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Michael Forgac - One of the best experts on this subject based on the ideXlab platform.

  • structure mechanism and regulation of the clathrin Coated Vesicle and yeast vacuolar h atpases
    The Journal of Experimental Biology, 2000
    Co-Authors: Michael Forgac
    Abstract:

    The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-Coated Vesicles and yeast vacuoles. The Coated-Vesicle V-ATPase undergoes trafficking to endosomes and synaptic Vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification.

  • subunit interactions in the clathrin Coated Vesicle vacuolar h atpase complex
    Journal of Biological Chemistry, 1999
    Co-Authors: Elena Vasilyeva, Michael Forgac
    Abstract:

    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.

  • inhibition and labeling of the Coated Vesicle v atpase by 2 azido 32p atp
    Journal of Biological Chemistry, 1995
    Co-Authors: Jimin Zhang, Yu Feng, Elena Vasilyeva, Michael Forgac
    Abstract:

    Abstract Previous studies have indicated that the 73-kDa A subunit of the Coated Vesicle V-ATPase possesses a nucleotide-binding site essential for activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M.(1987) Biochemistry 26, 6632-6638) and have identified a cysteine residue (Cys254) whose modification leads to complete loss of activity (Feng, Y., and Forgac, M.(1992) J. Biol. Chem. 267, 5817-5822). To further characterize the structure of the nucleotide-binding sites of the V-ATPase, labeling studies using the photoactivated analog 2-azido-[32P]ATP have been carried out. We have observed that 2-azido-[32P]ATP is hydrolyzed by the V-ATPase at a rate (at 1 mM) approximately 4-fold lower than observed for ATP, indicating that 2-azido-[32P]ATP is a good substrate for the V-ATPase. Irradiation of the V-ATPase in the presence of 0.5 mM 2-azido-[32P]ATP leads to inactivation of V-ATPase activity with a t of 3-5 min. The 73-kDa A subunit, the 58-kDa B subunit, and the 50-kDa subunit of the AP-2 adaptin complex (Myers, M., and Forgac, M.(1993) J. Biol. Chem. 268, 9184-9186) are all labeled in an ATP-protectable manner on irradiation of the purified V-ATPase with 2-azido-[32P]ATP. The time course for inactivation most closely correlates with labeling of the A subunit. Measurement of the stoichiometry of 2-azido-[32P]ATP incorporation into the A subunit as a function of inactivation indicates that complete loss of activity is obtained on incorporation of 1.2 mol of 2-azido-[32P]ATP/mol V-ATPase complex. 2-Azido-[32P]ATP labeling indicates that the V-ATPase possesses both rapidly (t 2 min) exchangeable nucleotide-binding sites. The A subunit is labeled upon modification of both rapidly and slowly exchangeable sites whereas the B subunit is labeled upon modification of only rapidly exchangeable sites. Inhibition of V-ATPase activity correlates with labeling of the rapidly exchangeable sites. Amino acid sequence analysis of peptides derived from the 2-azido-[32P]ATP-labeled A subunit indicates labeling of two peptides: a 12-kDa fragment which begins at residue 511 and contains Cys532 and a 3-kDa fragment which begins at residue 233 and contains the glycine-rich loop and Cys254. Only the 12-kDa fragment is labeled upon modification of the rapidly exchangeable sites. A 12-kDa V8 peptide, which begins at residue 375 of the B subunit is also labeled by 2-azido-[32P]ATP under conditions which modify the rapidly exchangeable sites. These results provide further information concerning the nucleotide-binding sites on the V-ATPase A and B subunits. The differences in nucleotide-binding sites between F- and V-ATPases are also discussed.

