Organellar pH Research Articles

Report on the Na+/H+ Exchanger Satellite Meeting at the 53rd Annual Meeting of the Canadian Society of Biochemistry, Molecular and Cellular Biology

The Satellite Meeting on Na+/H+ Exchangers, held on 17 April 2010, covered a range of new developments in this field. The symposium was chaired by Dr. Larry Fliegel, University of Alberta, and the speakers were Dr. John Orlowski of McGill University, Dr. Jan Rainey of Dalhousie University, Dr. Etana Padan of The Hebrew University of Jerusalem, Dr. Masa Numata of The University of British Columbia, Dr. Pavel Dibrov from the University of Manitoba, Dr. Todd Alexander of the University of Alberta, and Grant Kemp of the University of Alberta. Talks ranged from organellar pH homeostasis to structure and function of Na+/H+ exchanger proteins. Highlights of the symposium included elucidation of the structure of transmembrane regions of the NHE1 isoform and development of a new model of the NHE1 protein based on the E. coli Na+/H+ exchanger. The symposium brought together scientists from different corners of the world. The discussions that followed were lively and many scientists received constructive comments from their peers.

Saccharomyces cerevisiae glucose signalling regulator Mth1p regulates the organellar Na+/H+ exchanger Nhx1p

Organelle-localized NHEs (Na+/H+ exchangers) are found in cells from yeast to humans and contribute to organellar pH regulation by exporting H+ from the lumen to the cytosol coupled to an H+ gradient established by vacuolar H+-ATPase. The mechanisms underlying the regulation of organellar NHEs are largely unknown. In the present study, a yeast two-hybrid assay identified Mth1p as a new binding protein for Nhx1p, an organellar NHE in Saccharomyces cerevisiae. It was shown by an in vitro pull-down assay that Mth1p bound to the hydrophilic C-terminal half of Nhx1p, especially to the central portion of this region. Mth1p is known to bind to the cytoplasmic domain of the glucose sensor Snf3p/Rgt2p and also functions as a negative transcriptional regulator. Mth1p was expressed in cells grown in a medium containing galactose, but was lost (possibly degraded) when cells were grown in medium containing glucose as the sole carbon source. Deletion of the MTH1 gene increased cell growth compared with the wild-type when cells were grown in a medium containing galactose and with hygromycin or at an acidic pH. This resistance to hygromycin or acidic conditions was not observed for cells grown with glucose as the sole carbon source. Gene knockout of NHX1 increased the sensitivity to hygromycin and acidic pH. The increased resistance to hygromycin was reproduced by truncation of the Mth1p-binding region in Nhx1p. These results implicate Mth1p as a novel regulator of Nhx1p that responds to specific extracellular carbon sources.

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr(+/+) and cftr(-/-) mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase-generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease.

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na(+)/H(+) exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.

Defective organellar acidification as a cause of cystic fibrosis lung disease: reexamination of a recurring hypothesis.

The cellular mechanisms by which loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel produce cystic fibrosis (CF) lung disease remain uncertain. Defective organellar function has been proposed as an important determinant in the pathogenesis of CF lung disease. According to one hypothesis, reduced CFTR chloride conductance in organelles in CF impairs their acidification by preventing chloride entry into the organelle lumen, which is needed to balance the positive charge produced by proton entry. According to a different hypothesis, CFTR mutation hyperacidifies organelles by an indirect mechanism involving unregulated sodium efflux through epithelial sodium channels. There are reports of defective Golgi, endosomal and lysosomal acidification in CF epithelial cells, defective phagolysosomal acidification in CF alveolar macrophages, and organellar hyperacidification in CF respiratory epithelial cells. The common theme relating too high or low organellar pH to cellular dysfunction and CF pathogenesis is impaired functioning of organellar enzymes, such as those involved in ceramide metabolism and protein processing in epithelial cells and antimicrobial activity in alveolar macrophages. We review here the evidence for defective organellar acidification in CF. Significant technical and conceptual concerns are discussed regarding the validity of data showing too high/low organellar pH in CF cells, and rigorous measurements of organellar pH in CF cells are reviewed that fail to support defective organellar acidification in CF. Indeed, there is an expanding body of evidence supporting the involvement of non-CFTR chloride channels in organellar acidification. We conclude that biologically significant involvement of CFTR in organellar acidification is unlikely.

Unimpaired Lysosomal Acidification in Respiratory Epithelial Cells in Cystic Fibrosis

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

The multiple mechanisms of multidrug resistance and cellular pH.

