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M Forgac

Publications and source records attributed to M Forgac.

At least 55 records · Page 3Linked to original sources
PubMed1992

Characterization of the V0 domain of the coated vesicle (H+)-ATPase.

The coated vesicle (H+)-ATPase is composed of two domains, a peripheral V1 domain containing the 73 (A subunit)-, 58 (B subunit)-, 40-, 34-, and 33-kDa subunits and an integral V0 domain containing the 100-, 38-, 19-, and 17 (c subunit)-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). In the present manuscript we characterize the V0 domain with respect to its structural and activity properties. Glycerol density gradient separation of solubilized coated vesicle membrane proteins reveals the presence of an excess of V0 domains which migrate with a molecular weight of 250,000 and contain the V0 polypeptides in the same stoichiometry as in the intact V1V0 complex. Like the c subunit in V1V0, the c subunit of the free V0 domain is labeled by [14C]N,N'-dicyclohexylcarbodiimide (DCCD) and is extracted by chloroform:methanol. In addition, a monoclonal antibody specific for the 100-kDa subunit of the intact (H+)-ATPase recognizes the 100-kDa subunit of V0. Tryptic cleavage of the V0 complex gives the same pattern of fragments for the 100- and 38-kDa subunits as in the intact complex, but with an increase in sensitivity, suggesting greater exposure of these subunits in free V0. Proton conduction was measured in reconstituted vesicles containing the V0 domain and in native vesicles stripped of V1. No DCCD-inhibitable proton conduction was observed in either preparation, suggesting that unlike the corresponding F0 domain of F1F0, the free V0 domain is not an open proton channel.

Animals↗
PubMed1992

Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents.

The vacuolar class of (H+)-ATPases are highly sensitive to sulfhydryl reagents, such as N-ethylmaleimide. The cysteine residue which is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by N-ethylmalemide is located in subunit A and is able to form a disulfide bond with the cysteine moiety of cystine through an exchange reaction. This unique property distinguishes this cysteine residue from the remaining cysteine residues of the (H+)-ATPase. Using this reaction, we selectively labeled the cystine-reactive cysteine residue of subunit A with fluorescein-maleimide. After complete digestion of the labeled subunit A by V8 protease, a single labeled fragment of molecular mass 3.9 kDa was isolated and the amino-terminal sequence was determined. This fragment contains 2 cysteine residues, Cys240 and Cys254. Since Cys254 is conserved among all vacuolar (H+)-ATPases whereas Cys240 is not, it is likely that Cys254 is the residue which is responsible for the sensitivity of the vacuolar (H+)-ATPase to sulfhydryl reagents.

Amino Acid Sequence↗
PubMed1992

The 40-kDa subunit enhances but is not required for activity of the coated vesicle proton pump.

We have previously demonstrated reassembly of a functional vacuolar (H+)-ATPase from clathrin-coated vesicles using the dissociated peripheral domain (V1) and the membrane-bound integral domain (V0) (Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836-14841). We have used this reassembly procedure to test the function of the 40-kDa subunit of the coated vesicle (H+)-ATPase. In the absence of V0, a fraction of the peripheral subunits reassemble into a V1 subcomplex which contains the 73-kDa A subunit, the 58-kDa B subunit, and the 34- and 33-kDa subunits but lacks the 40-kDa subunit. This subcomplex, which sediments with a mass of approximately 500 kDa, can be separated from the remaining monomeric subunits (and the 40-kDa subunit) by density gradient sedimentation. When dissociated with 0.36 M KI, 2.5 mM ATP, and 2.5 mM MgSO4, and added to membranes from which V1 has been dissociated, this V1(-40 kDa) subcomplex is able to reassemble with V0 to give a (H+)-ATPase with a proton pumping activity approximately half that obtained in the presence of the 40-kDa subunit. The undissociated subcomplex is not competent for assembly of a functional (H+)-ATPase. Interestingly, the monomeric fraction obtained from density gradient sedimentation contains the 40-kDa subunit but lacks the 34-kDa subunit. This monomeric fraction is nevertheless also able to assemble with V0 to give a functional proton pump. The V1V0 complexes assembled in the absence of either the 40- or 34-kDa subunits, while active, are not stable to detergent solubilization and immunoprecipitation, suggesting that both of these subunits play a role in stabilization of the (H+)-ATPase complex. Evidence for interaction between the 40- and 33-kDa subunits is also presented.