  • proton conduction and bafilomycin binding by the v0 domain of the Coated Vesicle v atpase
    Journal of Biological Chemistry, 1994
    Co-Authors: Jimin Zhang, Yu Feng, Michael Forgac
    Abstract:

    Abstract We have previously demonstrated that the V0 domain of the Coated Vesicle V-ATPase, a 250-kDa integral complex, does not form a functional proton channel (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). In the present study we describe dissociation of the V0 complex and separation of the V0 subunits by gel filtration. Dicyclohexylcarbodiimide-inhibitable passive proton conductance of reconstituted Vesicles containing reassembled V0 subunits was measured in response to a K+/valinomycin-generated membrane potential. We observed that reconstituted Vesicles containing the 17/19-kDa subunits carried out passive proton transport, with the addition of the 38- and 100-kDa subunits increasing proton conductance. Reconstituted Vesicles containing the 38- and/or 100-kDa subunits showed no proton transport. Partial separation of the 17- and 19-kDa subunits revealed that the 17-kDa subunit alone carried out proton transport, with increased conductance on the addition of the 19-kDa subunit. These results indicate that the V0 domain possesses the information necessary to form a dicyclohexylcarbodiimide-inhibitable passive proton channel. Bafilomycin binding by native and reassembled V0 complexes was also measured by their ability to protect V-ATPase activity against bafilomycin inhibition. The native V0 domain, the isolated 100-kDa subunit and the 100/38-kDa subunits were able to protect against inhibition by bafilomycin, suggesting that the binding site for bafilomycin resides on the 100-kDa subunit.

  • the Coated Vesicle vacuolar h atpase associates with and is phosphorylated by the 50 kda polypeptide of the clathrin assembly protein ap 2
    Journal of Biological Chemistry, 1993
    Co-Authors: Melanie Myers, Michael Forgac
    Abstract:

    Abstract We have previously noted a 50-kDa polypeptide (p50) co-purifying with preparations of the bovine brain clathrin-Coated Vesicle vacuolar (H+)-ATPase (V-ATPase) (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). We show that p50 is also immunoprecipitated with the V-ATPase, further suggesting its specific association with the proton pump. To determine the identity of this 50-kDa polypeptide and the stoichiometry of its association with the V-ATPase, we performed N-terminal amino acid sequencing and quantitative amino acid analysis of the gel-purified protein. These results revealed the unknown polypeptide to be the 50-kDa subunit of the clathrin assembly protein AP-2 (AP50); we estimate the stoichiometry of association is one AP50 per V-ATPase complex. AP50 is an N-ethylmaleimide (NEM)-inhibitable autokinase and incubation of purified V-ATPase with [gamma-32P]ATP resulted in the NEM-sensitive phosphorylation of AP50 and the B subunit of the V-ATPase. The same phosphorylation pattern is seen if the labeling reaction is done with intact clathrin-Coated Vesicles and the V-ATPase subsequently immunoprecipitated from the solubilized Vesicles. This represents the first report of phosphorylation of one of the V-ATPase subunits. The functional significance of this phosphorylation for regulation or targeting of the V-ATPase in vivo remains to be determined.

Sandra L. Schmid - One of the best experts on this subject based on the ideXlab platform.

  • conserved functions of membrane active gtpases in Coated Vesicle formation
    Science, 2009
    Co-Authors: Thomas J Pucadyil, Sandra L. Schmid
    Abstract:

    Coated Vesicles concentrate and package cargo molecules to mediate their efficient transport between intracellular compartments. Cytosolic coat proteins such as clathrin and adaptor complexes and coat protein complex I (COPI) and COPII self-assemble to deform the membrane and interact directly with cargo molecules to capture them in nascent buds. The guanosine triphosphatases (GTPases) Arf, Sar1, and dynamin are core components of the Coated Vesicle machinery. These GTPases, which associate with and dissociate from donor membranes in a guanosine triphosphate-dependent manner, can also actively remodel membranes. Recent evidence suggests that, although structurally diverse, Arf family GTPases and dynamin may play mechanistically similar roles as fidelity monitors that govern cargo packaging and Coated Vesicle maturation and as components of the fission machinery to mediate Vesicle release.

  • clathrin Coated Vesicle formation from isolated plasma membranes
    Methods in Enzymology, 2005
    Co-Authors: Ishido Miwako, Sandra L. Schmid
    Abstract:

    Abstract Endocytic clathrin‐Coated Vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be defined. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors.

