Proton-translocating adenosine triphosphatase of chromaffin-granule membranes. The active site is in the largest (70 kDa) subunit.
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ABSTRACT:
SUBMITTER: Percy JM
PROVIDER: S-EPMC1147241 | biostudies-other | 1986 Oct
REPOSITORIES: biostudies-other
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Project description:Chromaffin granules, the secretory vesicles of the adrenal medulla, have a Na+/H+ exchange activity in their membranes which brings their proton gradient into equilibrium with a Na+ gradient. This explains why Na+ is mildly inhibitory to amine transport (which is driven by the H+ gradient) The activity can be demonstrated by using accumulation of 22Na+ in response to a pH gradient that is either imposed by diluting membrane 'ghosts' into alkaline media, or generated by ATP hydrolysis. It can also be monitored indirectly by fluorescence measurements in which the pH inside 'ghost' is monitored by quenching of a fluorescent weak base. This method has been used to monitor Na+ entry into acid-loaded 'ghosts' of H+ entry into methylamine accumulation. The exchanger appears to be reversible and non-electrogenic, with a stoichiometry of 1:1. Using an indirect assay we measured an apparent Km for Na+ of 4.7 mM, and a Ki for amiloride, a competitive inhibitor, of 0.26 mM. Direct assays using 22Na+ suggested a higher Km. Ethylisopropylamiloride was not inhibitory.
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Project description:1. Highly purified resealed chromaffin-granule ;ghosts' were assayed for ATPase and ATP-driven H(+)-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H(+)-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H(+)-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ;ghosts' with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H(+)-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ;ghosts', whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with (14)C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[(14)C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H(+)-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H(+)-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H(+)-gradient through OH(-)-anion exchange.
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Project description:The association of adenosine triphosphatase and ADP/ATP isotope-exchange activities with chromaffin-granule membranes was shown by sucrose-density-gradient centrifugation. The two activities were solubilized, and separated by differential sedimentation.
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Project description:Chromaffin-granule membranes contain two ATPases, which can be separated by (NH4)2SO4 fractionation after solubilization with detergents, or by phase segregation in Triton X-114. ATPase I (Mr 400000) is inhibited by trialkyltin, quercetin and alkylating agents, and hydrolyses both ATP and ITP. It contains up to five types of subunit, including a low-Mr hydrophobic polypeptide that reacts with dicyclohexylcarbodi-imide; these subunits are unrelated to those of mitochondrial F1F0-ATPase, as judged by size and reaction with antibodies. ATPase II (Mr 140000) is inhibited by vanadate, and is specific for ATP; it has not been extensively purified. Proton translocation by resealed chromaffin-granule 'ghosts', measured by uptake of methylamine or by quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine, is supported by the hydrolysis of ATP or ITP, and inhibited by quercetin or alkylating agents, but not by vanadate. ATPase I must therefore be the proton translocator involved in the uptake of catecholamines and possibly of other components of the chromaffin-granule matrix, whereas ATPase II does not translocate protons.
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Project description:Granule-plasma membrane docking and fusion can only occur when proteins that enable these reactions are present at the granule-plasma membrane contact. Thus, the mobility of granule membrane proteins may influence docking and membrane fusion. We measured the mobility of vesicle associated membrane protein 2 (VAMP2), synaptotagmin 1 (Syt1), and synaptotagmin 7 (Syt7) in chromaffin granule membranes in living chromaffin cells. We used a method that is not limited by standard optical resolution. A bright flash of strongly decaying evanescent field produced by total internal reflection was used to photobleach GFP-labeled proteins in the granule membrane. Fluorescence recovery occurs as unbleached protein in the granule membrane distal from the glass interface diffuses into the more bleached proximal regions, enabling the measurement of diffusion coefficients. We found that VAMP2-EGFP and Syt7-EGFP are mobile with a diffusion coefficient of ∼3 × 10-10 cm2/s. Syt1-EGFP mobility was below the detection limit. Utilizing these diffusion parameters, we estimated the time required for these proteins to arrive at docking and nascent fusion sites to be many tens of milliseconds. Our analyses raise the possibility that the diffusion characteristics of VAMP2 and Syt proteins could be a factor that influences the rate of exocytosis.
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Project description:Bovine chromaffin granules were lysed and their membranes resealed to give osmotically sensitive 'ghosts'. These swell in the presence of salts and MgATP. It is shown that this is due to proton entry accompanied by anions. The rate of swelling depends on the anion present, but swelling is not limited to media containing permeant anions. It is quite marked in solutions of sulphates, phosphates and acetates. It is not uncoupler-sensitive, suggesting that at least one component of swelling is due to coupled proton and anion entry (non-electrogenic proton translocation). Direct measurements of transmembrane pH and potential gradients generated in the presence of MgATP shows that these are rapidly established in sucrose media, and are rather little affected by the presence of salts. They contribute roughly equally to the total protonmotive force. The potential gradient is establihsed very rapidly, but the pH gradient is generated over several minutes. The gradients are not completely dissipated by uncoupler, and it is shown that, in media containing sulphate but no permeant anion, sulphate can be taken up by the 'ghosts'. There thus appear to be two mechanisms of proton translocation across the membrane, both dependent on ATP hydrolysis: an electrogenic transfer of protons, and proton movement linked to an anion transporter of broad specificity.
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Project description:ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.
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Project description:Proteins exposed on the cytoplasmic face of isolated chromaffin granules were labelled by lactoperoxidase-catalysed radioiodination and by non-enzymic biotinylation. Granule membranes were then prepared, and the H+-translocating ATPase isolated by fractionation with Triton X-114. The labelling of individual ATPase subunits was assessed by polyacrylamide-gel electrophoresis, followed by autoradiography or by blotting and decoration with 125I-labelled streptavidin. Subunits of 72, 57 and kDa were strongly labelled, and could be removed from the membrane at pH 11: they are therefore extrinsic proteins. The 120 kDa subunit was also labelled, but it was not solubilized at pH 11. Photolabelling with a hydrophobic probe indicated that this subunit penetrates the bilayer, and enzymic degradation studies showed the presence of N-linked oligosaccharides; this subunit therefore spans the chromaffin-granule membrane. Labelling of the 17 kDa subunit occurred predominantly on the extracytoplasmic (matrix) face of the granule membrane. These results are consistent with this V-type ATPase having a structure that is generally similar to that of mitochondrial (F-type) ATPases, although the attachment of the 120 kDa subunit may be asymmetrical.
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Project description:1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg(2+) and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.
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Project description:Catecholamines are accumulated by bovine chromaffin-granule "ghosts" in the presence of MgATP at 25 degrees C. With low concentrations of catecholamine, ratios of internal to external amine concentration of up to 20 000 were obtained. These values fit well with a transport model in which amine accumulation is both electrogenic and dependent on a pH gradient across the membrane.
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