Animals↗
PubMed1992

Differential expression of the "B" subunit of the vacuolar H(+)-ATPase in bovine tissues.

The B subunit is one of two nucleotide-binding polypeptides found in all members of the vacuolar class of H(+)-translocating ATPases. We have isolated aDNA clone encoding the bovine brain B (58 kDa) subunit and have deduced its amino acid sequence. The bovine brain amino acid sequence is 99% identical to a partial cDNA reported from human brain. Northern blot analysis of RNA isolated from bovine tissues and a bovine kidney cell line reveals that two messages of approximately 3.2 and 2.0 kilobases (kb) are expressed in all tissues examined except brain, where only the 3.2-kb message can be detected. Northern blotting of RNA isolated from human fibroblast and human lung tumor cell lines reveals that three messages of approximately 6.0, 3.2, and 2.0 kb are expressed, whereas only the 3.2-kb message is expressed in a human brain tumor cell line. This is the first demonstration of tissue-specific expression of multiple forms of a vacuolar H(+)-ATPase subunit. We have also isolated a partial cDNA clone from bovine brain which appears to encode an isoform of the B subunit. The deduced amino acid sequence is 82% identical to the major bovine brain B subunit sequence; it does not hybridize with either the 3.2- or 2.0-kb message on Northern blot. Southern blot analysis of bovine genomic DNA with probes derived from both isolated cDNAs indicates that the bovine B subunit is encoded by a multigene family.

Amino Acid Sequence↗
PubMed1992

Structure and properties of the coated vesicle (H+)-ATPase.

Clathrin-coated vesicles play an important role in both receptor-mediated endocytosis and intracellular membrane traffic in eukaryotic cells. The coated vesicle (H+)-ATPase functions to provide the acidic environment within endosomes and other intracellular compartments necessary for receptor recycling and intracellular membrane traffic. The coated vesicle (H+)-ATPase is composed of nine different subunits which are divided into two distinct domains. The peripheral V1 domain, which has the structure 73(3):58(3):40(1):34(1):33(1), possesses the nucleotide binding sites of the (H+)-ATPase. The integral V0 domain, which has the composition 100(1):38(1):19(1):17(6), contains the pathway for proton conduction across the membrane. Topographical analysis indicates a structure for the coated vesicle (H+)-ATPase very similar to that of the F-type ATPases. Reassembly studies have allowed us to probe the function of particular subunits in this complex and the activity properties of the separate domains. These studies have led to insights into possible mechanisms of regulating vacuolar acidification.

Proton Pumps↗
PubMed1992

Structure, function and regulation of the coated vesicle V-ATPase.

The coated vesicle V-ATPase plays an important role in both receptor-mediated endocytosis and intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling. The coated vesicle V-ATPase is a macromolecular complex of relative molecular mass 750,000 composed of nine subunits arranged in two structural domains. The peripheral V1 domain, which has a relative molecular mass of 500,000, has the subunit structure 73(3)58(3)40(1)34(1)33(1) and possesses all the nucleotide binding sites of the V-ATPase. The integral Vo domain of relative molecular mass 250,000 has a subunit composition of 100(1)38(1)19(1)17(6) and possesses the pathway for proton conduction across the membrane. Reassembly studies have allowed us to probe the role of specific subunits in the V-ATPase complex while chemical labeling studies have allowed us to identify specific residues which play a critical role in catalysis. From both structural analysis and sequence homology, the vacuolar-type H(+)-ATPases resemble the F-type H(+)-ATPases. Unlike the F1 and Fo domains of the F-type ATPases, however, the V1 and Vo domains do not appear to function independently. The possible relevance of these observations to the regulation of vacuolar acidification is discussed.