Multidrug resistance (MDR) describes a variety of strategies that tumour cells develop to evade the cytotoxic effects of anti-cancer drugs. Tumour cells that have become MDR show decreased cellular sensitivity to the drug employed in chemotherapy as well as a broad spectrum of drugs without obvious common targets or structural homology. MDR is a major obstacle to the successful treatment of tumours. Our understanding of multidrug resistance is limited, in part, by our limited knowledge of what makes tumour cells more sensitive to chemotherapeutics. Drug resistance may subvert the same mechanisms that make tumour cells hypersensitive or use molecular modifications unrelated to the mechanisms that lead to hypersensitivity in tumour cells. Indeed, multidrug resistance is likely to be the consequence of a multitude of mechanisms. Some drug-resistant and drug-sensitive mammalian cell lines have shown aberrations in regulation of cellular pH. These changes can contribute to some of the physiological and cell biological changes that are observed both in drug-sensitive and MDR tumour cell lines. As such, at least in some tumour cell lines, the changes of pH contribute both to the physiology of transformation and the physiology of drug-resistance. The observation that these variations from normal pH are not universally observed in either tumour or MDR cells suggests that these mechanisms cannot be generalized to all transformed or MDR cells. It remains to be resolved to what extent these changes are a consequence of studying cell lines grown in vitro or if they reflect the in situ physiology of human tumours. There are no detectable changes in cytosolic or organellar pH in cells transfected with P-glycoprotein, a plasma membrane-based protein implicated in drug resistance. Thus, there are likely to be multiple mechanisms contributing to multiple drug-resistance.

Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca(2+) from the acidic Ca(2+) stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca(2+) release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca(2+) wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca(2+) stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca(2+).

Does the proteome encode organellar pH?

Inherent to the proteome itself, may be information that enables proteins to buffer pH at a level that promotes their own function within a specialized compartment. We observe that the distribution of computed isoelectric points in the yeast proteome matches experimentally derived organellar pH estimates across distinct subcellular compartments. This raises an interesting evolutionary question: did the pI of proteins and the pH of organelles co-evolve to optimize function?

Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

Secretory carrier membrane proteins interact and regulate trafficking of the organellar (Na+,K+)/H+ exchanger NHE7

The mammalian (Na(+),K(+))/H(+) exchanger NHE7 resides chiefly in the trans-Golgi network (TGN) and post-Golgi vesicles where it is thought to contribute to organellar pH homeostasis. However, the mechanisms that underlie the targeting and regulation of NHE7 are unknown. To gain insight into these processes, yeast two-hybrid methodology was used to screen a human brain cDNA library for proteins that interact with the cytoplasmic C-terminus of NHE7. One binding partner we identified was SCAMP2, a member of the secretory carrier membrane protein (SCAMP) gene family. Direct association of these two proteins was further supported by co-immunolocalization and co-immunoprecipitation analyses using transfected cells, by their co-sedimentation in membrane fractions resolved on sucrose density gradients, and by in vitro protein binding assays. Other members of the SCAMP family, such as SCAMP1 and SCAMP5, also associated with NHE7. The majority of the NHE7-SCAMP complexes accumulated at the TGN, but a minor fraction also resided in recycling vesicles. Biochemical analyses indicated that the C-terminal cytoplasmic tail of NHE7 bound preferentially to a highly conserved cytoplasmic loop between the second and the third transmembrane segments (TM2-TM3 loop) of SCAMP2. A deletion mutant of SCAMP2 lacking this region (SCAMP2/Delta184-208) bound weakly to NHE7, but caused a significant fraction of NHE7 and wild-type SCAMP2 to redistribute to a pool of scattered recycling vesicles without noticeably affecting the location of other resident TGN (syntaxin 6) or Golgi cisternae (GM130) proteins. Conversely, a GFP-tagged TM2-TM3 construct of SCAMP2 interacted with NHE7, but also led to the redistribution of NHE7 to dispersed vesicular structures. We propose a model wherein SCAMPs participate in the shuttling of NHE7 between recycling vesicles and the TGN.

ClC-3 Chloride Channels Facilitate Endosomal Acidification and Chloride Accumulation

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton.