  • a dynamic actin cytoskeleton functions at multiple stages of clathrin mediated endocytosis
    Molecular Biology of the Cell, 2004
    Co-Authors: Defne Yarar, Clare M Watermanstorer, Sandra L. Schmid
    Abstract:

    Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-Coated Vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-Coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-Coated Vesicle formation, including Coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.

  • clathrin and dynamin dependent Coated Vesicle formation from isolated plasma membranes
    Traffic, 2003
    Co-Authors: Ishido Miwako, Thomas Schroter, Sandra L. Schmid
    Abstract:

    We have developed a new rapid cell-free assay for endocytic clathrin-Coated Vesicle formation using highly purified rat liver plasma membrane sheets. After incubation in the presence of cytosol and nucleotides, released Vesicles were collected by high-speed centrifugation and incorporated cargo receptors were detected by Western blotting. Three different cargo receptors were internalized into Vesicles while a receptor, known to be excluded from Coated pits, was not. The recruitment of cargo receptors into the Vesicle fraction was cytosol, ATP and temperature-dependent and was enhanced by addition of GTP. Vesicle formation in this assay was confirmed by subcellular fractionation and EM analysis. Plasma membranes stripped of their endogenous coat proteins with 0.5 m Tris retained Vesicle formation activity, which was highly dependent on clathrin and dynamin. Coat proteins and dynamin were not sufficient for clathrin-Coated Vesicle formation, and other peripheral membrane proteins recruited from the cytosol are required. The nonhydrolyzable ATP analogue, AMPPNP did not support clathrin-Coated Vesicle formation; however, surprisingly, GTPγS was as effective as GTP. This assay will provide a powerful tool to dissect the minimum machinery and to probe the hierarchy of events involved in cargo selection and endocytic clathrin-Coated Vesicle formation.

  • dominant interfering hsc70 mutants disrupt multiple stages of the clathrin Coated Vesicle cycle in vivo
    Journal of Cell Biology, 2001
    Co-Authors: Sherri L Newmyer, Sandra L. Schmid
    Abstract:

    Within the clathrin-Coated Vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

Dennis K Stone - One of the best experts on this subject based on the ideXlab platform.

  • Ciba Foundation Symposium 139 - Proton Passage Across Cell Membranes - Structural properties of the proton translocating complex of the clathrin-Coated Vesicle.
    Ciba Foundation symposium, 2007
    Co-Authors: Dennis K Stone
    Abstract:

    : The clathrin-Coated Vesicle proton pump is a representative member of the new class of endomembrane proton ATPases that share an inhibitor profile which distinguishes them from classic F1F0 and E1E2-type proton pumps. The Coated Vesicle proton pump is a large (530 kDa) heteroligomer composed of eight polypeptides with molecular masses of 116, 70, 58, 40, 38, 34, 33 and 17 kDa. The 200-fold purified enzyme catalyses ATP-generated proton pumping when reconstituted in liposomes composed of pure lipids. Subunit function has been determined by partial reaction analysis of subunit and subcomplex activities. The isolated 17 kDa subunit, when co-reconstituted with bacteriorhodopsin, forms a dicyclohexylcarbodiimide-inhibitable proton channel. Selective removal of the 116 kDa subunit transforms the proton ATPase from a Mg2+-activatable to a Ca2+-activatable ATPase. Subsequent dissociation and reconstitution of subunits reveals that the 70, 58, 40 and 33 kDa components are required, in composite, to form a functional ATP-hydrolytic core, and that no single subunit or subcomplex deficient in these subunits can catalyse ATP hydrolysis.

  • identification of a 14 kda subunit associated with the catalytic sector of clathrin Coated Vesicle h atpase
    Journal of Biological Chemistry, 1996
    Co-Authors: Sheng Bin Peng, Bill P Crider, Xiao Song Xie, Sue Jean Tsai, Dennis K Stone
    Abstract:

    Abstract The clathrin-Coated Vesicle H-ATPase is composed of a peripheral catalytic sector (V) and an integral membrane proton channel (V), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13,370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-Coated Vesicles and also isolated V. When stripped clathrin-Coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the Coated vacuolar proton pump confirmed its identity as a subunit F homologue.

