Acidification and Protein Traffic

Acidification and Protein Traffic Ora A. Weisz Renal-Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Acidification of some organelles, including the Golgi complex, lysosomes, secretory granules, and synaptic vesicles, is important for many of their biochemical functions. In addition, acidic pH in some compartments is also required for the efficient sorting and trafficking of proteins and lipids along the biosynthetic and endocytic pathways. Despite considerable study, however, our understanding of how pH modulates membrane traffic remains limited. In large part, this is due to the diversity of methods to perturb and monitor pH, as well as to the difficulties in isolating individual transport steps within the complex pathways of membrane traffic. This review summarizes old and recent evidence for the role of acidification at various steps of biosynthetic and endocytic transport in mammalian cells. We describe the mechanisms by which organelle pH is regulated and maintained, as well as how organelle pH is monitored and quantitated. General principles that emerge from these studies as well as future directions of interest are discussed. KEY WORDS: Proton, V-ATPase, trans-Golgi network, Polarity, Ratio-imaging, Influenza M2. ß 2003 Elsevier (USA).

I. Introduction The compartmentalization of membrane-bound compartments with unique microenvironments enables eukaryotic cells to perform myriad reactions simultaneously with precision and speed. The maintenance of an acidic pH milieu within a subset of these organelles is key to the optimal performance of the sorting and processing events that normally occur within these compartments. Acidified organelles in cells include the Golgi complex, endosomes, lysosomes, secretory granules, and synaptic vesicles. In most cells, the pH of these organelles ranges from 4.5 to 6.5. Acidification is regulated International Review of Cytology, Vol. 226 0074-7696/03 $35.00

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Copyright 2003, Elsevier (USA). All rights reserved.

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and maintained by the concerted action of a vacuolar-type ATP-ase (V-ATPase) in combination with various ion channels and transporters. Acidification of intracellular compartments plays a role in protein degradation, release of ligands from receptors, and proteolytic processing of precursor proteins into mature products, and provides a driving force for the accumulation of hormones or transmitters into secretory vesicles, synaptic vesicles, or synaptic-like microvesicles. However, in addition to these functions, acidification appears to regulate the molecular mechanism of membrane traYc through some compartments, as the sorting and transport of many proteins are less eYcient when acidification is disrupted. Cellular invasion by many viruses and toxins requires functional organelle acidification; conversely, perturbation of organelle acidification has been postulated to contribute to the pathology of several diseases, including cancer, Dent’s disease, and cystic fibrosis. This review focuses on the role of acidification in membrane transport along the biosynthetic and endocytic pathways. We first describe the mechanisms by which organelle pH is regulated and maintained, as well as traditional methods and new tools that have been used to monitor and quantitate organelle pH. Subsequently, we will review evidence for the role of acidification in various steps of biosynthetic and endocytic transport in mammalian cells, and finally, discuss general principles that emerge from these studies as well as new directions of study that are warranted.

A. Trafficking Routes and pH along the Endocytic and Biosynthetic Pathways Eukaryotic cells have elaborate membranous pathways through which proteins are targeted for distinct fates (Fig. 1). Sorting along these routes is generally thought to occur by the selective incorporation of subsets of proteins into distinct sets of coated vesicles that then bud from the membrane and are selectively targeted to and fuse with the appropriate donor compartment. Numerous signals on proteins have been identified that specify targeting: these include linear amino acid sequences, nonlinear patches of tertiary structure, and posttranslational modifications such as phosphorylation, glycosylation, and the covalent addition of lipid(s). Consequently, there are multiple mechanisms to recognize these distinct targeting signals, some of which are sensitive to organelle pH. 1. The Endocytic Pathway Endocytosed proteins are delivered to early endosomes (also called sorting endosomes), where low pH induces the dissociation of ligands from

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SG

pH 6.0−6.5 pH 5.5−6.0

ISG

pH < 5.5

ER CCV TGN

SE

Golgi

RE

LE

LYS

FIG. 1 Acidified compartments along the biosynthetic and endocytic pathways. The compartments and flow of membrane traYc that constitute the biosynthetic and endocytic pathways are depicted. The ‘‘typical’’ range of pH that has been measured for each compartment is encoded by the color scheme; however, it should be noted that variations from these ranges have also been observed in some cases. ER, endoplasmic reticulum; TGN, trans-Golgi network; ISG, immature secretory granule; SG, secretory granule; CCV, clathrin-coated vesicle; SE, sorting endosome; RE, recycling endosome; LE, late endosome; LYS, lysosome.

receptors. Recycling membrane proteins can be routed directly to the plasma membrane from sorting endosomes or transported deeper into the cell to a specialized early endosomal compartment termed the recycling endosome. By contrast, fluid-phase proteins, including released ligands, are delivered to late endosomes and ultimately to lysosomes. The origin of late endosomes is controversial: some argue that these compartments arise by maturation of the residual early endosomes after removal of recycling components, whereas other groups suggest that these organelles exist independently and receive cargo from early endosomes via carrier vesicles. The pH encountered by internalized ligands decreases gradually along the endocytic pathway. Early endosomes have a pH of approximately 6.2–6.3 (Yamashiro and Maxfield, 1987). The pH of sorting endosomes is lower than that of early endosomes (
6.0, and possibly as low as 5.4 in some cells) (Van Renswoude et al., 1982; Sipe and Murphy, 1987; Yamashiro and Maxfield, 1987). By contrast, recycling endosomes are more alkaline than sorting endosomes (pH 6.4–6.5; Yamashiro et al., 1984; Yamashiro and Maxfield, 1987), whereas late endosomes are more acidic, with a measured pH of between 5.0

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and 5.8 (Tycko et al., 1983; Tycko and Maxfield, 1982; Yamashiro and Maxfield, 1987). Lysosomes are the most acidic cellular compartment, with pHs between 4.6 and 5.3 measured in mammalian cells (Tycko and Maxfield, 1982; Yamashiro and Maxfield, 1987; Ohkuma and Poole, 1978). 2. The Biosynthetic Pathway Newly synthesized membrane and secretory proteins in mammalian cells are cotranslationally inserted into the endoplasmic reticulum (ER), where they may be glycosylated on arginine residues (N-linked glycosylation) within a consensus sequence. In the ER, chaperone proteins facilitate protein folding and assembly into multimeric complexes. This process is monitored by an extensive quality control machinery, which directs the removal and degradation of misfolded polypeptides. Fully assembled proteins or protein complexes are then transported via carrier vesicles to an intermediate compartment [also called the vesiculotubular compartment (VTC) or the cisGolgi network]. From there, proteins enter the Golgi complex from the cis-face, and sequentially traverse the stacked cisternae that form this organelle, ultimately emerging from the trans-Golgi network (TGN). Transport between Golgi cisternae occurs via coated vesicles, but there is considerable debate as to whether it is the cargo or Golgi enzymes that are transported in these carriers. Regardless, itinerant proteins undergo a series of posttranslational modifications in the Golgi complex. For example, N-linked oligosaccharides are processed by the sequential action of various glycosyltransferases and glycosidases, and O-linked glycans attached to serine or threonine residues are elongated. In addition, the phosphorylation of mannose residues on soluble lysosomal hydrolases also occurs in the cis/ medial Golgi; proteins with this modification are selectively sorted in the TGN for delivery to lysosomes. Terminal steps in oligosaccharide processing, such as addition of the negatively charged sugar sialic acid and sulfation of some sugar residues, occur late during transport through the Golgi complex, in the trans-Golgi and TGN. The TGN is a major sorting station along the biosynthetic pathway: from this compartment, signal-mediated sorting of proteins destined for delivery to a variety of intracellular compartments, including endosomes, lysosomes, and regulated secretory granules, occurs. In addition, a retrograde transport pathway exists from the TGN to earlier compartments along the biosynthetic pathway. It is generally believed that the default pathway for proteins that do not contain sorting signals is transport to the plasma membrane (or secretion into the extracellular media for soluble proteins). However, many polarized cells maintain direct delivery pathways to their diVerentiated apical and basolateral plasma membrane domains, and thus must be able to selectively sort proteins from the TGN into distinct carriers destined for delivery to either surface.

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Along the biosynthetic pathway, only the TGN and secretory granules have been convincingly demonstrated to be acidified. Recent studies in live cells using targeted fluorescent probes measured ER pH to be between 7.1 and 7.4, similar to the pH of cytoplasm (Kim et al., 1998; Wu et al., 2001). The general consensus based on diVerent approaches by several groups is that TGN pH is between 5.9 and 6.3 (Seksek et al., 1995; Miesenbock et al., 1998; Demaurex et al., 1998), although some groups have reported significantly lower (Grambas and Hay, 1992) or higher (Poschet et al., 2001) pHs. Some of this variability may be due to diVerences in cell type; for example, Grambas and Hay (1992) reported a significant diVerence in the steady state TGN pH of chick embryo fibroblasts (pH
5.2) and Madin– Darby canine kidney (MDCK) cells (pH
5.6) measured using the same method and Chandy et al. (2001) found a large diVerence in the pH of HeLa versus respiratory epithelial cells (pH 6.7 vs 6.4, respectively), although other groups have found only small diVerences in TGN pH between diVerent cell types (Seksek et al., 1996; Chandy et al., 2001). In addition, TGN pH can be dramatically increased by elevation of cAMP or activation of protein kinase C (Seksek et al., 1995). Other methods using probes localized more generally to the Golgi complex but including the TGN have typically measured somewhat higher pHs (between 6.3 and 6.7; Schapiro and Grinstein, 2000; Kim et al., 1996; Chandy et al., 2001; Llopis et al., 1998; Farinas and Verkman, 1999), consistent with selective acidification of the TGN. The pH of immature secretory granules measured in live cells is about 6.3 (Urbe et al., 1997), and decreases to 5.0–5.6 in mature secretory granules (Wu et al., 2001; Miesenbock et al., 1998; Urbe et al., 1997; Blackmore et al., 2001).

II. Regulation of Organelle pH Steady-state organelle pH is regulated and maintained by a balance between the rates of intralumenal proton pumping, counterion conductance, and intrinsic proton leak (Fig. 2). As protons are pumped into compartments against their concentration gradients by the V-ATPase, the ability of this electrogenic pump to function further is theoretically limited by the accumulation of a positive membrane potential. In mammalian cells, passive ion flow through anion channels neutralizes this potential and allows acidification to continue (Glickman et al., 1983; Xie et al., 1983; Arai et al., 1989). In addition, all membranes are intrinsically permeable to protons, and the extent of this permeability also contributes to the steady-state pH that can be attained in a given compartment. Several excellent reviews have discussed the relative contributions of these activities to pH regulation in detail (Futai et al., 1998; Grabe and Oster, 2001). A briefer treatment of the subject is given below.

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FIG. 2 Regulation of organelle pH in acidified compartments. The pH of acidified compartments is generated and maintained by the balance between proton pump, counterion conductance, and proton eZux. Protons are pumped into membranous compartments by the V-ATPase. Pumping is inhibited by the buildup of excess positive charge, which can be neutralized by passive influx of a counterion (typically chloride) or by eZux of another cation such as Na+ (not shown). Alternatively, Na+ entry (mediated by the Na+, K+-ATPase) can increase membrane potential to further inhibit V-ATPase activity and limit acidification. Finally, the rate of intrinsic or regulated hydrogen ion leakage from each compartment also contributes significantly to the steady-state concentration of protons in some organelles.