Adenosine Triphosphatases↗
PubMed1991

A single gene encodes the catalytic "A" subunit of the bovine vacuolar H(+)-ATPase.

We have previously demonstrated that the 73-kDa (A) subunit of the bovine coated vesicle (H+)-ATPase possesses a nucleotide binding site required for catalytic activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). Here we report the cDNA sequence of the coding region of the bovine brain A subunit. Comparison of the deduced amino acid sequence with those previously reported for the A subunits of vacuolar ATPases from lower eukaryotes, plants, and archaebacteria reveals significant homology, especially in sequences implicated in nucleotide binding. The message encoding the bovine brain A subunit is relatively large, approximately 4.6 kilobases; Northern blotting of RNA isolated from rat brain and human brain tumor cells reveals a message of similar size. Northern analysis of several bovine tissues indicates that only one message for this subunit is expressed. Southern blot analysis of bovine genomic DNA indicates that the bovine A subunit is encoded by a single gene.

Amino Acid Sequence↗
PubMed1991

Modulation of coated vesicle chloride channel activity and acidification by reversible protein kinase A-dependent phosphorylation.

We have previously shown that activity of a Cl- channel is required for acidification of clathrin-coated vesicles by the coated vesicle (H+)-ATPase (Arai, H., Pink, S. and Forgac, M. (1989) Biochemistry 28, 3075-3082). We demonstrate that activity of the coated vesicle Cl- channel is modulated by phosphorylation. Cl- conductance was measured in a reconstituted preparation of coated vesicle membrane proteins using the Cl(-)-sensitive fluorescence probe, 6-methoxy-N-(3-sulfopropyl)quinolinium. Treatment of coated vesicle membranes with alkaline phosphatase resulted in a 25 +/- 5% decrease in Cl- channel activity. A parallel decrease in ATP-dependent acidification of coated vesicles was also observed. The decrease in Cl- conductance and ATP-dependent acidification was reversed by treatment with protein kinase A and MgATP; the alkaline phosphatase inhibitor, sodium orthovanadate, blocked the inhibition of acidification. These results indicate that Cl- conductance in coated vesicles is modulated by a protein kinase A-dependent phosphorylation and that this modulation in turn affects ATP-dependent acidification.

Adenosine Triphosphate↗
PubMed1991

Immunolocalization of the vacuolar-type (H+)-ATPase from clathrin-coated vesicles.

Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.

Animals↗
PubMed1990

Functional reassembly of the coated vesicle proton pump.

We have shown previously that treatment of the coated vesicle proton-translocating adenosine triphosphatase (H(+)-ATPase) with chaotropic agents results in the release of a set of peripheral polypeptides which includes the 73-, 58-, 40-, 34-, and 33-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973), with a coordinate loss of H(+)-ATPase activity. In the present paper we report the functional reassembly of the coated vesicle proton pump following dissociation of the peripheral subunits. Reassembly was demonstrated by restoration of ATP-driven proton transport using both native membranes and reconstituted vesicles and by Western blot analysis using a monoclonal antibody specific for the 73-kDa subunit. Reassembly occurs by attachment of a peripheral subcomplex containing the 73-, 58-, 34-, and 33-kDa subunits together with the 40-kDa polypeptide. The reassembled H(+)-ATPase, like the native proton pump, is inhibited by N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and N,N'-dicyclohexylcarbodiimide. Reassociation shows a biphasic time dependence, with restoration of 50-60% of the starting proton transport activity in the 1st h followed by recovery of a further 20-30% of the activity after 24 h. Reassembly also shows a marked dependence on protein concentration but, unlike solubilization of the intact H(+)-ATPase complex, does not require the presence of glycerol. Despite the ability of nucleotides to promote dissociation of the peripheral complex by chaotropic agents, reassociation is not blocked by the presence of 1 mM ATP. These results thus provide the first evidence for functional reassembly of a vacuolar H(+)-ATPase complex and should be useful in further analysis of the role of individual subunits in the assembly and activity of these ATP-driven proton pumps.

