1 exhibit decreased K+ content, increased cytosolic acidification, and fail to properly acidify the medium. These effects, as well as the growth defect, are counteracted by the deletion of NHA1 gene, which encodes a plasma membrane Na+, K+/H+ antiporter. The beneficial effect of a lack of Nha1 on the growth vanishes as the pH of the medium approaches neutrality, is not eliminated by the expression of two non-functional Nha1 variants (D145N or D177N), and is exacerbated by a hyperactive Nha1 version (S481A). All our results show that high levels of Ppz1 overactivate Nha1, leading to an excessive entry of H+ and efflux of K+, which is detrimental for growth.

• Keywords
• K+ transport, Nha1, Ppz1 phosphatase, Saccharomyces cerevisiae, cation homeostasis, intracellular pH,
• Publication type
• Journal Article MeSH

Nutrient availability controls the landscape of nutrient transporters present at the plasma membrane, notably by regulating their ubiquitylation and subsequent endocytosis. In yeast, this involves the Nedd4 ubiquitin ligase Rsp5 and arrestin-related trafficking adaptors (ARTs). ARTs are targeted by signaling pathways and warrant that cargo ubiquitylation and endocytosis appropriately respond to nutritional inputs. Here, we show that glucose deprivation regulates the ART protein Csr2/Art8 at multiple levels to trigger high-affinity glucose transporter endocytosis. Csr2 is transcriptionally induced in these conditions through the AMPK orthologue Snf1 and downstream transcriptional repressors. Upon synthesis, Csr2 becomes activated by ubiquitylation. In contrast, glucose replenishment induces CSR2 transcriptional shutdown and switches Csr2 to an inactive, deubiquitylated form. This glucose-induced deubiquitylation of Csr2 correlates with its phospho-dependent association with 14-3-3 proteins and involves protein kinase A. Thus, two glucose signaling pathways converge onto Csr2 to regulate hexose transporter endocytosis by glucose availability. These data illustrate novel mechanisms by which nutrients modulate ART activity and endocytosis.

• MeSH
• Arrestin genetics   metabolism   MeSH
• Time Factors MeSH
• Endocytosis * MeSH
• Transcription, Genetic MeSH
• Glucose deficiency   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Mutation MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Protein Phosphatase 1 metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• Monosaccharide Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• Repressor Proteins metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Ubiquitination MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arrestin MeSH
• BMH1 protein, S cerevisiae MeSH Browser
• BMH2 protein, S cerevisiae MeSH Browser
• Csr2 protein, S cerevisiae MeSH Browser
• Glucose MeSH
• Hxt6 protein, S cerevisiae MeSH Browser
• HXT7 protein, S cerevisiae MeSH Browser
• Nuclear Proteins MeSH
• MIG1 protein, S cerevisiae MeSH Browser
• Mig2 protein, S cerevisiae MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Protein Phosphatase 1 MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• Monosaccharide Transport Proteins MeSH
• Repressor Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SNF1-related protein kinases MeSH Browser

Transport across the plasma membrane is the first step at which nutrient supply is tightly regulated in response to intracellular needs and often also rapidly changing external environment. In this review, I describe primarily our current understanding of multiple interconnected glucose-sensing systems and signal-transduction pathways that ensure fast and optimum expression of genes encoding hexose transporters in three yeast species, Saccharomyces cerevisiae, Kluyveromyces lactis and Candida albicans. In addition, an overview of GAL- and MAL-specific regulatory networks, controlling galactose and maltose utilization, is provided. Finally, pathways generating signals inducing posttranslational degradation of sugar transporters will be highlighted.

• MeSH
• Gene Regulatory Networks genetics   MeSH
• Yeasts genetics   metabolism   physiology   MeSH
• Monosaccharide Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal * MeSH
• Signal Transduction * MeSH
• Environment MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Monosaccharide Transport Proteins MeSH

BACKGROUND: The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. RESULTS: The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. CONCLUSION: We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.

• MeSH
• Metals, Alkali metabolism   MeSH
• Candida genetics   growth & development   metabolism   pathogenicity   MeSH
• Potassium metabolism   MeSH
• Microscopy, Fluorescence MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Cations metabolism   MeSH
• Lithium metabolism   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Sodium-Hydrogen Exchangers genetics   metabolism   MeSH
• Cation Transport Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   growth & development   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Base Sequence MeSH
• Salts metabolism   MeSH
• Substrate Specificity MeSH
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Metals, Alkali MeSH
• CNH1 protein, Candida albicans MeSH Browser
• Potassium MeSH
• Fungal Proteins MeSH
• Cations MeSH
• Lithium MeSH
• Membrane Proteins MeSH
• Sodium-Hydrogen Exchangers MeSH
• NHA1 protein, S cerevisiae MeSH Browser
• Cation Transport Proteins MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Salts MeSH
• Superoxide Dismutase-1 MeSH
• Superoxide Dismutase MeSH