A. The Vacuolar H+-ATPase V-ATPases in eukaryotic cells are multisubunit complexes containing 13 polypeptides with molecular masses ranging from 13 to 100 kDa; the molecular mass of the entire complex approaches 1106 kDa. Functionally, the V-ATPase can be divided into two domains, a 570-kDa peripheral complex (V1) that hydrolyzes ATP and an integral membrane complex (V0) that contains the proton pore. The structure, function, and regulation of V-ATPases have been the subject of several recent reviews (Stevens and Forgac, 1997; Forgac, 1999; Schoonderwoert and Martens, 2001; Nishi and Forgac, 2002). The number and activity of V-ATPases in a membrane play key roles in regulating the steady-state pH of organelles, vesicles, and lumens. For example, the maintenance of distinct traYcking routes for V-ATPases in diVerent cell types of the kidney is thought to be important in the regulation of transepithelial acid–base transport (Brown and Breton, 2000). In yeast, a family of chaperones, the VTC proteins, is thought to regulate the relative rates of synthesis and degradation of V-ATPases as well as their intracellular distribution (Nelson et al., 2000). However, there are numerous other mechanisms by which V-ATPases can regulate organelle pH. These mechanisms,

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which illustrate the complexity behind the generation and maintenance of acidification, are described briefly below. 1. Subunit Composition Heterogeneity There are multiple isoforms of some V-ATPase subunits. Four isoforms of the a subunit (part of the V0 domain) have been cloned from mouse (Nishi and Forgac, 2000; Oka et al., 2001a), three from chicken (Mattsson et al., 2000), and two from bovine (Peng et al., 1994, 1999); these are expressed in a highly tissue-specific manner, and several isoforms also have splice variants. This subunit is thought to play a crucial role in targeting V-ATPases to distinct compartments. Saccharomyces cerevisiae and Caenorhabditis elegans also have multiple forms of the a subunit (Pujol et al., 2001; Manolson et al., 1992, 1994; Oka et al., 2001b) and in yeast, these subunits were recently demonstrated to impart diVerential regulation to V-ATPase complexes (Kawasaki-Nishi et al., 2001). Alternative splicing and multiple isoforms of several subunits in the V1 domain have also been demonstrated (Puopolo et al., 1992; Hernando et al., 1995; Nelson et al., 1992; Umemoto et al., 1991; Crider et al., 1997; Sun-Wada et al., 2002). Particularly compelling evidence for the selective role of distinct isoforms in pH regulation in diVerent tissues comes from recent studies of the a3 knockout mouse, which has severe osteopetrosis (inability to resorb bone due to loss of osteoclast-mediated extracellular acidification), but which retains normal lysosomal V-ATPase function (Li et al., 1999). Thus, there is potential for tremendous diversity in the composition of the murine V-ATPase. In addition to the heterogeneity that can arise by combination of diVerent subunit isoforms into functional domains, it is possible that V-ATPase function could be regulated by removal of individual subunits from the complex. For example, apical endosomes isolated from principal cells in kidney papillae, which are unable to acidify, contain abundant levels of the V1 domain B subunit, but lack the A subunit, as well as a transmembrane component of the V0 domain (Sabolic et al., 1992). It is possible, however, that the B subunit associates with these membranes via an independent mechanism (Breton et al., 2000). Finally, there is some evidence that V-ATPase subunits may have redundant functions. In addition to its role as a major constituent of the VATPase, the 16-kDa proteolipid (c subunit) has also been shown to function as a gap junction in insects and crustaceans (Finbow et al., 1994), and has been suggested to mediate membrane fusion events in yeast (Peters et al., 2001). Moreover, this subunit has also been demonstrated to function as neurotransmitter release channels in Torpedo (Brochier and Morel, 1993). In mammalian cells, the 16-kDa subunit interacts with b1 integrin in focal adhesions, and may participate in the signaling pathways important for cell

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growth control (Skinner and Wildeman, 1999; Miura et al., 2001). Finally, a regulatory subunit of the V-ATPase that modulates catalytic function also interacts with components of the endocytic transport machinery (Carroll et al., 2001; Geyer et al., 2002). Such multifunctionality has potential implications for the interpretation of studies in which V-ATPase activity is inhibited using macrolide antibiotics that may bind to this subunit (see Section IV). 2. Reversible Dissociation of Functional Domains In yeast, a fraction of the V0 and V1 domains normally exists in unassembled pools. Moreover, assembled V-ATPase complexes reversibly dissociate from one another in response to glucose depletion (Kane, 1995). The assembly and disassembly of these domains are controlled by the RAVE [regulator of the (H+)-ATPase of the vacuolar and endosomal membranes] complex and may be mediated by direct binding of the V1 domain E subunit to the glycolytic enzyme aldolase (Lu et al., 2001; Seol et al., 2001; Kane, 2000). Interestingly, the eYciency of assembly of V0 domains with V1 domains containing diVerent a subunits is variable, as is their degree of disassembly in response to glucose deprivation (Kawasaki-Nishi et al., 2001). Dissociation of the V0 and V1 domains of the yeast ATPase may be important for homotypic vacuolar fusion to occur (Peters et al., 2001), and has also been reported to occur in insect cells (Sumner et al., 1995). DiVerences in the stoichiometry of V0 and V1 domains in V-ATPase preparations from diVerent tissues have led to the suggestion that mammalian V-ATPase may be regulated in this manner as well (Peng et al., 1999). Moreover, it is interesting to speculate that the apparent presence of partial V-ATPase complexes on some intracellular compartments isolated from kidney could reflect regulated assembly of the pump (Sabolic et al., 1992). Clearly, the ability to reversibly uncouple the ATPase domain from the proton pore would impart a powerful means to rapidly regulate organelle pH in response to metabolic and physiological stresses. 3. Modulation of Coupling Efficiency V-ATPase activity can also be controlled by modulating the ratio of proton transport to ATP hydrolysis (Moriyama and Nelson, 1988). In some organelles such as the vacuole of the lemon fruit, which maintain a pH of near 2, coupling is generally high, but varies with the seasonal frequency (Muller et al., 1999). In vitro, mild proteolysis or high levels of ATP have been shown to aVect mammalian V-ATPase coupling eYciency (Adachi et al., 1990; Arai et al., 1989). Moreover, yeast V-ATPase complexes containing diVerent a subunits have been shown to diVer in their coupling eYciency, suggesting that these subunits may play a role in this type of regulation (KawasakiNishi et al., 2001).

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4. Disulfide Bond Modulation of Catalytic Activity Recent studies demonstrated that reversible disulfide bond formation between two A subunits or between E subunits in the V1 domain of the V-ATPase can modulate pump activity, such that reduced V-ATPase is less active (Feng and Forgac, 1992; Tavakoli et al., 2001). For example, whereas approximately half of the V-ATPase in clathrin-coated vesicles is present in the oxidized, disulfide-bonded state, essentially all of the V-ATPase associated with synaptic vesicles is reduced (Feng and Forgac, 1992; Rodman et al., 1994). Given that the V1 domain projects into the cytoplasm, it is unclear how such diVerential disulfide bonding is generated or maintained. 5. Soluble Modulators of V-ATPase Activity Approximately 10 years ago, two groups identified small proteins that diVerentially aVected purified V-ATPase activity. These include a small protein (
6000 MW) that inhibits V-ATPase activity (Zhang et al., 1992a) and two proteins (MW
6000 and 35,000) that stimulate proton pumping (Zhang et al., 1992b; Xie et al., 1993). Small proteins have also been shown to modulate the activity of the related mitochondrial F0F1 ATPase (Pullman and Monroy, 1963). The diversity of mechanisms that regulate V-ATPase activity emphasizes the key role of acidification in cellular homeostasis. Moreover, this complexity may explain why the role of acidification in protein traYc remains murky, as diVerent approaches to perturb pH may have variable eVectiveness in diVerent cell types or compartments (see Section IV).

B. Counterion Conductance and Proton Leak As described above, the steady-state pH of organelles results from the balance between the rates of intralumenal proton pumping, counterion conductance, and intrinsic proton leak. As the V-ATPase pumps protons into a compartment, its ability to function further is theoretically limited by the accumulation of a positive membrane potential. In mammalian cells, passive anion flow through a chloride channel neutralizes this potential to allow acidification to continue (Glickman et al., 1983; Xie et al., 1983; Arai et al., 1989; Sonawane et al., 2002). The importance of this counterion conductance in regulating organelle pH is not clear, and probably varies for diVerent compartments (Rybak et al., 1997). In phagosomes, the intrinsic counterion permeation is considerably higher than proton pumping activity, suggesting that the membrane potential in these organelles is insignificant (Lukacs et al., 1991). It was proposed instead that V-ATPase activity is intrinsically

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inhibited as acidification proceeds, possibly by a kinetic or allosteric eVect, so that acidification does not proceed beyond a certain point (Lukacs et al., 1991). However, in some systems, it appears that counterion conductance can modulate pH. For example, maximal acidification of endosomes isolated from proximal tubules occurred only when chloride ions were present, and inhibitors of chloride channel activity reduced acidification (Marshansky and Vinay, 1996). Intriguingly, the chloride conductance in endosomes (Lukacs et al., 1992; Bae and Verkman, 1990) and coated vesicles (Mulberg et al., 1991) can be modulated by protein kinase A-dependent phosphorylation, suggesting that this could be a means to regulate the extent of organelle acidification. Moreover, the secretagogue thyrotropin was shown to induce acidification of secretory granules in parafollicular cells by increasing chloride permeability (Barasch et al., 1988). Additional evidence for the role of counterion conductance in regulating organelle pH comes from studies of CLC chloride channel family members. A primary manifestation of Dent’s disease, which is caused by mutations in the CLC-5 chloride channel, is the inability to reabsorb low-molecularweight proteins and albumin in the kidney (Marshansky et al., 2002). This phenotype is consistent with a defect in endosomal acidification, and indeed, endosomes from CLC-5 knockout mice acidify more slowly than wild-type endosomes (Piwon et al., 2000). In addition, the CLC-3 chloride channel has recently been suggested to play a role in lysosome acidification (Li et al., 2002). Finally, Barasch et al. (1991) have suggested that chloride permeability of some organelles in epithelial cells is diminished in cystic fibrosis cells, which lack a functional chloride channel (the cystic fibrosis transmembrane conductance regulator), although the eVect of this on organelle pH is in dispute (Weisz, 2003). In addition to a chloride counterion conductance, the Na+, K+-ATPase has also been suggested to modulate acidification. In this case, the extent of acidification would be limited by electrogenic Na+ pumping into the lumen of organelles. Inhibition of acidification by Na+, K+-ATPase in early endosomes but not in later endocytic compartments has been proposed to explain the diVerence in pH between these organelles (Cain et al., 1989; Fuchs et al., 1989). However, this mechanism does not appear to operate in all cells (Sipe et al., 1991; Anbari et al., 1994). In fact, this pump may enhance acidification in some cases, as other investigators have suggested that the Na+, K+-ATPase is required to supply lumenal Na+ for NHE-3-mediated acidification of endosomal compartments in some cell types (D’Souza et al., 1998; Marshansky and Vinay, 1996). More recent studies and models have stressed the role of intrinsic proton leak rates in the steady-state maintenance of organelle pH (Grabe and Oster, 2001; Wu et al., 2001; Schapiro and Grinstein, 2000; van Dyke and Belcher, 1994). Using organelle-specific pH-sensitive probes, Wu et al.

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(2001) recently measured the pH of various compartments along the regulated secretory pathway. A successive decrease in pH was found between the ER (pH
7.4), Golgi (pH
6.2), and mature secretory granules (pH
5.5); modeling studies suggested that the decrease in pH in each compartment was due to a corresponding decrease in proton leak rate coupled with an increased V-ATPase pump density (Wu et al., 2001). The proton eZux rate in the TGN has also been measured using other techniques and found to be quite high, but lower than that of earlier Golgi compartments, consistent with the greater acidity of the TGN (Demaurex et al., 1998). Interestingly, the leak rate was sensitive to inhibition by Zn2+, suggesting that a membrane protein rather than lipid composition may regulate the rate of proton eZux in diVerent compartments (Schapiro and Grinstein, 2000). C. Proton Flux and pH Changes The pH of a compartment is classically defined as the negative log of the hydrogen ion concentration; for example, the proton concentration in a solution of pH 6.0 is 106M, or 1 mM. Given the small volume of cellular organelles, theoretical calculations suggest that sizable changes in pH might be achieved by the influx or eZux of only a few protons. For example, in an aqueous solution, approximately 60 free protons would generate a pH of 6 in a compartment with a volume of 1016 liters (roughly the size of an endosome). Similarly, because the pH scale is logarithmic,
600 free protons would result in a pH of 5. However, these simplistic calculations fail to take into account the sizable concentration of proteins, lipids, and other molecules in cellular compartments that bind to protons and buVer organelle pH. For example, the pKa of histidine residues is
6, and at lower pHs carboxyl and phosphate groups begin to be protonated. In addition, bicarbonate and other membrane-permeant molecules contribute significantly to the buVering capacity within organelles. In this context, a pH of 6 in an endosome that contains 10 mM buVer means that there might be approximately 600,000 pH buVer molecules that are buVering about 60 free protons at any given time. This intrinsic buVering capacity thus stabilizes organelle pH and prevents wild swings in the pH of a compartment when the balance between proton pumping and leak rates is altered.

III. Tools to Measure Organelle pH Numerous techniques to quantitate organelle pH have been developed over the past several decades. Below, we summarize some of the past and current methodologies that have been adapted to measure the pH of organelles along

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the biosynthetic and endocytic pathways. There have been recent dramatic advancements in our ability to selectively target pH-sensitive probes to specific intracellular compartments, allowing measurement of pH at the suborganellar level. These new approaches continue to support the earlier results made using various other methods. Thus, we have a clear idea of the steady-state pH of most intracellular organelles. Nevertheless, our understanding of how pH is regulated, both at steady-state conditions and by signaling pathways is only just beginning to be investigated. The recent advancements in pH measurement should allow more rapid progress in these areas.