Adenosine Triphosphate↗
PubMed1990

Proteolysis and orientation on reconstitution of the coated vesicle proton pump.

We recently proposed a structural model for the ATP-dependent proton pump from clathrin-coated vesicles (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802). To test this model further, we have carried out additional structural analysis of the (H+)-ATPase in both the detergent-solubilized and reconstituted states in this and the following paper (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973). The orientation of the reconstituted proton pump was determined by analyzing the effect of detergent on ATP hydrolysis and by quantitating the extent of labeling of luminally oriented subunits using a membrane-impermeant reagent. Greater than 90% of the reconstituted (H+)-ATPase is oriented with the cytoplasmic surface facing outward. Treatment of the reconstituted (H+)-ATPase with trypsin results in rapid cleavage of the 100-, 73-, 58-, 38-, and 34-kDa subunits and slower cleavage of the 40- and 33-kDa subunits, consistent with our previous results indicating that all of these polypeptides have some portion of their mass exposed to the cytoplasmic surface. The 19- and 17-kDa subunits, by contrast, appear resistant to cleavage by trypsin in both the detergent-solubilized and reconstituted states, consistent with their being buried extensively in the hydrophobic phase of the bilayer. Treatment of the enzyme with trypsin under conditions in which the 100-, 73-, 58-, 38-, and 34-kDa subunits have been cleaved results in a species which is virtually inactive with respect to proton transport but retains 50% of the original ATPase activity, suggesting that proteolysis has resulted in uncoupling of these two activities. Cleavage of both the 73- and 58-kDa subunits by trypsin at a site 1-2 kDa from the amino terminus is inhibited in the presence of 2',3'-O-(2,4,6-trinitrophenyl)-ATP, consistent with the suggestion that both the 73- and 58-kDa subunits may be nucleotide binding proteins.

Adenosine Triphosphate↗
PubMed1990

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump.

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.

Biological Transport, Active↗
PubMed1989

Interaction of anions and ATP with the coated vesicle proton pump.

ATP-driven proton transport in intact clathrin-coated vesicles requires the presence of a permeant anion, such as Cl-, to provide charge compensation during the electrogenic movement of protons. Using the purified (H+)-ATPase from clathrin-coated vesicles in both the detergent-solubilized and reconstituted states, we have studied the direct effects of anions on the activity of this enzyme. Both proton transport and ATP hydrolysis by the purified enzyme are independent of the presence of Cl-. In addition, proton transport does not occur even at high Cl- concentrations unless K+ and valinomycin are present to dissipate the membrane potential generated. These results indicate that the anion channel which provides for Cl- flux in intact coated vesicles is not a component of the purified (H+)-ATPase. Inhibition of ATPase activity is observed in the presence of I-, NO3-, or SO4(2-), with 50% inhibition occurring at 350 mM I-, 50 mM NO3-, or 40 mM SO4(2-). The presence of ATP lowers the concentration of I- required for 50% inhibition from 350 mM to 100 mM and increases the maximal inhibition observed in the presence of NO3- from 65% to 100%. Two separate mechanisms appear to be responsible for anion inhibition of the (H+)-ATPase. Thus, I- and high concentrations of NO3- (in the presence of ATP) cause inhibition by dissociation of the (H+)-ATPase complex, while SO4(2-) and NO3- (in the absence of ATP) cause inhibition without dissociation of the complex, suggesting the existence of an inhibitory anion binding site on the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Topography and subunit stoichiometry of the coated vesicle proton pump.