  • isolation of a protein activator of the clathrin Coated Vesicle proton pump
    Journal of Biological Chemistry, 1993
    Co-Authors: Xiao Song Xie, Bill P Crider, Dennis K Stone
    Abstract:

    Abstract An activator of the clathrin-Coated Vesicle proton translocating ATPase has been purified 1600-fold from bovine brain. The activator, which requires detergent (polyoxyethylene 9-lauryl ether) for release from clathrin-Coated Vesicles, is heat-stable, trypsin-sensitive, and has an apparent molecular mass of about 6 kDa as determined by high performance liquid chromatography. The activator stimulates the purified H(+)-ATPase of Coated Vesicles over 50-fold under acidic conditions. Similarly, the activator stimulates proton pumping catalyzed by the reconstituted proton pump. Importantly, this stimulation of proton pumping is observed only when the activator is reconstituted into the interior of the proteoliposomes. Moreover, the activator protein is demonstrated to protect, and co-sediment with, purified proton pump during glycerol gradient centrifugation performed in the presence of ATP. These observations support the notion that this activator serves to determine the pH set point of acidic endomembranes through interactions with the transmembranous sectors of the proton pump.

Elizabeth Smythe - One of the best experts on this subject based on the ideXlab platform.

  • regulation of the clathrin Coated Vesicle cycle by reversible phosphorylation
    Biochemical Society Symposia, 2005
    Co-Authors: Alexander Flett, Sophia Semerdjieva, Antony P Jackson, Elizabeth Smythe
    Abstract:

    Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the Coated Vesicle cycle.

  • Clathrin-Coated Vesicle formation: a paradigm for Coated-Vesicle formation.
    Biochemical Society Transactions, 2003
    Co-Authors: Elizabeth Smythe
    Abstract:

    Clathrin-Coated pits are the major ports of entry into the cell and are responsible for the internalization of a variety of biologically important macromolecules. These transport intermediates form as a result of the co-ordinated assembly of a number of cytosolic proteins on to the membrane which results in specific cargo recruitment. We have used a variety of approaches including permeabilized cell assays and light and electron microscopy to identify and characterize the proteins and enzymes involved in Coated Vesicle formation.

  • regulating the clathrin Coated Vesicle cycle by ap2 subunit phosphorylation
    Trends in Cell Biology, 2002
    Co-Authors: Elizabeth Smythe
    Abstract:

    Abstract Clathrin-Coated pits form from the concerted assembly of many different protein components that interact with each other and with the lipid bilayer. When the Coated pit pinches off to form a Coated Vesicle, the coat is removed to allow fusion of the endocytic Vesicle with an early endosome, and the disassembled coat components can then recycle through the cytosol to participate in new rounds of Coated pit formation. One potential mechanism to regulate the cycles of coat protein assembly and disassembly is reversible phosphorylation, particularly given that many of the coat components, such as the AP2 adaptor complex, are phosphorylated on multiple sites both in vitro and in vivo . Recent studies have provided particular insight into the role of phosphorylation of the cargo-binding μ2 subunit of AP2.

  • the role of dynamin and its binding partners in Coated pit invagination and scission
    Journal of Cell Biology, 2001
    Co-Authors: Elaine Hill, Jeroen Van Der Kaay, Peter C Downes, Elizabeth Smythe
    Abstract:

    Plasma membrane clathrin-Coated Vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-Coated Vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of Coated Vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in Coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of Coated pit invagination and Coated Vesicle scission. Dynamin has previously been shown to have a role in scission of Coated Vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-Coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated Coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the Coated Vesicle cycle.