A. Visualization of Acidic Compartments Using DAMP The use of 3-(2,4-dinitroanilino)- 30 -amino-N-methyldipropylamine (DAMP) to identify acidic organelles was pioneered by Anderson et al. in 1984 (see Dunn et al., 1994 for review). This reagent is a weak base that rapidly diVuses into intact cells and concentrates in acidic organelles. Upon fixation, antibodies directed against dinitrophenol can be used to localize the accumulated DAMP, and acidified compartments can then be visualized after incubation with secondary antibodies coupled to fluorophores, gold, or peroxidase. In addition to labeling all components of the endocytic pathway, DAMP was found to label trans-Golgi cisternae, and provided the first demonstration that this compartment is normally acidified (Schwartz et al., 1985; Anderson and Pathak, 1985). However, this method is generally considered to be an unreliable reporter of pH because although DAMP accumulates in organelles in a pH-dependent fashion, the eYciency of DAMP fixation varies depending on the amount of protein in each organelle.

B. Acid-Sensitive Proteins as pH Probes Early attempts to quantitate the pH of organelles along the biosynthetic pathway made use of proteins whose conformation or posttranslational modifications are sensitive to pH. For example, recognition by conformation-sensitive antibodies and the acquisition of trypsin susceptibility have been used to establish the lowest pH to which newly synthesized influenza hemagglutinin (HA) is exposed en route to the cell surface (pH 5.2–5.6: Grambas and Hay, 1992; below pH 6.0: Boulay et al., 1987). Similarly, the extent of processing of secretogranin II in isolated immature secretory granules (ISGs) incubated under physiological conditions was compared with its processing in ISGs equilibrated at a range of pH values; using this standard curve, the intragranular pH of this compartment was estimated to be
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(Urbe et al., 1997). Although the results obtained using these elegant approaches generally agree with those obtained using other methods, these techniques are quite cumbersome, and more recent advances in selectively targeting pH-sensitive fluorophores to intracellular compartments have made it far simpler to assess pH in these organelles.

C. Fluorescence Ratio Imaging Most methods to measure organelle pH involve some version of the ratio imaging approach described by Ohkuma and Poole (1978). In this technique, a probe whose fluorescence is pH sensitive is staged or accumulated in the compartment of interest. Traditionally, fluorescein has been the molecule of choice with which to quantitate pH in acidified compartments, as the emission intensity produced upon fluorescence excitation at 490 nm is highly dependent on pH. Because the fluorescence exited by 450-nm light is pH independent, the ratio of the fluorescence excited by 490-nm light divided by that excited by 450-nm light provides a quotient that is sensitive to pH, but independent of the other variables that aVect the amount of detected fluorescence, e.g., the concentration of the probe, photobleaching, and focal length. The actual pH of a compartment can then be determined by fitting the obtained ratios against a calibration curve constructed by measuring the fluorescence ratio of the probe at various defined pH values. Because the pH-sensitive emission of fluorescein decreases significantly as pH rises, ratio imaging using this fluorophore is most accurate for measuring pHs between
5.0 and 6.0. Other pH probes with lower pKas, such as those based on Oregon Green (pKa
5), can also be used to quantitate the pH of very acidic compartments, such as phagosomes (Vergne et al., 1998; Lin et al., 1999). The general principles behind ratio fluorescence microscopy are described more fully in Dunn et al. (1994). 1. Intracellular Targeting of Fluorescent Probes Several methods have been used to stage fluorophores in individual compartments to allow pH measurements of individual compartments in live cells. Fluorophore-conjugated diethylaminoethyl (DEAE) dextran, a fluid phase marker that is eYciently delivered to lysosomes and accumulates without degradation, is commonly used to assess the pH of that compartment. Similarly, expression of the transferrin receptor (TfR) has been widely exploited to measure the pH of various endocytic compartments. This is typically accomplished by internalizing fluorescein-rhodamine-coupled transferrin for various periods to stage the fluorophores in either sorting or recycling endosomes (Yamashiro and Maxfield, 1987; Yamashiro et al.,

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1984). Alternatively, internalized fluorescent conjugated antibodies directed against proteins can be used to measure the pH of acidified compartments: for example, antibodies against a2-macroglobulin and the asialoglycoprotein receptor (ASGPR) have been used to measure lysosomal and endosomal pH, respectively (Tycko et al., 1983; Tycko and Maxfield, 1982). Several laboratories have developed elegant approaches to accumulate fluorescent conjugates in various biosynthetic compartments. To measure Golgi pH, Kim et al. (1996) exploited the retrograde traYcking route taken by verotoxin B subunit. This toxin binds to glycolipid receptors at the plasma membrane and is eYciently transported to the Golgi complex, where it transiently accumulates. The Golgi pH measured by ratio imaging of internalized fluorescein isothiocyanate (FITC)-conjugated verotoxin B was 6.45: however, this reflects contributions from all Golgi cisternae and thus represents an average value (Kim et al., 1996). To selectively measure the pH of the TGN, Demaurex et al. (1998) generated a cell line expressing a chimeric protein encoding the luminal domain of the interleukin 2 receptor (CD25) coupled to the transmembrane and cytoplasmic domains of TGN38. Like wild-type TGN38, this chimeric protein resides in the TGN at steady state, but rapidly recycles through the plasma membrane. Internalization of FITC-conjugated anti-CD25 antibodies by Chinese hamster ovary (CHO) cells expressing this chimera resulted in their accumulation in the TGN and ratio imaging was used to obtain a pH of 5.95 for this compartment (Demaurex et al., 1998). In another creative approach, Wu et al. (2000) engineered chimeras of avidin fused to various targeting signals to localize the protein to the ER, Golgi complex, and secretory granules. Cells expressing this protein were then incubated with a membrane-permeant FITC-biotin conjugate to selectively label the compartment of interest and allow pH quantitation (Wu et al., 2000, 2001). In a similar technique, Verkman’s group expressed single chain antibodies directed against 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one and fused to intracellular targeting sequences in CHO cells (Farinas and Verkman, 1999). Incubation of these cells with membrane-permeant fluorescent conjugated antihapten antibodies resulted in their specific accumulation at intracellular sites and was used to measure an average Golgi pH of 6.25 (Farinas and Verkman, 1999). 2. Phluorins: pH-Sensitive Green Fluorescent Proteins More recently, the intrinsic pH-sensitive fluorescence of green fluorescent protein (GFP) has been exploited to generate sensitive probes with to measure pH (Kneen et al., 1998; Miesenbock et al., 1998). In particular, mutant GFPs with reversible excitation ratio changes between pH 7.5 and 5.5 have been developed that allow more accurate measurement of pH at near neutral

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pH, where standard FITC-based ratio imaging is not feasible. Several groups have now reported the use of targeted GFP-based constructs to measure the pH of various compartments, including the ER, Golgi, TGN, mitochondria, peroxisomes, and secretory granules (Kneen et al., 1998; Miesenbock et al., 1998; Blackmore et al., 2001; Llopis et al., 1998; Jankowski et al., 2001; Poschet et al., 2001; Chandy et al., 2001). Given the tremendous flexibility that such probes oVer, it is likely that this approach will become the method of choice to measure pH in the near future.

D. Liposome-Mediated Delivery of Fluorescent Probes An alternative approach to measure pH makes use of the propensity of liposomes microinjected into cells to fuse selectively with the Golgi complex. Using this approach, Seksek et al. (1995, 1996) were able to deliver soluble FITC and rhodamine into the lumen of the Golgi complex and measure the pH of this organelle. The fluorophores colocalized with a trans-Golgi marker leading the authors to conclude that the liposomes selectively fused with this region of the Golgi complex; however, colocalization with markers of earlier Golgi compartments was not tested to confirm this definitively (Seksek et al., 1995, 1996).

IV. Methods to Perturb Organelle pH A variety of reagents and techniques have been used to perturb the pH of acidified organelles. The majority of these treatments aVect the pH of all acidified compartments in a cell, and thus cause significant disruption of multiple cellular functions. In addition, many of these reagents have additional eVects on cells that are unrelated to changes in organelle pH. The variability in the extent of pH disruption and in the side eVects caused by diVerent treatments certainly contributes to the discrepant results obtained experimentally using distinct methods to perturb pH, and hinders our ability to dissect the role of acidification in membrane traYc (described in later sections).

A. Weak Bases and Lysosomotropic Agents Weak bases are amine-containing compounds with a pKa in the physiological range. Most of the molecules exist as neutral, nonprotonated forms at alkaline or neutral pHs, and can freely diVuse across cell membranes. However,

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at acidic pHs the molecules are rapidly protonated and become unable to permeate membranes. Mass action drives the accumulation of weak bases in acidified compartments, where protons are readily available. Protonation of the weak bases in these compartments consumes the available protons, resulting in alkalinization. Changes in organelle pH occur very rapidly (<1 min) upon weak base addition, whereas the drugs continue to accumulate within acidified compartments for minutes to hours (Ohkuma and Poole, 1978). This slow concentration of weak bases probably reflects the neutralization of the intrinsic buVering capacity of a compartment (by driving the dissociation of labile protons from amino acids and sugars from resident proteins) as well as consumption of protons that continue to be pumped in by the V-ATPase. Weak bases that accumulate in acidic compartments have been collectively referred to as lysosomotropic agents (de Duve et al., 1974). Those that are commonly used to perturb organelle pH include chloroquine, ammonium chloride, methylamine, monodansylcadaverine, and primaquine. Typically, these drugs are added to cells at millimolar concentrations (generally between 5 and 50 mM), except for the chloroquine, which is dibasic and requires only micromolar concentrations to act. It should be noted, however, that many other membrane-permeant amine-containing drugs that are meant to target specific cellular processes can also disrupt organelle acidification when added to cells at high concentrations. Two notable examples of this include the antiviral agent amantadine (Ohkuma and Poole, 1978) and the sodium channel/sodium-hydrogen exchanger inhibitor amiloride (Dubinsky and Frizzell, 1983). Although incubation with weak bases is an easy way to perturb the pH of intracellular acidified compartments, these reagents have many other eVects on cell function that can indirectly disrupt membrane traYcking. The rate and eYcacy with which these drugs penetrate membranes and accumulate in intracellular compartments vary, and can lead to diVerences in the rate and extent of alkalinization that are achieved (Ohkuma and Poole, 1978; Young et al., 1981). In addition, osmolarity increases as protonated weak bases accumulate in acidified compartments, causing dramatic swelling and vacuolation (Wibo and Poole, 1974; Seglen and Reith, 1977). Moreover, whereas the eVects of ammonium chloride and methylamine on organelle pH are rapidly reversible (Tietze et al., 1980; Ohkuma and Poole, 1978), chloroquine is only slowly reversible (Ohkuma and Poole, 1978). In addition to their direct eVects on organelle pH, weak bases also perturb other cellular functions. For example, chloroquine has been demonstrated to inhibit lysosomal phospholipases A1 and A2 (Hostetler and Richman, 1982; Kubo and Hostetler, 1985); these eVects were observed in vitro and are independent of chloroquine’s eVects on pH. In addition, chloroquine inhibits insulin-independent insertion of GLUT4 into the plasma membrane of

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adipocytes via a pH-independent mechanism (Romanek et al., 1993), and primaquine has been suggested to aVect membrane recycling in a pHindependent fashion (van Weert et al., 2000; Dunn et al., 1989). Other amines, such as methylamine, have been found to inhibit transglutaminase activity (Davies et al., 1980) and the AE2 anion exchanger, a key regulator of cytoplasmic pH in some cells, can be activated by low concentrations of ammonium chloride (Bonay et al., 1996). Although these enzymes may not directly regulate membrane traYc, diVerential eVects of weak bases on the function of these and other proteins could indirectly contribute to the discrepancies observed in studies using diVerent weak bases. Addition of weak bases to cells (ammonium chloride in particular) causes a rapid but transient elevation in intracellular pH that is followed by significant acidification of the cytosol (overshoot) when the drug is removed (Boron and De Weer, 1976). In fact ammonium chloride is often used experimentally to acidify cytosolic pH either transiently (Chen and Boron, 1991, 1995a,b) or tonically (Sandvig et al., 1987, 1988). Acidification of the cytosol can result in defective assembly of clathrin coats at the plasma membrane (Heuser, 1989) as well as the redistribution of endocytic compartments within some cell types (Parton et al., 1991). Functionally, acidification of the cytoplasm has been demonstrated to disrupt membrane traYc along the endocytic and biosynthetic pathways (Sandvig et al., 1987, 1988; Samuelson et al., 1988; Cosson et al., 1989). Given the considerable variability in how cells are exposed to weak bases, the regimen of drug treatment could contribute to the eVects on membrane traYc observed by diVerent experimenters using the same reagent.

