To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.IMPORTANCE With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L per se as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.

Vacuolar H+ -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by μCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc.

We have characterized cresyl violet as a membrane-permeant fluorophore that localizes to lysosomes and acidic vacuoles of budding yeast, Drosophila, human, murine and canine cells. An acidotropic weak base, cresyl violet is shown to be virtually insensitive to physiological alkali and divalent cations. Because of its unique spectral properties, it can be used in combination with green, red and far-red fluorophores, is less susceptible to photobleaching than alternative acidotropic probes, and does not undergo photoconversion. At concentrations that yield bright labeling of acidic compartments, cresyl violet does not alter the organellar pH nor does it affect the buffering capacity. Its affordability, together with its chemical and spectral properties, make cresyl violet a superior lysosomal marker devoid of many of the negative characteristics associated with other lysosomal probes.

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog l-leucyl-l-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

Non‐technical summary Most cellular processes are exquisitely sensitive to pH. Consequently our cells have a range of processes directed to control cellular pH. Plasma membrane transport proteins move acid or base across the plasma membrane to regulate pH precisely. We studied AE1 (also called Band 3) of erythrocytes and kidney cells, which rapidly transports the base, bicarbonate. AE1's high transport rate, combined with the surprisingly slow rates of H+ diffusion in cytosol, led us to wonder whether AE1 changes the pH of its local environment. The key findings were that H+ diffusion through the cytosol occurs at 0.6 μm s−1, and along the inner surface of the plasma membrane at only 0.01 μm s−1. We estimated that the size of the region of altered pH (H+ microdomain) around AE1 is 0.3 μm in diameter. pH‐regulatory transporters, like AE1, have differential effects on their immediate environment, with implications for the regulation of nearby pH‐sensitive proteins.

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+ (1.09 ± 0.11 or 1.18 ± 0.32 mm min−1, respectively). This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H+-coupled transport proteins.

This chapter contains sections titled: Biochemistry of Bicarbonate Bicarbonate Transport Proteins The Bicarbonate Transport Metabolon Other Acid/Base Transporter Carbonic Anhydrase Metabolons Noncatalytic Role of the CA Metabolon References

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside co‐transport, with a low turnover rate (10 nucleoside molecules s−1). hCNT3 also supports H+‐coupled uridine transport, as measured in X. laevis oocytes. We used a new approach to monitor H+/uridine co‐transport in mammalian cells. We fused mNectarine, a new H+‐sensitive red fluorescent protein, to the N‐terminus of hCNT3 to generate mNect‐hCNT3. Fusion of the fluorescent H+ sensor enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in human embryonic kidney 293 cells expressing mNect‐hCNT3 or, as a negative control, cytosolic mNectarine and catalytically dead mutant of the AE1 Cl−/HCO3− exchanger, AE1‐P652C. mNectarine fluorescence values were calibrated for pH using the nigericin/high K+ clamping method. Cells were incubated at the permissive pH for H+‐coupled nucleoside transport, pH 5.5, under Na+‐free conditions. At this pH there was a continuous cytosolic acidification. In mNect‐hCNT3 expressing cells (but not under negative control conditions) the rate of acidification increased upon addition of 0.5 mM uridine, consistent with H+‐coupled uridine transport. We conclude that mNect‐hCNT3 acts as a sensitive self‐referencing sensor of nucleoside transport. Research is supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada.

Cl−/HCO3− exchange proteins (AEs) interact with carbonic anhydrases (CAs) to maximize HCO3− transport. CAs catalyse the hydration of CO2 to HCO3− and H+. We hypothesize that rapid AE1‐mediated HCO3− transport causes differences in pH between the region around AE1 and the rest of the cell (pH micro‐environments). To examine this possibility we have measured intracellular pH during HCO3− transport using pH‐sensitive fluorescent proteins (FPs) localized to unique cellular locations. We have identified a FP, deGFP4, that reports on pH without interference from other ions; deGFP4 had a Stern‐Volmer (Ksv) quenching constant of 5×107 M−1 for H+ and 0.5 M−1 for Cl−. We constructed deGFP4 fusion proteins to: AE1 N‐terminus (measures pH at the cytosolic surface of AE1), the nucleoside transporter hCNT3 (a spectator protein), calnexin C‐terminus (to probe pH at the cytosolic ER surface), a GPI‐linked construct (to probe pH at the extracellular surface), and deGFP4 alone (cytosolic pH). The AE1‐deGFP4 and cytosolic deGFP4 exhibit an average transport rate of 0.5 pH units/min with an overall change in pH of 0.8 pH units. A new pH‐sensitive red FP (RFP) was recently developed and has been used as a cytosolic pH sensor, and as a fusion to hCNT3. pH was measured simultaneously in distinct regions of the cell using both GFP and RFP fusion proteins. This research is supported by the Canadian Institutes of Health Research.

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+–H+ exchanger (NHE1) and Cl−–HCO3− exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3−, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 ± 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane‐anchored isoform, CAIV. The membrane‐permeant CA inhibitor, 6‐ethoxyzolamide (ETZ), both prevented and reversed PE‐induced hypertrophy in a concentration‐dependent manner in neonate cardiomyocytes (IC50= 18 μm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 ± 0.3 μm and 2.7 ± 0.3 μm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 ± 0.4 Hz to 0.77 ± 0.04 Hz following 24 h treatment; these Ca2+‐handling abnormalities were completely prevented by ETZ (0.28 ± 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane [Formula: See Text] anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 [Formula: See Text] transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and [Formula: See Text] co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) [Formula: See Text] exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."