We have previously shown that the coated vesicle (H+)-ATPase contains nine polypeptides of molecular weight 17,000-100,000 which form a single, macromolecular complex that can be immunoprecipitated using monoclonal antibodies which recognize the native enzyme (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M. (1987) Biochemistry 26, 6632-6638). In the present paper, we have calculated from quantitative amino acid analysis that these polypeptides are present in the native complex in a stoichiometry of three copies each of the 73,000- and 58,000-dalton subunits, six copies of the 17,000-dalton subunit, and one copy each of the 100,000-, 40,000-, 38,000-, 34,000-, 33,000-, and 19,000-dalton subunits. To determine the disposition of the (H+)-ATPase subunits with respect to the membrane, we have carried out labeling studies using the membrane impermeant reagents Na125I/lactoperoxidase and 125I-sulfo-succinimidyl-3-(4-hydroxyphenyl)propionate and the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID). Polypeptides exposed to the cytoplasmic surface were identified by labeling with impermeant reagents in intact vesicles from which clathrin had been dissociated followed by immunoprecipitation of the native enzyme. Polypeptides exposed to the luminal surface were identified by increased labeling by these reagents following detergent solubilization under nondenaturing conditions. Labeling by [125I]TID was used to indicate which polypeptides are embedded in the lipid bilayer. Results of these experiments indicate that the principal polypeptides labeled from the cytoplasmic surface are those of molecular weight 73,000 and 58,000, although some cytoplasmic labeling of the 100,000, 40,000, 38,000 and 34,000/33,000 polypeptides was also observed. The polypeptides which show the greatest increase in labeling following detergent solubilization are those of molecular weight 100,000, 19,000, and 17,000, with some increase observed for the 40,000, 38,000, and 34,000/33,000 polypeptides. [125I]TID labeled the 17,000-dalton subunit most heavily, with significant labeling of the 100,000- and 40,000-dalton subunits also observed. In addition, we find that the 73,000-dalton polypeptide can be dissociated from the complex with 0.5 M KI in the absence of detergent, indicating a peripheral association of this subunit with the membrane. We have combined these results to construct a structural model of the coated vesicle (H+)-ATPase.

Amino Acids↗
PubMed1987

Subunit composition and ATP site labeling of the coated vesicle proton-translocating adenosinetriphosphatase.

The partially purified proton-translocating adenosinetriphosphatase [(H+)-ATPase] from clathrin-coated vesicles has been reported to contain eight polypeptides of molecular weights 15,000-116,000 [Xie, X.S., & Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495]. To determine whether these polypeptides form a single macromolecular complex, we have isolated three monoclonal antibodies which recognize the reconstitutively active (H+)-ATPase in the native, detergent-solubilized state. All three monoclonal antibodies precipitate the same set of polypeptides from either the partially purified enzyme or the detergent-solubilized coated vesicle membrane proteins. The immunoprecipitated polypeptides have molecular weights of 100,000, 73,000, 58,000, 40,000, 38,000, 34,000, 33,000, 19,000, and 17,000. These results thus indicate that this set of polypeptides forms a single macromolecular complex and suggest that they correspond to subunits of the coated vesicle (H+)-ATPase. To identify the ATP-hydrolytic subunit of the coated vesicle (H+)-ATPase, the purified enzyme was reacted with N-ethylmaleimide (NEM) and 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), both of which inhibit activity in an ATP-protectable manner. Labeling was carried out by using [3H]NEM or [14C]NBD-Cl, and the specificity of the reaction was increased by prelabeling of the protein with the nonradioactive reagents in the presence of ATP and by taking advantage of the nucleotide specificity of protection. The principal polypeptide labeled by both [3H]NEM and [14C]NBD-Cl had a molecular weight of 73,000. In addition, this protein was the only polypeptide whose labeling was significantly reduced in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloro-7-nitrobenzofurazan↗