B. Perturbation of pH by Proton Ionophores, Exchangers, and Symporters The proton-selective ionophore gramicidin, as well as nigericin (an ionophore permeable to both K+ and H+), rapidly dissipates proton gradients across lysosomal membranes of isolated lysosomes (Reijngoud et al., 1976). In addition, the Na+/H+ exchanger monensin has been demonstrated to raise the pH of acidified compartments, and aVects protein transport through the biosynthetic and endocytic pathways (TartakoV et al., 1978; Maxfield, 1982; TartakoV, 1983). The action of monensin is rapidly reversible; Maxfield (1982) demonstrated that intracellular endocytic vesicles reacidified to steady-state levels within 1 min of monensin removal. In addition to these ionophores, the prodigiosins, a family of bacterially produced pigments, have also been demonstrated to raise lysosomal pH and aVect the biosynthetic processing of glycoproteins (Kataoka et al., 1995; Ohkuma et al., 1998). These and related compounds appear to act as H+/Cl symporters

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that serve to nonselectively uncouple proton pumping from ATPase activity (Ohkuma et al., 1998; Matsuya et al., 2000). Interestingly, the ionophores, exchangers, and symporters can cause diVerential swelling of acidified organelles including the Golgi complex and endosomes, most likely as a result of diVerences in the intrinsic ion permeabilities of these compartments. For example, monensin has a dose-dependent eVect: at low concentrations (1 mM ), only the Golgi complex is dilated (Danielsen et al., 1983), whereas at higher concentrations (10 mM), endosomal swelling has also been observed (Stein et al., 1984). By contrast, the prodigiosins cause dramatic swelling of mitochondria as well as the Golgi complex, although which ions cause these eVects is not yet known (Kataoka et al., 1995; Ohkuma et al., 1998). Although these reagents provide a rapid means of disrupting organelle pH, they are incorporated nonselectively into all membranes, and can also disequilibrate the organellar concentrations of other ions; thus, they are of limited use for examining the role of pH in membrane traYcking. More specific perturbants are described below.

C. Inhibition of V-ATPase Activity by Macrolide Antibiotics and Other Drugs Bafilomycin A1 and concanamycins A and B are related membrane-permeant microbial metabolites that inhibit V-ATPase activity at low concentration and with high selectivity (Bowman et al., 1988; Dro¨ se et al., 1993; Dro¨ se and Altendorf, 1997). The exact mechanism by which these drugs disrupt V-ATPase function is not yet clear; however, proton conduction by the pore is inhibited by bafilomycin A1 (Crider et al., 1994; Zhang et al., 1994). This drug binds tightly though noncovalently to the V-ATPase, although the identification of the target subunit has been controversial, with diVerent groups reporting the 100- to 115-kD a subunit and the 14- to 17-kDa proteolipid c subunit as the binding sites (Zhang et al., 1994; Rautiala et al., 1993; Bowman and Bowman, 2002). Concanamycin A had also recently been reported to bind to the c subunit of V0 (Huss et al., 2002). Unlike lysosomotropic agents or ionophores, these agents do not typically cause swelling of acidified compartments, although vacuolization of the Golgi has been noted in one case (Palokangas et al., 1994). Another class of V-ATPase inhibitors is the destruxins: several members of this family of fungal metabolites exhibit reversible, uncompetitive inhibition of some V-ATPases (Bandani et al., 2001; Muroi et al., 1994). However, these drugs are of limited use for pH perturbation studies, as they require somewhat higher concentrations than the macrolide antibiotics and are not as selective as other V-ATPase inhibitors (Muroi et al., 1994; Murata et al., 1997). More promising are some recently described members of another structural

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class of compounds, the benzolactone enamides, which have recently been shown to potently inhibit mammalian V-ATPase with high selectivity (Boyd et al., 2001). Despite their exquisite selectivity and other advantages, the use of macrolide antibiotics to inhibit V-ATPases presents several complications. First, the eVects of these inhibitors are generally irreversible: bafilomycin A1 binds to purified V-ATPase with a dissociation constant of approximately 10 nM and remains bound to V-ATPase upon gradient fractionation of membranes. Thus, de novo synthesis of subunits may be required to restore V-ATPase function (Hanada et al., 1990; Mattsson and Keeling, 1996). Nevertheless, although many investigators find no recovery of membrane transport rates upon removal of macrolide antibiotics (Yilla et al., 1993; van Weert et al., 1995), some studies have reported rapid reversal of the drug’s eVects when low concentrations (100 nM; 5- to 10-fold lower than the standard concentration) of bafilomycin A1 were added to cultured cells (Yoshimori et al., 1991; Harada et al., 1996). Second, there is accumulating evidence that the heterogeneous subunit composition and localization of V-ATPases confer diVerent pharmacological sensitivity to inhibitors. One striking example in mammalian cells is the diVerential sensitivity organelle V-ATPases to inhibition by megalomicin, a macrolide antibiotic that disrupts protein traYc. Megalomicin added to cells at micromolar concentrations (50 mM ) has dramatic eVects on both biosynthetic and endocytic traYc (Bonay et al., 1996, 1997). However, whereas this drug significantly inhibits lysosomal V-ATPase activity at submicromolar concentrations, it has little eVect on the V-ATPase activity of a Golgienriched membrane fraction even at 500-fold higher concentrations (Bonay et al., 1996, 1997). DiVerential pharmacological sensitivities of V-ATPases isolated from osteoclasts and tobacco cell organelles have also been described (Chatterjee et al., 1992; Matsuoka et al., 1997; Farina and Gagliardi, 2002). Third, as with other pH perturbants, some eVects of macrolide antibiotics are independent of organelle pH changes. Kinoshita et al. (1996) reported that bafilomycin A1, but not other pH perturbants, induces apoptosis in PC12 cells independently of its eVects on organelle pH. In addition, as many cells express V-ATPase at the plasma membrane, treatment with these drugs can alter cytoplasmic as well as organelle pH (Heming et al., 1995; Wu and Delamere, 1997; Nordstrom et al., 1995). Recent data suggest that the role of V-ATPases in regulating membrane traYc may be more complex than originally envisioned. For example, the V-ATPase has been found to associate with synaptobrevin and synaptophysin in a detergent-resistant complex, suggesting a potential role in regulating SNARE complex formation (Galli et al., 1996). Moreover, a model in which homotypic vacuolar fusion is mediated by the yeast V-ATPase V0 pore complex has recently been proposed (Peters et al., 2001; Muller et al., 2002). The 16-kDa proteolipid subunit of V0 can

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also bind to b1 integrin (Skinner and Wildeman, 1999), and has also been reported to function as a gap junction and neurotransmitter release channel in some organisms (Brochier and Morel, 1993; Finbow et al., 1994). Although it is not known whether macrolide antibiotics can aVect these diverse functions of V-ATPase subunits, there is a significant possibility that treatment with these drugs could perturb pH-independent functions of this complex. Such side eVects may account for some of the diVerence observed upon treatment with macrolide antibiotics compared with other pH perturbants. Fourth, in some cases, these drugs may prove ineVective at chronically altering pH. For example, Dictyostelium discoideum overcame the eVects of concanamycin A after only 1 h of drug treatment (Temesvari et al., 1996). This recovery did not require protein synthesis, and included the reacidification of endolysosomal compartments and the restoration of normal endocytic and exocytic rates (Temesvari et al., 1996). Similarly, Plant et al. (1999) demonstrated that yeast lacking V-ATPase activity were able to regulate vacuolar pH under some conditions, suggesting the existence of alternative pathways to acidify this compartment. Although these examples come from lower eukaryotes, it is possible that mammalian cells may also be able to adapt under some conditions.

D. Selective Perturbation of Organelle pH by Expression of a Proton-Selective Channel A significant problem with all of the pH perturbants discussed above is that they nonselectively aVect the pH of all acidified subcellular compartments; in this sense, they can be classified as ‘‘global’’ pH perturbants. This nonselectivity makes it is impossible to gauge the eVect of acidification on a single step in protein transport along a pathway that contains multiple acidified compartments. Because no mechanism exists to target pH perturbing drugs to particular organelles, our laboratory initiated a diVerent strategy to attempt to perturb the pH of subsets of intracellular acidified compartments. We reasoned that cellular expression of a potentially targetable protein that disrupts pH gradients could provide a means of controlling pH at distinct intracellular sites. To this end, we used replication-defective recombinant adenovirus to express an acid-activated ion channel, the M2 protein of influenza virus type A in mammalian cells (Ogden et al., 1999; Whittaker et al., 1996). This 97 amino acid nonglycosylated integral membrane protein forms tetrameric pores that are highly selective for protons over other small cations, and several laboratories have examined the mechanism of proton conduction by this channel in detail (Sugrue and Hay, 1991; Shimbo et al., 1996; Chizhmakov et al., 1996; Lin and Schroeder, 2001; Mould et al., 2000a,b; Wang et al., 1995a; Pinto et al., 1992, 1997; Sansom et al.,

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1997; Tang et al., 2002). M2 conductance significantly increases as luminal pH decreases but is much less sensitive to changes in cytoplasmic pH (Mould et al., 2000a; Pinto et al., 1992; Holsinger et al., 1994; Shimbo et al., 1996; Chizhmakov et al., 1996). Thus, in essence, M2 selectively increases the protein leak rate across acidified compartments, thereby increasing their luminal pH. Given the established role of proton leak rate in regulating organelle pH, targeted expression of M2 would seem to be a logical strategy to eVect significant change in the pH of a subgroup of acidified compartments. M2 activity is readily inhibited by the drugs amantadine (at concentrations far below this drug’s use as a weak base) and BL-1743; therefore the eVects of M2 on organelle pH can be rapidly and reversibly modulated (Pinto et al., 1992; Wang et al., 1995b; Tu et al., 1996). Furthermore, amantadine-sensitive acid-activated proton conductance is reconstituted when synthetic peptides encoding the transmembrane region of M2 are incorporated into lipid bilayers (DuV and Ashley, 1992). This suggests the possibility that mutagenesis of other domains of M2 to incorporate known targeting motifs may be used to retarget the protein without a dramatic eVect on M2 function. High levels of M2 expression have been demonstrated to cause vacuolation of the Golgi complex (Cmarkova et al., 1995; Sakaguchi et al., 1996) and to alter the pH of this organelle (Ciampor et al., 1992a,b; Grambas and Hay, 1992). Functionally, M2 expression aVects the biosynthetic transport in a manner consistent with alkalinization of the TGN (Takeuchi and Lamb, 1994; Ciampor et al., 1992a; Ohuchi et al., 1994; Sakaguchi et al., 1996; Henkel and Weisz, 1998; Henkel et al., 1999, 2000). M2 expressed in polarized MDCK cells by infection with replicationdefective recombinant adenovirus colocalizes with markers of two intracellular acidified compartments: the TGN and a slightly acidic subapical endocytic compartment termed the apical recycling endosome (ARE; Henkel et al., 1998; Apodaca et al., 1994; Wang et al., 2000). It is not clear yet whether M2 is concentrated in these compartments or whether the observed localization pattern reflects constitutive transport of itinerant M2 en route to other destinations, such as the plasma membrane, where M2 clearly accumulates (Henkel et al., 1998; Lamb et al., 1985). It is worth noting that the cytoplasmic tail of the Rostock isoform of M2 (which has more profound eVects on Golgi pH than other isoforms; Grambas and Hay, 1992; Grambas et al., 1992; Ciampor et al., 1992b) contains the sequence YRRL, which like the YQRL motif essential for targeting of the TGN resident protein TGN38 can bind avidly to the m2 subunit of AP-2 (Boll et al., 1996). However, whether this motif is important for Rostock M2 localization is not known. Early experiments demonstrated that expression of M2 inhibits the rate of recycling of an apical but not a basolateral marker in polarized cells, and showed that lysosomal function is unaVected by even very high levels of M2 expression, thus validating the use of M2 as a selective pH perturbant

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(Henkel et al., 1998, 1999). In subsequent studies, we have used this approach to examine multiple aspects of biosynthetic and endocytic membrane traYc. These experiments are described in more detail in later sections. Although the use of M2 to perturb cellular pH oVers some significant advantages over other methods, there are several issues that remain to be addressed more fully and technical hurdles that must to be overcome. First, it should be noted that at reasonable expression levels, M2 accumulates at the cell surface. Because the cells are bathed in neutral to slightly alkaline medium, M2 should be minimally active at the plasma membrane; nevertheless, expression of M2 has been reported to alter cytosolic pH (Shimbo et al., 1996; Ciampor et al., 1992b). In addition, to date we have been unsuccessful in retargeting M2 to other organelles by site-directed mutagenesis to incorporate known targeting motifs. Although we have not investigated this in detail, one possibility is that the tetrameric structure of M2 hinders signal recognition by adaptor-based sorting machinery. Another potential complication is that significant alteration of the M2 cytoplasmic tail sequence might aVect its ion channel gating, as recent studies have found decreases in channel open times in M2 constructs with truncated cytoplasmic tails (Tobler et al., 1999). Thus, although promising, the broad applicability of influenza M2 expression as an organelle-selective pH perturbant remains to be demonstrated. V. Role of Acidification in Endocytic Membrane Traffic Internalization of proteins, lipids, and soluble ligands and their transport along the endocytic pathway is critical for numerous cell functions, including uptake of essential nutrients, regulation of plasma membrane receptors, and modulation of signal transduction. This process has been the subject of an outstanding and very comprehensive review by Mukherjee et al. (1997). In this section, we will discuss the role of acidification at each stage along the endocytic pathway (internalization, recycling, delivery to late endosomes and lysosomes, and degradation). In addition, we will examine the role of acidification in retrograde traYc of proteins from the cell surface to the TGN. Finally, recent advances that provide insight as to the mechanism by which acidification perturbs endocytic traYcking will be reviewed. A. Internalization from the Plasma Membrane 1. Specialized Forms of Internalization In addition to the internalization pathways described below, some cells, particularly cells of the immune system, have specialized mechanisms to ingest large particles by phagocytosis, a process in which a membranous

