Vesicular trafficking: molecular tools and targets.

Intracellular trafficking of membrane-coated vesicles represents a fundamental process that controls the architecture of different intracellular compartments and communication between the cell and its environment. Major trafficking pathways consist of an inward flux of endocytic vesicles from the plasma membrane and an outward flux of exocytic vesicles to the plasma membrane. This overview describes a number of molecular biology tools commonly used to analyze endocytic and exocytic pathways. The overall emphasis is on major proteins responsible for vesicle formation, recognition, and fusion. These include components of vesicle coats, adaptor complexes, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and Rab guanosine 5'-triphosphatases (GTPases), which represent attractive targets for genetic manipulation aimed at unraveling mechanisms of endocytosis and exocytosis.

Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity.

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

Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases.

The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c'') of molecular mass 100-17 kDa. The V1 domain is responsible for ATP hydrolysis whereas the V0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.

Biochemical evidence for ATPase activity in CFTR-enriched apical membrane vesicles from tracheal epithelium.

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.

Localization of a class II phosphatidylinositol 3-kinase, PI3KC2alpha, to clathrin-coated vesicles.

We have analysed phosphatidylinositol 3-kinase activity associated with subcellular fractions prepared from rat brains. Phosphatidylinositol 3-kinase activity is not markedly enriched with synaptic vesicle purification; whilst the activity associated with the most pure fractions is inhibited at low concentrations of wortmannin (IC50 approximately 4-5 nM). In contrast, clathrin-coated vesicle (CCV) fractions showed increased enzyme activity compared to light membrane fractions from which they are purified. In addition to a wortmannin-sensitive activity, we also detected an activity that could only be inhibited at higher concentrations of wortmannin (IC50 approximately 400 nM), characteristic of certain class II enzymes (including phosphatidylinositol 3-kinase C2alpha) to be highly enriched in CCV fractions. Immunoblotting with an antibody raised against phosphatidylinositol 3-kinase C2alpha, confirmed that this enzyme is highly enriched in CCVs and displays an enrichment profile during the purification that mirrors enrichment of the low nanomolar wortmannin-insensitive activity. If the CCV purification protocol is adapted to favour nerve terminally derived vesicles, we find reduced levels of the C2alpha enzyme in the CCV fractions, suggesting that the enzyme may principally reside on vesicles associated with the cell body.

Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex.

The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.

Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases.

The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake. The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane. The V-ATPases are composed of two functional domains. The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases. A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification. Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo. One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity. Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

The vacuolar H+-ATPase of clathrin-coated vesicles is reversibly inhibited by S-nitrosoglutathione.

It has been previously demonstrated that the vacuolar H+-ATPase (V-ATPase) of clathrin-coated vesicles is reversibly inhibited by disulfide bond formation between conserved cysteine residues at the catalytic site on the A subunit (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13224-13230). Proton transport and ATPase activity of the purified, reconstituted V-ATPase are now shown to be inhibited by the nitric oxide-generating reagent S-nitrosoglutathione (SNG). The K0.5 for inhibition by SNG following incubation for 30 min at 37 degreesC is 200-400 microM. As with disulfide bond formation at the catalytic site, inhibition by SNG is reversed upon treatment with 100 mM dithiothreitol and is partially protected in the presence of ATP. Also as with disulfide bond formation, treatment of the V-ATPase with SNG protects activity from subsequent inactivation by N-ethylmaleimide, as demonstrated by restoration of activity by dithiothreitol following sequential treatment of the V-ATPase with SNG and N-ethylmaleimide. Moreover, inhibition by SNG is readily reversed by dithiothreitol but not by the reduced form of glutathione, suggesting that the disulfide bond formed at the catalytic site of the V-ATPase may not be immediately reduced under intracellular conditions. These results suggest that SNG inhibits the V-ATPase through disulfide bond formation between cysteine residues at the catalytic site and that nitric oxide (or nitrosothiols) might act as a negative regulator of V-ATPase activity in vivo.

Basolateral distribution of caveolin-1 in the kidney. Absence from H+-atpase-coated endocytic vesicles in intercalated cells.

In kidney epithelial cells, a variety of physiological processes are dependent on the active recycling of membrane proteins between intracellular vesicles and the cell surface. Although clathrin-mediated endocytosis occurs in several renal cell types, endocytosis can also occur by non-clathrin-coated vesicles, including pinocytotic structures known as caveolae that contain a novel coat protein, caveolin. Exo- and endocytosis of a vacuolar H+-ATPase in intercalated cells also occurs via specialized "coated" vesicles that do not contain clathrin. The aim of this study was to localize caveolin in the kidney and, in addition, to determine whether it could be a component of the H+-ATPase recycling process. Using an antibody against the alpha- and beta-isoforms of caveolin-1, our immunocytochemical data show a marked heterogeneity in the cellular expression of this isoform of caveolin in kidney. In contrast, caveolin-3 was not detectable in renal epithelial cells. Caveolin-1 was abundant in endothelial cells and smooth muscle cells and was present in the parietal cells of Bowman's capsule. Distal tubule cells, connecting tubule cells, and collecting duct principal cells exhibited marked punctate basolateral staining, corresponding to the presence of caveolae detected by electron microscopy, whereas all intercalated cells were negative in both cortex and medulla. These data indicate that although caveolin-1 may participate in basolateral events in some kidney epithelial cell types, it does not appear to be involved in the regulated recycling of H+-ATPase in intercalated cells. Therefore, these cells recycle H+-ATPase by a mechanism that involves neither clathrin nor caveolin-1.

