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The electroneutral exchange of protons for Na ؉ and K ؉ across the mitochondrial inner membrane contributes to organellar volume and Ca 2؉ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na ؉ /H ؉ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4,6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22 Na ؉ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na ؉ /H ؉ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na ؉ /H ؉ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.

Background: mTORC1 is dysregulated in human disease, and there is an interest in the development of mTORC1 inhibitors. Niclosamide inhibits mTORC1 signaling, but its mode of action remains unclear. Results: Niclosamide extrudes protons from lysosomes, thus lowering cytoplasmic pH and inhibiting mTORC1 signaling. Conclusion: Cytoplasmic acidification inhibits mTORC1 signaling. Significance: Our findings may aid the design of niclosamide-based anticancer therapeutic agents.

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BackgroundAcidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known.Methodology/FindingsWe examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1–TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1–TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles.ConclusionsThis study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.