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structure encapsulates the particle and retrieves it into a cytoplasmicbounded vacuole called a phagosome (Tjelle et al., 2000). Subsequently, phagosomes undergo a maturation process that involves fusion with endocytic organelles and ultimately with lysosomes. During this process, they gradually become acidic (the pH of phagosomes in macrophages has been variously reported to be between
4.5 and >6.0; Lukacs et al., 1990; Vergne et al., 1998). This V-ATPase-mediated acidification likely plays a role in bacterial killing. Many intracellular pathogens, including Mycobacteria, Legionella, and Toxoplasma, live in and multiply in phagosomes, and appear to limit the acidification of these compartments (Sinai and Joiner, 1997). In addition to phagocytosis, some cells of the immune system as well as other motile cells are able to capture large volumes of extracellular fluid in large irregular vesicles (macropinosomes) that are formed by fusion of lamellipodia. Interestingly, macropinosomes appear to have distinct fates in diVerent cell types. Whereas in many cells these structures ultimately release their contents extracellularly after fusion with the plasma membrane, macropinosomes in macrophages gradually shrink and acidify, and like phagosomes, ultimately merge with lysosomes (Racoosin and Swanson, 1993; Swanson and Watts, 1995). The role of acidification in macropinosome formation is not known but could be related to the role of macrophages in antigen capture and presentation. 2. Clathrin-Dependent and -Independent Endocytosis The first step in endocytosis of small molecules is internalization from the plasma membrane. This can occur via three known mechanisms: a clathrin-dependent pathway mediated by the formation of clathrin-coated vesicles (sometimes called micropinosomes), a clathrin-independent pathway involving formation of uncoated vesicles, and internalization of caveolae. A large proportion of internalization of receptors and ligands occurs via clathrin-coated vesicles. Receptors are recruited into clathrin-coated pits and drive the membrane invagination and clathrin rearrangement required to pinch oV 100-nm vesicles into the cytoplasm. These vesicles are then uncoated and can acidify prior to fusion with early endosomes (Xie et al., 1983; Yamashiro et al., 1983). Clathrin-coated vesicles (CCVs) can be purified to near homogeneity from several sources. CCVs from brain have been demonstrated to contain V-ATPase subunits (Forgac et al., 1983), although another report found no evidence for V-ATPase-dependent acidification in CCVs isolated from rat liver (Fuchs et al., 1994). Although there does not appear to be a direct role for acidification in clathrin-mediated internalization (Harford et al., 1983a,b; Schwartz et al., 1984; Ciechanover et al.,

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1983a), the sustained block of acidification of other endocytic compartments ultimately leads to inhibition of ligand uptake (see below). A clathrin- and dynamin-independent internalization pathway has also been demonstrated in many cell types. Interestingly, this type of internalization is upregulated in cells that are blocked in clathrin-mediated endocytosis, suggesting that it can serve as an alternative compensatory pathway (Damke et al., 1995). To date, no role for acidification in this endocytic mechanism has been described. Caveolae are small omega-shaped invaginations associated with the coat protein caveolin. Although these structures have been implicated in a wide variety of signaling events at the plasma membrane, in many cell types these invaginations can also pinch oV to form discrete cytoplasmic vesicles. Internalization of 5-methyltetrahydrofolate mediated by the folate receptor appears to occur via caveolae, and several toxins and viruses have been demonstrated to be internalized primarily in these structures (Norkin, 2001). In addition, caveolae have been suggested to function as transcytotic carriers in endothelial cells (Mineo and Anderson, 2001). The term potocytosis is now generally used to describe internalization mediated by the caveolae (Mineo and Anderson, 2001). Isolated caveolae were shown to contain the 70-kDa subunit of the V-type ATPase (Mineo and Anderson, 1996) and it appears likely that caveolae can acidify; however, the role of acidification in caveolae-mediated traYc is unclear. Although the eVect of pH perturbants on internalization of toxins and viruses has not yet been examined, treatment of monkey kidney MA104 cells with bafilomycin A1 blocked folate receptormediated transport of 5-methyltetrahydrofolate, demonstrating a role for acidification in internalization via this route (Mineo and Anderson, 1996). However, the requirement for acidification in this case is probably due to the requirement for a proton gradient to drive the folate transporter that mediates cytoplasmic uptake of folate (Prasad et al., 1994).

B. Protein and Lipid Recycling to the Cell Surface Historically the role of acidification in endocytosis and recycling has been characterized by examining the eVect on traYcking of both receptors and ligands when organelle pH is perturbed. Many receptors undergo multiple rounds of internalization and recycling prior to degradation. After internalization, ligand dissociation from receptors is induced by the low pH encountered in sorting endosomes. The ligand then behaves as a fluid-phase marker and is eYciently delivered to lysosomes via late endosomes, whereas the receptor traYcs via tubular recycling endosomes back to the plasma membrane (Yamashiro et al., 1984; Mukherjee et al., 1997). Two receptors that have been used to characterize this route in detail are the hepatic

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ASGPR and the transferrin receptor (TfR). The mammalian ASGPR binds to terminal galactose residues that gradually become exposed on serum glycoproteins and clears these proteins from the circulation (Ashwell and Morell, 1974). Proteins bound to the ASGPR dissociate from the receptor at endosomal pH and are delivered to lysosomes for degradation, whereas the ASGPR eYciently recycles to the plasma membrane (Spiess, 1990; Ciechanover et al., 1983b). The TfR is a ubiquitous receptor whose function is to regulate the cellular supply of iron (Lok and Loh, 1998). This receptor binds tightly to iron-loaded transferrin at neutral pH, e.g., at the cell surface (Dautry-Varsat et al., 1983). Upon internalization of the transferrin–TfR complex, the low pH of early (sorting) endosomes causes rapid dissociation of iron from transferrin. TfR binds to apotransferrin (transferrin that has lost its iron) more tightly at acidic pH than at neutral pH, thus ensuring that the apotransferrin is eYciently recycled to the plasma membrane, where the increased pH induces its dissociation and promotes binding of iron-loaded transferrin. How does perturbing pH aVect the traYcking of recycling proteins? Because ligand binding to and subsequent dissociation from many receptors is dependent on pH, understanding the role of pH in traYcking of these receptors requires examination of the fate of the receptor itself as opposed to the ligand. Numerous studies have demonstrated that blocking organelle acidification by lysosomotropic agents disrupts the uptake of transferrin (Klausner et al., 1983; Ciechanover et al., 1983a; Morgan, 1981) and inhibits uptake and degradation of asialoglycoproteins (Harford et al., 1983a,b; Schwartz et al., 1984). This is not surprising, as in the absence of any other eVect, inhibiting the dissociation of prebound ligands would block binding of new ligands upon receptor recycling to the plasma membrane. However, in some cases, pH perturbation also has a profound eVect on the traYcking and distribution of the receptors themselves. In the case of the ASGPR, the primary eVect of ammonium chloride, chloroquine, or primaquine was a rapid and profound loss of cell surface receptors and the intracellular accumulation of receptor and ligand in an endocytic compartment (Harford et al., 1983a; Schwartz et al., 1984; Zijderhand-Bleekemolen et al., 1987; Tolleshaug and Berg, 1979). The rate of internalization of asialoglycoprotein was unaVected, suggesting an eVect on receptor recycling (Harford et al., 1983a; Schwartz et al., 1984). More recently, Johnson et al. (1993) have documented a similar eVect of pH disruption on the recycling of the transferrin receptor. Similar eVects on the recycling of other receptors in diVerent cell types has also been observed; for example, monensin treatment inhibited recycling of the low-density lipoprotein receptor (Basu et al., 1981), chloroquine and ammonium chloride slowed recycling of the mannose receptor in macrophages (Tietze et al., 1980), and methylamine and monodansylcadaverine inhibited the recycling of the a2-macroglobulin receptor in fibroblasts

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and macrophages (Van Leuven et al., 1980; Kaplan and Keogh, 1981). Furthermore, scavenger uptake of secreted lysosomal hydrolases mediated by the mannose-6-phosphate receptor (M6PR) was also blocked by chloroquine, apparently because receptor recycling was impaired (Gonzalez-Noriega et al., 1980). From the above data, we might conclude that there is a general role for acidification in the eYcient membrane protein recycling to the plasma membrane. The importance of acidification in this traYcking step is less universal than initially envisaged however, as not all receptors respond to pH identically; for example, neither monensin nor ammonium chloride blocked ligand-associated Fc receptor recycling (Mellman et al., 1984). Moreover, diVerent pH perturbants appear to have distinct eVects on this pathway. This has been examined in greatest detail by Stoorvogel and colleagues, who compared the eVects of primaquine versus bafilomycin A1 on TfR (van Weert et al., 1995, 2000). Whereas both agents neutralized endosomal pH, only primaquine interfered with TfR recycling to the plasma membrane and it was concluded that the eVect of primaquine on recycling was pH independent (van Weert et al., 2000; Stoorvogel et al., 1987). Although the cause of the inhibitory eVect of primaquine was not identified, osmotic swelling of the endosomes by primaquine, which might alter iterative sorting of membrane proteins by disrupting endosomal morphology, was ruled out as a factor (van Weert et al., 2000). Thus, the importance of acidification in membrane recycling remains elusive.

C. Retrograde Transport to the TGN and Golgi Complex In many cell types, a fraction of internalized proteins is targeted via a retrograde pathway to the TGN. The extent to which proteins traverse this retrograde pathway depends on the protein as well as the cell type (Green and Kelly, 1990). The TGN resident proteins furin, TGN38, and VIP36/ MAL are localized to the TGN at steady state, but are constantly cycling through the plasma membrane (Ladinsky and Howell, 1992; Bos et al., 1993; Molloy et al., 1994; Chapman and Munro, 1994; Puertollano and Alonso, 1999). Similarly, a fraction of internalized TfR and ASGPR appears to pass through the TGN prior to their return to the cell surface (Snider and Rogers, 1985; Hull et al., 1991); however this appears to be a relatively minor pathway for TfR (Snider and Rogers, 1985) as well as most other recycling proteins (Reichner et al., 1988; Green and Kelly, 1990). Several studies have examined the eVect of pH perturbation on retrograde traYc to the Golgi complex. Stein and Sussman demonstrated the existence of two recycling pathways for TfR in K562 cells, one of which

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was sensitive to monensin. Their conclusion, though not directly tested, was that the monensin-sensitive pathway involved TfR recycling through the Golgi complex (Stein et al., 1984; Stein and Sussman, 1986). In an interesting twist, a more recent study found that pH perturbation with bafilomycin A1 induced retrograde transport of transferrin deeper into the Golgi complex in a subset of cells, and concluded that acidification of the TGN may normally function to limit such retrograde transport (van Weert et al., 1997). Other experiments have used morphological approaches to examine the eVect of pH perturbation on the traYcking of TGN-resident proteins, which normally recycle via the cell surface at significant rates. Treatment of NRK cells with either chloroquine or bafilomycin A1 resulted in redistribution of TGN38 (Chapman and Munro, 1994; Reaves and Banting, 1994) and furin (Chapman and Munro, 1994) to swollen early endosomal compartments. Endocytosis of the proteins was qualitatively similar to control in pHperturbed cells, however, the internalized proteins accumulated in endocytic compartments and failed to return to the TGN (Chapman and Munro, 1994; Reaves and Banting, 1994). Similar results were obtained for the TGN resident VIP36/MAL proteolipid transiently expressed in monensin, chloroquine, or ammonium chloride-treated COS-7 cells (Puertollano and Alonso, 1999). This block is similar to the failure of internalized receptors to recycle to the cell surface from early endosomal compartments (see above). Interestingly, however, glycolipids internalized from the plasma membrane do not appear to require acidic pH to traYc back to the Golgi complex. Schapiro et al. (1998) found that transport of verotoxin 1B, which traYcs from the plasma membrane to the Golgi in a complex with its glycolipid receptor globotriaosyl ceramide, was unaVected by addition of 100 nM concanamycin A plus 50 mM chloroquine. Similar results were obtained for cholera toxin, which is internalized with its receptor (the ganglioside GM1) via caveolae (Schapiro et al., 1998). Control experiments confirmed that furin was redistributed to vesicular compartments as expected by this treatment. Thus, retrograde traYc of proteins and glycolipids appears to be diVerentially sensitive to acidification. One diVerence between these two transport processes is that unlike TGN38 or the other proteins described above, toxin-glycolipid complexes do not recycle back to the plasma membrane. Thus, it is possible that determinants of anterograde traYcking somehow contribute to the requirement for endosomal acidification during return to the TGN. In summary, pH perturbation results in a general block for most proteins to exit early endosomes; however, transport of some proteins and lipids is unaVected. In addition, pH–perturbing drugs have variable eVects on this step, suggesting that multiple factors, not all of which are related to organelle acidity, can be diVerentially aVected by these treatments.