Identification of novel vesicles in the cytosol to vacuole protein degradation pathway.

The key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30-40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.

Japanese encephalitis virus infection in Vero cells: the involvement of intracellular acidic vesicles in the early phase of viral infection was observed with the treatment of a specific vacuolar type H+-ATPase inhibitor, bafilomycin A1.

The involvement of intracellular acidic vesicles in the early phase of Japanese encephalitis (JE) virus infection in Vero cells was observed by adding a specific vacuolar type H+-ATPase (V-ATPase) inhibitor (bafilomycin A1) in the cell culture medium. Studies with the detection of viral envelope (E) protein suggested that bafilomycin A1 inhibited virus infection in the cells. Subcellular distribution of incoming biotinylated virions and 3H-uridine-labeled viral RNA were observed in fractions of a Percoll density gradient. At 10 min of the chasing period, virions and viral RNA were found mainly in fractions with a mean density of 1.04 g/ml corresponding to the endosome both in the control and bafilomycin A1-treated cells. At 60 min of the chasing period, the peak of biotin activity was detected in fractions with a mean density of 1.08 g/ml corresponding to the lysosome, whereas the peak of radioactivity did not run parallel with that of biotin and shifted to fractions with a mean density of 1.05 g/ml and higher than 1.084 g/ml, respectively. At 60 min of the chasing period in bafilomycin A1-treated cells, the peak of biotin and radioactivity were still found mainly in the fraction with a density of 1.04 g/ml, representing the endosome. Subcellular fractionation by a Percoll density gradient revealed that bafilomycin A1 treatment resulted in the accumulation of virions in the endosome fraction and suggested the prevention of intracellular translocation of the virions which occurs during the early entry process of an infecting virus to the cells.

Reconstitution of ATPase activity from individual subunits of the clathrin-coated vesicle proton pump. The requirement and effect of three small subunits.

The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (VC) and a transmembranous proton channel (VB). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas VC, when separated from VB, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of VC results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the VC complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of VC, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca2+ but also in the presence of Mg2+. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of VC but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with VB and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.

Evidence for a selective and electroneutral K+/H(+)-exchange in Saccharomyces cerevisiae using plasma membrane vesicles.

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H(+)-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Localization and properties of kinases in clathrin-coated vesicles from zucchini hypocotyls.

Five major polypeptides of 70, 50, 47, 19 and 17 kDa and four minor polypeptides (100, 65, 45 and 39 kDa) become phosphorylated when clathrin-coated vesicles (CCV) from zucchini hypocotyls are incubated in [gamma 32P]Mg-ATP. After dissociation with 0.5 M Tris/HCl the CCV coat polypeptides were subjected to gel filtration in order to separate clathrin triskelions from beta-adaptin-containing fractions. Only the latter bore kinase activities, with phosphorylated polypeptides of 39 kDa in addition to the 50, 19-kDa and 17-kDa polypeptides just mentioned. Heparin, an inhibitor of casein kinase II, permitted the phosphorylation of only the 19-kDa and 17-kDa polypeptides. Staurosporine, an inhibitor of protein kinase c-like activities, prevented the phosporylation of the 70-kDa polypeptide. When recombined with the triskelions the beta-adaptin fractions achieved the phosphorylation of the 45-kDa and 70-kDa polypeptides. Because of its heat stability and calcium-binding properties we interpret the 45-kDa polypeptide as being a clathrin light chain. Antibodies raised against the 70-kDa group of heat-shock proteins (Hsp70) recognize a 70-kDa polypeptide in the beta-adaptin-containing fractions. Because this polypeptide only phosphorylates in the presence of triskelions we consider it to be the uncoating ATPase, which is known to aggregate upon dissociation of the CCV coat. Our results therefore indicate that zucchini CCV contain a number of phosphorylable polypeptides equivalent to the beta, mu and sigma adaptins of bovine brain. Just as in bovine brain CCV a casein-kinase-II-like activity is associated with the zucchini CCV 50/47-kDa polypeptides, further pointing to their identity as plant mu2/mu1 adaptin equivalents.

Activity and in vitro reassembly of the coated vesicle (H+)-ATPase requires the 50-kDa subunit of the clathrin assembly complex AP-2.

We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.

Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase.

Vacuolar-type proton pumps are complex heterooligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and transmembranous proton flow can be assigned to isolated domains. Data suggest that the molecular site of ATP hydrolysis resides within the 70-kDa subunit but that ATPase activity likely requires at least three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned and sequenced the 70-kDa subunit from bovine brain and have expressed the protein in insect Sf9 (Spodoptera frugiperda) cells with a recombinant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [alpha 32P]ATP and UV irradiation with an apparent Kd of 35 microM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.6 mumol P(i).mg protein-1.min-1, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.