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

Defective Acidification of Intracellular Organelles Results in Aberrant Secretion of Cathepsin D in Cancer Cells

Aberrant secretion of lysosomal hydrolases such as (pro)cathepsin D (proCD) is a common phenotypic change in many human cancers. Here we explore the underlying molecular defect(s) and find that MCF-7 breast and CaCo-2 colorectal cancer cells that are unable to acidify their endosomal compartments secreted higher amounts of proCD than did acidification-competent cancer cell types. The latter secreted equivalent amounts of proCD only after dissipation of their organellar pH gradients with NH(4)Cl. Assessing the critical steps that resulted in proCD secretion revealed that the Golgi-associated sorting receptor for CD, i.e. the cation-independent mannose-6-phosphate receptor (MPR300), was aberrantly distributed in acidification-defective MCF-7 cells. It accumulated mainly in late endosomes and/or lysosomes as a complex with its ligand (proCD or intermediate CD), as evidenced by its co-localization with both CD and LAMP-2, a late endosome/lysosome marker. Our immunoprecipitation analyses also showed that MCF-7 cells possessed 7-fold higher levels of receptor-enzyme complexes than did acidification-competent cells. NH(4)Cl induced similar receptor redistribution into LAMP-2-positive structures in acidification-competent cells but not in MCF-7 cells. The receptor also recovered its normal Golgi localization upon drug removal. Based on these observations, we conclude that defective acidification results in the aberrant secretion of proCD in certain cancer cells and interferes mainly with the normal disassembly of the receptor-enzyme complexes and efficient receptor reutilization in the Golgi.

Chemical genetic screening identifies sulfonamides that raise organellar pH and interfere with membrane traffic.

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469-6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279-286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin-mediated iron uptake. These inhibitors do not block endoplasmic reticulum-to-Golgi transport, but do disrupt Golgi-to-cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down-regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V-ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V-ATPases in particular.

Regulation of Tyrosinase Processing and Trafficking by Organellar pH and by Proteasome Activity

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.

Multiple modes of regulation of Na+/H+ exchangers.

Mammalian Na(+)/H(+) exchangers (NHE) mediate electroneutral countertransport of H(+) for Na(+) across the plasmalemmal and organellar membranes. They contribute to cellular and organellar pH and volume regulation and transepithelial Na(+) transport. The aim of this review is to illustrate the complex regulation of these transporters by focusing on the multiple mechanisms controlling the epithelial isoform, NHE3. A variety of agents and conditions (e.g., hormones, growth factors, cellular pH, and medium osmolarity) act in concert to achieve short-term and long-term regulation of this isoform. The underlying mechanism involves changes in the number of transporters on the cell surface and/or altered activity of the individual exchangers due to allosteric activation by intracellular protons, phosphorylation and interaction with accessory proteins and the cytoskeleton. A similar regulatory versatility probably applies to other NHE isoforms, and the lessons learned from studying members of the NHE family could serve as a useful reference when exploring the modes and levels of regulation of other transporters.

Molecular Cloning and Characterization of a Novel (Na+,K+)/H+ Exchanger Localized to the trans-Golgi Network

The luminal pH of organelles along the secretory and endocytic pathways of mammalian cells is acidic and tightly regulated, with the [H+] varying up to 100-fold between compartments. Steady-state organellar pH is thought to reflect a balance between the rates of H+ pumping by the vacuolar-type H+-ATPase and H+ efflux through ill-defined pathways. Here, we describe the cloning of a novel gene (NHE7) in humans that is homologous to Na+/H+ exchangers, is ubiquitously expressed, and localizes predominantly to the trans-Golgi network. Significantly, NHE7 mediates the influx of Na+ or K+ in exchange for H+. The activity of NHE7 was also found to be relatively insensitive to inhibition by amiloride but could be antagonized by the analogue benzamil and the unrelated compound quinine. Thus, NHE7 displays unique functional and pharmacological properties and may play an important role in maintaining cation homeostasis of this important organelle.

Characterization of herpes simplex virus-containing organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles.

The cytoplasmic compartments occupied by exocytosing herpes simplex virus (HSV) are poorly defined. It is unclear which organelles contain the majority of trafficking virions and which are occupied by virions on a productive rather than defective assembly pathway. These problems are compounded by the fact that HSV-infected cells produce virus continuously over many hours. All stages in viral assembly and export therefore coexist, making it impossible to determine the sequence of events and their kinetics. To address these problems, we have established assays to monitor the presence of capsids and enveloped virions in cell extracts and prepared HSV-containing organelles from normally infected cells and from cells undergoing a single synchronized wave of viral egress. We find that, in both cases, HSV particles exit the nucleus and accumulate in organelles which cofractionate with the trans-Golgi network (TGN) and endosomes. In addition to carrying enveloped infectious virions in their lumen, HSV-bearing organelles also displayed nonenveloped capsids attached to their cytoplasmic surface. Neutralization of organellar pH by chloroquine or bafilomycin A resulted in the accumulation of noninfectious enveloped particles. We conclude that the organelles of the TGN/endocytic network play a key role in the assembly and trafficking of infectious HSV.