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D. Delivery to Late Endosomes and Lysosomes Most internalized proteins that are not recycled to the plasma membrane from early endosomes are transported to late endosomes. The mechanism by which cargo reaches late endosomes is controversial and may involve intermediate carrier vesicles that shuttle cargo between the two organelles or the gradual maturation of early endosomes into late endosomes (Morales et al., 1999). Once in late endosomes, proteins can be delivered to lysosomes for degradation or routed to the TGN. The eVect of pH perturbation on transport of internalized proteins to late endosomes and lysosomes has been examined by several laboratories during the past 10 years. Unfortunately, no clear consensus regarding the function of acidification in these traYcking steps has emerged from these experiments. As in the case of the endocytic markers discussed above, this is likely due to diVerential contributions of acid pH to the sorting of diVerent proteins. In addition, as most lysosomal hydrolases have acidic pH optima, inhibition of lysosomal degradation does not necessarily signify inhibition of lysosomal delivery. For example, Yoshimori et al. (1991) examined the eVect of bafilomycin A1 on the delivery of EGF and the fluid phase marker FITC-dextran to lysosomes, and concluded that this drug did not interfere with lysosomal delivery but potently blocked EGF degradation. However, in this study, delivery to lysosomes was assessed only qualitatively by immunofluorescence and immunogold electron microscopy, so an eVect on the rate of delivery could have been missed. By contrast, several groups have found that acidification is required for transport of membrane and fluid phase markers to late endosomes. Bayer et al. (1998) found that even low concentrations of bafilomycin A1 dramatically inhibited the egress of horseradish peroxidase (HRP; a fluid phase marker) as well as internalized virus particles from early endosomes. Similarly, the Gruenberg laboratory showed in an elegant study that in vitro formation of endosomal carrier vesicles (ECVs) from purified early endosomes is inhibited by bafilomycin A1 (Clague et al., 1994). These ECVs could represent intermediates in early endosome to late endosome transport or they might be rudimentary late endosomes that result from the removal of recycling membrane. The inability of ECVs to bud from early endosomes could cause the extensive tubulation of this compartment that has been observed by some groups (D’Arrigo et al., 1997; Clague et al., 1994). By contrast to these data, however, van Weert et al. (1995) concluded that transport of HRP to late endosomes in the hepatoma cell line HepG2 was unaVected by bafilomycin A1 treatment, but that a later step, delivery from late endosomes to lysosomes, was inhibited. Similarly, van Deurs et al. (1996) concluded that bafilomycin A1 treatment did not aVect transport to late endosomes, but decreased the eYciency of delivery of cationized gold to lysosomes, and Yamamoto et al. (1998) also found that fusion of

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autophagosomes and lysosomes in rat hepatoma cells was selectively inhibited by this drug. Finally, Furuchi et al. (1993) examined the eVect of bafilomycin A1 on cholesterol traYc in macrophages and concluded that the eZux of this lipid from lysosomes was dramatically inhibited upon pH disruption. How can these discrepant data be reconciled? Unfortunately, although numerous factors have been invoked (diVerences in cell type, endogenous versus heterologous marker expression, experimental approach used) there is no single satisfying explanation to resolve this issue. The observation that bafilomycin A1 has eVects on retrograde transport of transferrin in only a small subset of cells for each cell type examined provides an important hint that clonal cell lines respond heterogeneously to pH disruption (van Weert et al., 1997). Thus, morphological approaches to examine membrane traYc will likely lead to diVerent conclusions than biochemical methods. This may be compounded by the possibility that the steady-state distribution of markers typically used to identify endocytic organelles may be significantly disrupted in pH-perturbed cells. However, it would not be surprising if ultimately acidification is demonstrated to regulate sorting in each compartment of the endocytic pathway.

E. Molecular Basis of Transport Inhibition by pH Perturbants Although there have been numerous descriptive studies of how pH perturbation alters membrane traYc, the root causes of these eVects have not yet been pinpointed. Recent work has begun to address the molecular role of acidification in membrane dynamics. One consistent finding in these studies by several groups is that changes in organelle pH generated by treatment with bafilomycin A1 or other pH perturbants aVect events occurring on the cytoplasmic face of vesicles. This suggests that a pH gradient across the membrane of acidic compartments (rather than simply acidic lumenal pH) is required for eYcient membrane flow through acidified organelles. There may be multiple steps in protein transport that respond to pH; currently, there is good evidence that recognition of targeting signals and binding of coat proteins are both regulated by acidic endosomal pH. Because the traYcking of some proteins is more sensitive to pH than others, it is tempting to speculate that the ability of proteins to partition into membrane domains in intracellular compartments is modulated by pH. This might be mediated by changes in organelle morphology, lipid dynamics, or composition, or by protein interactions with the adaptor or sorting machinery that directs them through the cell. This has been examined in greatest detail by Fred Maxfield’s group, which has compared the eVect of pH perturbation on traYcking of wild-type TfR, mutant TfRs, and bulk lipid flow

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(Presley et al., 1997). Presley et al. (1997) found that the rate constant for exit of TfR from recycling endosomes was reduced by approximately 45% in bafilomycin A1-treated cells. By contrast, the rate of bulk membrane traYc, measured using a fluorescent lipid probe, was decreased by only
25% upon bafilomycin A1 treatment (Presley et al., 1997). This was similar to the eVect of bafilomycin A1 on the rate of traYcking of mutant receptors that lack a functional tyrosine-containing internalization motif and that also presumably mark the bulk flow pathway (Johnson et al., 1993). Thus, acidification appears to be important not only for the eYciency of bulk membrane flow through the recycling pathway, but also specifically for the selective recognition of at least some targeting motifs in recycling endosomes. However, it is not yet known whether intracellular recognition of these tyrosine-based signals by adaptor complexes is pH dependent. Studies using CHO mutants defective in acidification support and extend this idea. Although several such lines have been isolated (Merion et al., 1983; Colbaugh et al., 1988; Robbins et al., 1983; RoV et al., 1986), the best characterized to date are members of the end2 complementation group, a temperature-sensitive mutant with a partial defect in endosomal acidification (RoV et al., 1986). Although the mutant protein responsible for the end2 phenotype has not been characterized, it is likely to be membrane associated (Martys et al., 1995). Internalization of transferrin and LDL in end2 cells is inhibited by approximately 30% compared with the parental line; however, the most dramatic phenotype that has been found is a 45% reduction in transferrin recycling to the cell surface (Johnson et al., 1994; Presley et al., 1993). Notably, this eVect is identical to that induced by bafilomycin A1 treatment (Presley et al., 1997). However, in contrast to the eVect of bafilomycin A1 (Presley et al., 1997), the rate of bulk membrane flow was unaltered in end2 cells (Presley et al., 1993). It is tempting to speculate that this diVerence reflects the milder change in endosomal pH in end2 cells compared with bafilomycin A1-treated cells: the average pH of the recycling compartment in control cells was 6.43 0.03 versus 6.67 0.05 in mutant cells; by contrast, bafilomycin A1 treatment increased the pH of this compartment to 7.5 0.04 (Presley et al., 1993, 1997). Thus, one might imagine that recognition of targeting motifs is impaired at the intermediate pH of end2 cells, whereas defects in global membrane traYc appear only when endosomal pH is profoundly disrupted. In addition, an elegant series of experiments by Gruenberg’s laboratory has implicated a role for acidification in eYcient coat assembly on endosomes. Neutralization of endosomal pH either with specific inhibitors of V-ATPases or with the ionophore nigericin prevented binding of coat proteins (COPs) to isolated endosomal membranes and inhibited the formation of endosomal carrier vesicles (shuttle vesicles believed to mediate transport from early to late endosomes) in an in vitro system (Aniento et al., 1996;

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Clague et al., 1994); however, neutralization of endosomal pH did not cause the release of prebound COPs (Gu et al., 1997). Interestingly, these treatments had no eVect on COP binding to membranes of the early biosynthetic pathway (Aniento et al., 1996; Gu et al., 1997), nor did they aVect other steps in endocytic traYc, such as homotypic fusion of early endosomes (Clague et al., 1994; Jones et al., 1999). This selectivity may reflect intrinsic diVerences in the subunit composition of COPs that bind to these compartments, as - and -COPs are not associated with endosomal membranes (Aniento et al., 1996; Whitney et al., 1995). More recent experiments have shown that ADP ribosylation factor 6 (ARF) and its GTP-exchange factor ARNO bind in a pH-dependent manner to endosomal membranes (Maranda et al., 2001; Gu and Gruenberg, 2000), and that ARF regulates COP binding, implicating this protein as the cytoplasmic sensor of organelle pH (Gu and Gruenberg, 2000). Interestingly, a previous study had demonstrated acidification-dependent ARF1 binding to an ER/Golgi-enriched membrane fraction in vitro (Zeuzem et al., 1992). ARF binding to Golgi membranes causes activation of phospholipase D activity, which has been implicated in the recruitment of coatomer to membranes (Ktistakis et al., 1996); however ARF1 recruitment of COPs to endosomes does not require phospholipase D activity (Gu and Gruenberg, 2000). How ARFs and ARNO are able to sense intraendosomal pH remains a mystery.

VI. Acidification and the Secretory Pathway Despite the general (though not exclusive) opinion that the TGN is the sole acidified compartment along the biosynthetic pathway, there is a large literature on the role of pH in the transport and processing of newly synthesized proteins. Although one might suppose that the role of acidification would be relatively simple to decipher in this case, in fact, the opposite is true. In part, this is because delivery of newly synthesized proteins destined for endosomal and lysosomal compartments obviously involves their passage through acidified compartments along the endocytic pathway; this complication makes it diYcult to examine the role of acidification in TGN sorting of these proteins. In addition, disrupting the pH gradient in the TGN may have downstream eVects that perturb earlier steps in biosynthetic transport. In this section, we describe the postulated roles for acidification in constitutive anterograde transport, retrograde transport of Golgi complex proteins, formation of secretory granules, sorting of lysosomal enzymes, and polarized biosynthetic membrane traYc.

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A. Role of pH in Intra-Golgi Transport The pH of organelles along the secretory pathway is known to be important for proper processing and sorting of proteins and lipids. Whereas most studies examining the role of pH have demonstrated decreased rates of protein secretion, the compartments aVected are in dispute. Although only the trans-Golgi and TGN are thought to be acidified, some pH perturbants paradoxically aVect transport through the early Golgi complex. For example, it has long been known that the Na+/H+ ionophore monensin causes extensive swelling of all Golgi compartments and blocks biosynthetic transport of some proteins at a relatively early step, prior to the acquisition of endoglycosidase H (endo H) resistance in the cis/medial Golgi (GriYths et al., 1983; TartakoV, 1983; Alonso-Caplen and Compans, 1983). Treatment with concanamycin A in some studies had a similar eVect on the processing of N-linked oligosaccharides on VSV G (Muroi et al., 1993a,b). By contrast, Yilla et al. (1993) found that concanamycin B treatment did not aVect acquisition of endo H resistance, but slowed the rate of protein secretion. As in monensin-treated cells, the extent of protein sialylation was impaired; however, the rate of sialylation was not examined (Yilla et al., 1993). More recent experiments using prodigiosin 25-C, a V-ATPase inhibitor that acts by uncoupling proton transport from ATPase activity (Ohkuma et al., 1998), found a defect in the rate of sialylation and cell surface transport of VSV G, although the aVected steps were not identified (Kataoka et al., 1995). In yet another variation, a study comparing the eVects of various pH perturbants on vesicular transport concluded that biosynthetic transport of the Semliki Forest virus E1-p62 glycoprotein complex to the TGN was blocked by bafilomycin A1, but not delivery from the TGN to the PM (Palokangas et al., 1994). Ammonium chloride had a similar eVect, whereas monensin blocked acquisition of endo H resistance as expected based on previous observations (Palokangas et al., 1994). In this study, bafilomycin A1 treatment was found to cause extensive vacuolization of the Golgi complex, similar to the eVect observed with monensin (Palokangas et al., 1994). Thus, the literature is highly divergent regarding the role of acidification in intra-Golgi transport. Because there is little evidence that Golgi compartments other than the TGN are acidified, we suspected that indirect eVects of pH perturbation on membrane traYcking through normally acidified compartments might be responsible for these discrepant results. To test this idea, we took advantage of the ability to rapidly and reversibly regulate the activity of the protonselective influenza M2 channel. In a detailed study by Sakaguchi et al. (1996) on the eVects of high level expression of influenza M2 on intraGolgi transport, a similar but puzzling inhibition of intra-Golgi transport had been noted. Both the acquisition of endo H-resistant oligosaccharides

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and the proteolytic processing of a marker protein (influenza HA) in the TGN were significantly delayed in M2-expressing cells. Normal processing kinetics could be restored by including the M2 pore blocker amantadine in the medium to block M2 activity (Sakaguchi et al., 1996). Several studies have shown that anterograde and retrograde transport through the Golgi are inextricably linked, as the coat proteins and fusion machinery required for forward traYc through the secretory pathway must be continually recycled (Pelham, 1994; Letourneur et al., 1994; Lewis and Pelham, 1996). Thus, a selective delay in transport to or from the TGN could result in accumulation of components of the transport machinery in this compartment and might ultimately aVect the ability of early biosynthetic traYc to proceed eYciently. We hypothesized that a gradual M2-mediated accumulation of common components of the protein transport machinery in the TGN might ultimately slow transport through earlier Golgi compartments, and could be responsible for the eVects on intra-Golgi traYc observed by Sakaguchi et al. (1996). If this were the case, then the initial round of protein transport through the Golgi complex upon acute activation of preaccumulated, inactive M2 should be normal. To test this idea, we used BL1743, a potent but rapidly reversible inhibitor of M2 (Tu et al., 1996) to acutely regulate M2 activity. Influenza M2 and HA were expressed using the vaccinia T7 system, and allowed to accumulate in the presence of BL1743. The cells were metabolically labeled for a brief period, then rapidly chilled and the inhibitor washed out to activate M2. Upon warming, we measured the rate of transport of radiolabeled HA. Both the kinetics of endo H resistance and sialylation were normal in these cells, whereas M2expressing cells not treated with BL-1743 showed markedly delayed HA transport kinetics (Henkel and Weisz, 1998). Importantly, cells expressing acutely activated M2 still had decreased kinetics of HA cell surface delivery, suggesting that transport through later compartments (presumably the TGN) were directly aVected by acute M2 activation (Henkel and Weisz, 1998). Together, these data suggested that M2 slows steps in early Golgi transport only after protein traYc is allowed to proceed for extended periods in the presence of active M2. These findings are consistent with the idea that the direct target(s) of M2 activity is downstream of the early Golgi, and that early eVects on transport in pH-perturbed cells in general may be an indirect result of eVects on downstream acidified compartments.

B. Retrograde Transport of Golgi Proteins Interestingly, there are several instances where the localization of Golgiresident proteins themselves appears to depend on proper cellular acidification. In the best studied example to date, Linstedt and colleagues

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demonstrated that steady-state localization of the type II membrane proteins GPP130 and GP73 in the cis-Golgi are disrupted by pH perturbation (Linstedt et al., 1997; Puri et al., 2002). Treatment with chloroquine or monensin resulted in rapid accumulation of these proteins in enlarged endosomal structures, and the protein rapidly redistributed to its normal pattern upon drug washout (Bachert et al., 2001; Linstedt et al., 1997; Puri et al., 2002). Dissection of the protein revealed that a lumenal coiled-coil domain adjacent to the transmembrane region of GPP130 conferred pH-dependent targeting to endosomes: in the absence of this region, protein localization to the cis-Golgi was insensitive to pH-perturbing drugs (Bachert et al., 2001). Thus, steady-state distribution of the full length protein appears to be maintained despite rapid cycling through the cell surface and endosomal compartments; the relevance of this complex traYcking route to GPP130 and GP73 function is not known. In a similar vein, Axelsson et al. (2001) recently reported diVerential redistribution of three glycosyltransferases in cells treated with ammonium chloride or bafilomycin A1. These results are somewhat more diYcult to interpret, as long-term treatments (>12 h) with these agents were used and diVerent eVects were observed with each drug. In another example, bafilomycin A1 was reported to perturb the brefeldin A-induced retrograde transport to the ER of the Golgi enzyme mannosidase II (Palokangas et al., 1998). In addition, this drug caused redistribution of b-COP to pre-Golgi compartments and tubulation of the intermediate compartment; however, anterograde traYc from the ER to the Golgi complex was qualitatively unaVected (Palokangas et al., 1998). Weak DAMP staining was detected in a small fraction (<20%) of these pre-Golgi elements (Palokangas et al., 1998). Together the data were taken to demonstrate a weak acidification of the intermediate compartment and a requirement for functional V-ATPase in retrograde transport to the ER. However, although the eVect of bafilomycin A1 on brefeldin-stimulated retrograde transport was reported in NRK cells, we have been unable to reproduce this striking result in COS-7 cells; thus, this phenomenon and its relevance may be somewhat cell-type specific. In summary, there is significant evidence that steady-state Golgi enzyme distribution is disrupted in pH-perturbed cells. However, there are few data to suggest that these eVects result directly from changes in Golgi pH rather than indirectly due to changes in other (acidified) compartments.

C. Regulated Secretion In addition to a constitutive secretory pathway, many cell types are capable of directing newly synthesized proteins (typically hormones or enzymes) into storage granules whose exocytosis can be stimulated in response to

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extracellular signals. Aggregation of propeptides in the TGN is thought to drive their sorting into large immature secretory granules (ISGs) that bud from this compartment (Chanat and Huttner, 1991), although other sorting mechanisms may also be necessary (Dannies, 2001). Contaminating proteins (typically lysosomal proteins) and excess membrane are then removed from these ISGs in clathrin-coated vesicles that bud from these structures (Klumperman et al., 1998). The pH of ISGs is thought to be
6.3, similar to that of the TGN (Urbe et al., 1997). There is some debate as to whether endoproteolytic conversion of the granule contents to their mature forms occurs in the TGN or in the ISG: this may depend on the particular propeptide in question (Tooze et al., 1987; Schnabel et al., 1989; Xu and Shields, 1994; Orci et al., 1987; Urbe et al., 1997). Final maturation involves condensation of the granule contents and further acidification to a final pH of approximately 5.0–5.5 (Arvan and Castle, 1986; Orci et al., 1986, 1987; Urbe et al., 1997). The biogenesis of secretory granules has been the subject of excellent recent reviews (Tooze et al., 2001; Dannies, 1999). Acidification plays a role in multiple steps in secretory granule formation, and has recently been demonstrated to be important in priming preexisting granules for exocytosis (Barg et al., 2001). In addition, V-ATPase inhibitors block the formation of mature secretory granules and cause the accumulation of large vacuolar structures near the TGN (Henomatsu et al., 1993; Schoonderwoert et al., 2000), demonstrating the importance of acidification in granule maturation. The role of acidic pH in earlier steps in prohormone sorting and processing has been studied more extensively, but is somewhat confusing. It has long been known that many prohormones as well as their processing enzymes aggregate in vitro under acidic conditions (Chanat and Huttner, 1991; Rindler, 1998; Yoo, 1996; Freedman and Scheele, 1993), and this has been suggested to be a key step in their incorporation into forming ISGs. However, the requirement for acidic pH in prohormone aggregation in vivo is not definitive. For example, whereas prolactin aggregates under acidic conditions in solution (Thompson et al., 1992; Rindler, 1998), a recent study found that treatment with chloroquine or bafilomycin A1 slowed, but did not block aggregation of prolactin in GH4C1 neuroendocrine cells (Lee et al., 2001). Regardless, a generally consistent finding has been that pH perturbation with weak bases or V-ATPase inhibitors diverts prohormones into the constitutive secretory pathway and blocks their proteolytic processing (Moore et al., 1983; Stoller and Shields, 1989; Xu and Shields, 1994; Back and Soinila, 1996; Urbe et al., 1997; Tanaka et al., 1997; Schoonderwoert et al., 2000), although again there are some exceptions. In two cases, discrepant eVects on the processing of the same propeptides [pro-ACTH and proopiomelanocortin (POMC)] in AtT-20 cells were observed depending on the pH perturbant used (Mains and May, 1988; Tanaka et al., 1997), and in other

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studies, processing of POMC and prolactin was inhibited by bafilomycin A1, but the prohormones were retained intracellularly (Henomatsu et al., 1993; Schoonderwoert et al., 2000). By contrast, prohormone convertase enzyme was diverted into the constitutive secretion pathway in these cells (Henomatsu et al., 1993). Thus, although the data point to multiple roles for acidification in secretory granule biogenesis, there is significant variability in the eVect of pH perturbation on the fate of individual granule components. This is likely due to diVerences in pH optima of proteolytic enzymes that process these hormones, the relative role of aggregation versus other sorting pathways, and the cell type in which they are expressed.

D. Delivery of Newly Synthesized Proteins to Lysosomes Although traditionally the lysosome has been viewed as a static, terminal organelle that receives and degrades proteins and lipids, it has become increasingly clear that lysosomal residents can reversibly traYc out of lysosomes. Indeed, in some specialized cell types, subsets of lysosomes exist that can be stimulated to fuse with the plasma membrane to release their contents (Blott and GriYths, 2002); this process may be stimulated in part by changes in lysosomal pH (Tapper and Sundler, 1995b). Moreover, lysosomal membrane proteins continually cycle through endosomes and the cell surface (Lippincott-Schwartz and Fambrough, 1987). The traYcking of newly synthesized lysosomal proteins is complex, as their delivery involves passage through both the biosynthetic and endocytic pathways. As a result, pH perturbation has varied eVects on the fates of individual lysosomal proteins. Targeting of lysosomal resident proteins can be divided into two general categories: delivery of soluble hydrolases and transport of membrane proteins. The eVect of acidification on the transport of these classes of proteins is described below. 1. Lysosomal Membrane Proteins Several cytoplasmic tail signals that direct membrane proteins to lysosomes have been described, including tyrosine- and dileucine-containing motifs (Hunziker and Geuze, 1996). Newly synthesized lysosomal membrane proteins make use of two pathways to reach their destination: in the direct pathway, they traYc from the TGN to late endosomes and subsequently to lysosomes. The indirect pathway involves initial delivery to the cell surface, upon which the proteins are internalized and delivered to lysosomes via an endocytic route. The extent to which a particular lysosomal membrane protein utilizes the direct versus indirect pathway is a function of its lysosomal sorting motif as well as cell type.

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That loss of acidification disrupts the traYcking of lysosomal membrane proteins should come as no surprise, given the eVects of pH perturbation on the endocytic pathway. This was strikingly observed in studies on an avian lysosomal-associated membrane protein called LEP100 that normally cycles through lysosomes, endosomes, and the plasma membrane (LippincottSchwartz and Fambrough, 1987). Chloroquine treatment blocked endosome-to-lysosome transport of LEP 100, resulting in the depletion of the lysosomal pool with its concomitant accumulation at the cell surface and in endosomes (Lippincott-Schwartz and Fambrough, 1987). However, few subsequent studies have explored the eVect of pH perturbation on lysosomal membrane protein delivery; presumably, this is because transport of lysosomal membrane proteins is expected to be disrupted due to inhibition of traYcking through earlier compartments along the endocytic pathway, just as transport of components destined for lysosomal degradation is aVected by pH perturbation (see Section V). 2. The Mannose 6-Phosphate Receptor and Soluble Hydrolases Soluble lysosomal hydrolases are generally synthesized as precursors that are processed into intermediate forms along the biosynthetic pathway, and to mature forms upon their arrival to lysosomes. Targeting of these enzymes to lysosomes is a multistep process. A subset of mannose residues on the N-linked oligosaccharides of soluble lysosomal hydrolases is modified in the cis/medial-Golgi complex by the two-step addition of a phosphate residue to generate mannose 6-phosphate (von Figura and Hasilik, 1986; Kornfeld, 1987). This moiety is recognized in the TGN by a selective receptor (the mannose 6-phosphate receptor, or MPR) that transports the hydrolase to late endosomes. The acid pH in late endosomes promotes dissociation of the hydrolase from its receptor, and the enzyme is subsequently delivered to lysosomes by an unknown mechanism. The MPR then recycles to the TGN either directly or via the plasma membrane (Munier-Lehmann et al., 1996). At the plasma membrane the MPRs can scavenge extracellular soluble hydrolases and direct their delivery to lysosomes (Munier-Lehmann et al., 1996). Two MPRs have been identified: a cation-independent 215-kDa protein and a cation-requiring 46-kDa form (Munier-Lehmann et al., 1996). Interestingly, the two MPRs have diVerential pH binding profiles: the cation-dependent MPR binds many substrates weakly at neutral pH whereas the cation-independent MPR is pH insensitive within a relatively broad range (Hoflack et al., 1987). Although the two MPRs have overlapping substrate specificities, they cannot completely substitute for each other when one is nonfunctional (Pohlmann et al., 1995; Kasper et al., 1996). In addition to this pathway, a MPR-independent mechanism also contributes to the transport of some lysosomal enzymes (von Figura and Hasilik, 1986).

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Numerous studies (with a few exceptions; Punnonen et al., 1993; Madden and Storrie, 1989) have shown that treatment with weak bases or V-ATPase inhibitors blocks lysosomal delivery of newly synthesized soluble hydrolases and leads to their aberrant secretion (Imort et al., 1983; Rosenfeld et al., 1982; Gonzalez-Noriega et al., 1980; Nishimura et al., 1988; Hasilik and Neufeld, 1980; Oda et al., 1991; Tapper and Sundler, 1995a; Pohlmann et al., 1984). This is due not only to defective sorting along the biosynthetic pathway but also to an inability to scavenge secreted hydrolases from the extracellular medium (Gonzalez-Noriega et al., 1980). The profound eVect of pH disruption on transport of most lysosomal hydrolases is due to the multiple steps that are aVected in drug-treated cells. For example, eYcient processing of hydrolases to their intermediate and mature forms is inhibited in pH-perturbed cells, making it diYcult to identify the step in traYc that is aVected. Moreover, pH perturbation aVects both substrate binding to and dissociation from the MPRs. Finally, traYcking of the MPRs themselves is disrupted, although no consistent eVect has been found. In one study, the cation-independent MPR, which is found in the TGN and endosomes of HepG2 cells at steady state, was redistributed to the TGN upon primaquine treatment (Geuze et al., 1985). By contrast, in another rat hepatocyte cell line both MPRs were found in endosomal compartments as well as the TGN (Matovcik et al., 1990). Regardless, these factors combine to prevent eVective diversion of newly synthesized hydrolases from the default constitutive secretion route. One lingering issue is whether acidification of lysosomes themselves is required for them to be recognized as appropriate targets for fusion by donor vesicles carrying newly synthesized lysosomal proteins. It is known, for example, that inactivation of lysosomes using HRP blocks the lysosomal delivery of internalized EGF–EGF receptor complexes (Futter et al., 1996). Until recently, this question was unanswerable, as lysosomal acidification could not be easily dissociated from that of other compartments. In one attempt, Park et al. (1991) examined lysosomal hydrolase delivery in End3 cells that are conditionally defective in endosomal but not lysosomal acidification. In these experiments, cell growth at the restrictive temperature caused a massive secretion of lysosomal hydrolase precursors and little accumulation of mature proteins in lysosomes. Although this interesting study confirmed a requirement for acidified endosomes for normal lysosome biogenesis, the key role of endosomal acidification in lysosomal hydrolase processing and delivery precluded analysis of the role of lysosomal acidification in enzyme delivery. We have taken advantage of the dose-dependent eVect of influenza M2 expression on organelle acidification to examine this question. In HeLa cells, we found that low level expression of M2 (mediated by recombinant adenovirus) maximally inhibited constitutive protein transport along the

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biosynthetic pathway but had no eVect on the recycling of transferrin. By contrast, a five-fold increase in adenoviral titer resulted in partial inhibition of transferrin recycling compared with bafilomycin A1 treatment. Importantly, the rate of degradation of epidermal growth factor was unaVected even at the higher level of M2 expression, suggesting that lysosomal pH was not significantly aVected (Henkel et al., 1999). We then tested the eVect of M2 on delivery of the newly synthesized lysosomal hydrolase cathepsin D. This protein is synthesized as a 53-kDa precursor (P) in the ER, clipped in the TGN and/or endosomes to an slightly smaller intermediate (I) form, then processed to a mature 30-kDa form (M) in lysosomes (Gieselmann et al., 1983). Conversion to the mature form is not dependent on prior cleavage to the intermediate form (Richo and Conner, 1994). As predicted, bafilomycin A1 blocked proteolytic maturation of cathepsin D and slightly increased secretion of the precursor. Low expression levels of M2 slightly delayed processing of cathepsin D to both the intermediate and mature forms, suggesting that at least some cleavage of cathepsin D to its intermediate form normally occurs in the TGN. Higher expression of M2 had a more dramatic eVect on both steps that was almost completely reversed when AMT was included. Because higher expression levels of M2 had a greater eVect on cathepsin D maturation than that observed at low expression levels, we conclude that a portion of cathepsin D traYcs through early endosomes in HeLa cells as has been previously suggested in other cell types (Ludwig et al., 1991; Rijnboutt et al., 1992). In neither case was cathepsin D delivery to lysosomes completely inhibited. The properly targeted fraction could represent cathepsin D transport via the pH-independent pathway (Diment et al., 1988; von Figura and Hasilik, 1986; Rijnboutt et al., 1991; Radons et al., 1994; Braulke et al., 1987) or cathepsin D binding to the cation-independent MPR, which may still occur at neutral pH. Regardless, these data demonstrate that ineYcient delivery of newly synthesized hydrolases in pH-perturbed cells is not simply due to the loss of recognition of the lysosome as an appropriate target, and also reveal the utility of selectively perturbing organelle pH in dissecting the complex role of acidification in protein transport.

E. Biosynthetic Traffic in Polarized Cells Polarized cells diVerentially sort newly synthesized proteins that are destined for the apical and basolateral cell surfaces into distinct vesicles that exit the TGN and selectively fuse with the appropriate target membrane. The targeting signals and mechanisms that direct proteins into apically or basolaterally destined vesicles have been the subject of numerous reviews (Aroeti et al., 1998; Keller and Simons, 1997; Nelson and Yeaman, 2001).

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In general, whereas apical sorting signals are generally localized to the lumenal and transmembrane regions of proteins, basolateral sorting signals tend to be present on the cytoplasmic tails of proteins. Few studies have focused on the function of acidification in protein sorting in polarized cells. Matlin (1986) found that ammonium chloride treatment slowed apical delivery of influenza hemagglutinin (HA), but did not aVect the ultimate polarity of its delivery. By contrast, Caplan et al. (1987) observed that polarized MDCK cells treated with the weak base ammonium chloride misdirected about half of the newly synthesized soluble basolateral markers laminin and heparan sulfate proteoglycan to the apical medium. In this study, the polarity of secretion of apical markers was unaVected by ammonium chloride treatment, as were delivery of apical and basolateral membrane proteins (Caplan et al., 1987; Matlin, 1986). In another study, treatment with monensin or chloroquine resulted in a two-fold increase in the ratio of apical to basolateral secretion of both an endogenous protein complex (gp80) that was directed apically and an exogenous marker (lysozyme) that was secreted equally from both plasma membrane domains (Parczyk and Kondor-Koch, 1989). Although these results are not completely reconcilable, together they suggest that increased organelle pH favors sorting (or missorting) of secreted proteins into the apical medium. It is not clear why only soluble proteins were aVected, although there is some evidence from other studies that membrane-bounded and secreted proteins are delivered to the PM in distinct populations of vesicles (Boll et al., 1991; de Almeida and Stow, 1991; Milgram et al., 1994; Saucan and Palade, 1994). Perhaps targeting of only one subset of these vesicles is sensitive to changes in pH. Furthermore, from these studies it is not apparent whether the missorting occurred because the molecules were misdirected into apically destined vesicles or whether fusion of vesicles with the basolateral surface was compromised. We examined the eVect of influenza M2 expression on biosynthetic delivery in polarized MDCK cells (Henkel et al., 2000). M2 expression significantly decreased the kinetics of apical cell surface delivery of HA, consistent with previous observations (Matlin, 1986), but had no eVect on basolateral delivery of the polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of -glutamyltranspeptidase were reduced with no eVect on the basolaterally secreted fraction. By contrast to the selective eVects we observed with M2, treatment with bafilomycin A1 slowed the kinetics of all proteins tested at multiple transport steps, including intra-Golgi traYc. We hypothesize that this generalized inhibition of transport reflects multiple consequences of drug treatment that are independent of or indirectly due to changes in organelle acidification (see above). M2 expression reduced the amount of HA released from the TGN in mechanically perforated cells, and inclusion of the M2 ion channel blocker

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amantadine restored transport to normal levels, suggesting that M2mediated alkanization of the TGN was responsible for the observed eVects. Interestingly, however, M2 expression had no eVect on the rate of secretion of a nonglycosylated protein (human growth hormone, hGH) that is normally secreted equally from both surfaces, but M2 slowed apical secretion of a glycosylated mutant of hGH that is secreted predominantly apically. These data suggest that acidification of the TGN may be important for the segregation of apically sorted cargo into forming vesicles, but not for vesicle formation itself. However, the mechanism by which cargo loading into vesicles could be disrupted by M2 activity is unknown. One possibility is that acidification could be important in regulating the size or composition of glycolipid rafts in the TGN. Association with glycolipid-enriched microdomains has been suggested to be important for eYcient apical sorting of transmembrane and lipid anchored proteins (Ikonen, 2001). Alternatively, acidic pH might be important for the recognition of signals that direct rafts to forming apical vesicles or for the active recruitment of rafts into forming vesicles. Finally, acidic pH may play a completely diVerent and as yet unanticipated role in modulating apically destined traYc.

VII. Concluding Remarks As the studies described above amply demonstrate, our understanding of the roles of acidification in cellular membrane dynamics remains murky. However, although the literature is confounding on the details, several general themes emerge regarding the function of organelle acidification in membrane traYc. First, organelle acidification is important for the eYcient transport, but not for the actual sorting, of membrane proteins. In most instances, disrupting organelle pH results in accurate targeting of membrane proteins, albeit at a reduced rate. By contrast, acidification regulates many receptor–ligand binding and dissociation events that are critical for sorting soluble proteins, such as secretory granule components, lysosomal hydrolases, and internalized proteins. Second, in addition to altering the lumenal microenvironment of a compartment, disruption of organelle acidification can also perturb events on the cytoplasmic face of compartments. For example, pH perturbation can alter the recruitment of coat proteins to a given compartment. Finally, a role for acidification in cargo loading has been observed in several systems. For example, pH-dependent recruitment of membrane proteins with tyrosine-containing motifs to the sites of vesicle formation may contribute to coat assembly on endosomes. Moreover, loading of presorted cargo into apically destined vesicles at the TGN appears to be pH sensitive.

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Our shallow learning curve with regard to how acidification regulates protein sorting and traYc is largely due to technical limitations. No pH perturbant is ideal, and the eYcacy, reproducibility, and side eVects of each method diVer. In addition, the diversity of experimental protocols used by diVerent laboratories to perturb pH as well as to quantitate membrane traYc makes it impossible to compare studies and certainly contributes to the confusion in the field. Finally, the requisite interrelatedness of cellular membrane traYcking pathways presents a formidable barrier to understanding the role of acidification in individual organelles. Perturbation of organelle pH can ultimately disrupt traYc through upstream nonacidified compartments, thus complicating the interpretation of experimental results. Conversely, the possibility that some perturbants of membrane traYc disrupt organelle pH is also frequently overlooked. The maintenance of acidic pH in a given organelle requires that the channels and pumps described above are continually present at consistent levels despite the large amount of membrane flow through the compartments. Indeed, disruption of membrane traYc can perturb the balance of these components and cause disequilibration of organelle pH. For example, a recent study found that inhibition of endocytosis by expression of dominant-negative dynamin resulted in a significant increase (0.4 pH units) in endosomal pH (Uchida, 2000). Given these formidable hurdles, how can we improve on our current understanding? Several recent technical advances promise to help. First, the approach we have taken in our laboratory represents a small step in our ability to selectively perturb organelle pH. Our success expressing M2 in a repressible manner coupled with the availability of rapidly reversible inhibitors for this channel demonstrates the basic feasibility of this approach. Second, the recent development of new, targeted fluorescent probes has significantly improved our ability to quantitate pH, particularly in mildly acidic compartments. Similarly, other probes have recently been developed that allow quantitation of counterions that may contribute to the maintenance of organelle pH. Finally, the rapid emergence of live-cell imaging as a mainstream technique will allow us to visualize in real time the eVects of pH perturbation on the traYcking of fluorescently tagged proteins and to concurrently monitor changes in the morphology of biosynthetic and endocytic compartments. Together, these new developments oVer the opportunity to dissect the role of acidification in membrane traYc with better resolution. Acknowledgments This review is dedicated to the past and present members of my laboratory whose work is described herein. These studies were funded by grants from the National Institutes of Helth (DK 54407) and the Cystic Fibrosis Foundation. I am very grateful to Ken Dunn for his helpful comments on the manuscript and to Fred Maxfield for enlightening dicussions on organelle pH homeostasis.

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