  • stage specific assays for Coated pit formation and Coated Vesicle budding in vitro
    Journal of Cell Biology, 1991
    Co-Authors: Sandra L. Schmid, Elizabeth Smythe
    Abstract:

    Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated Coated pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving Coated Vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via Coated pits and Coated Vesicles and resulted in delivery of ligand to endosomal structures.

Jimin Zhang - One of the best experts on this subject based on the ideXlab platform.

  • inhibition and labeling of the Coated Vesicle v atpase by 2 azido 32p atp
    Journal of Biological Chemistry, 1995
    Co-Authors: Jimin Zhang, Yu Feng, Elena Vasilyeva, Michael Forgac
    Abstract:

    Abstract Previous studies have indicated that the 73-kDa A subunit of the Coated Vesicle V-ATPase possesses a nucleotide-binding site essential for activity (Arai, H., Berne, M., Terres, G., Terres, H., Puopolo, K., and Forgac, M.(1987) Biochemistry 26, 6632-6638) and have identified a cysteine residue (Cys254) whose modification leads to complete loss of activity (Feng, Y., and Forgac, M.(1992) J. Biol. Chem. 267, 5817-5822). To further characterize the structure of the nucleotide-binding sites of the V-ATPase, labeling studies using the photoactivated analog 2-azido-[32P]ATP have been carried out. We have observed that 2-azido-[32P]ATP is hydrolyzed by the V-ATPase at a rate (at 1 mM) approximately 4-fold lower than observed for ATP, indicating that 2-azido-[32P]ATP is a good substrate for the V-ATPase. Irradiation of the V-ATPase in the presence of 0.5 mM 2-azido-[32P]ATP leads to inactivation of V-ATPase activity with a t of 3-5 min. The 73-kDa A subunit, the 58-kDa B subunit, and the 50-kDa subunit of the AP-2 adaptin complex (Myers, M., and Forgac, M.(1993) J. Biol. Chem. 268, 9184-9186) are all labeled in an ATP-protectable manner on irradiation of the purified V-ATPase with 2-azido-[32P]ATP. The time course for inactivation most closely correlates with labeling of the A subunit. Measurement of the stoichiometry of 2-azido-[32P]ATP incorporation into the A subunit as a function of inactivation indicates that complete loss of activity is obtained on incorporation of 1.2 mol of 2-azido-[32P]ATP/mol V-ATPase complex. 2-Azido-[32P]ATP labeling indicates that the V-ATPase possesses both rapidly (t 2 min) exchangeable nucleotide-binding sites. The A subunit is labeled upon modification of both rapidly and slowly exchangeable sites whereas the B subunit is labeled upon modification of only rapidly exchangeable sites. Inhibition of V-ATPase activity correlates with labeling of the rapidly exchangeable sites. Amino acid sequence analysis of peptides derived from the 2-azido-[32P]ATP-labeled A subunit indicates labeling of two peptides: a 12-kDa fragment which begins at residue 511 and contains Cys532 and a 3-kDa fragment which begins at residue 233 and contains the glycine-rich loop and Cys254. Only the 12-kDa fragment is labeled upon modification of the rapidly exchangeable sites. A 12-kDa V8 peptide, which begins at residue 375 of the B subunit is also labeled by 2-azido-[32P]ATP under conditions which modify the rapidly exchangeable sites. These results provide further information concerning the nucleotide-binding sites on the V-ATPase A and B subunits. The differences in nucleotide-binding sites between F- and V-ATPases are also discussed.

  • proton conduction and bafilomycin binding by the v0 domain of the Coated Vesicle v atpase
    Journal of Biological Chemistry, 1994
    Co-Authors: Jimin Zhang, Yu Feng, Michael Forgac
    Abstract:

    Abstract We have previously demonstrated that the V0 domain of the Coated Vesicle V-ATPase, a 250-kDa integral complex, does not form a functional proton channel (Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773-9778). In the present study we describe dissociation of the V0 complex and separation of the V0 subunits by gel filtration. Dicyclohexylcarbodiimide-inhibitable passive proton conductance of reconstituted Vesicles containing reassembled V0 subunits was measured in response to a K+/valinomycin-generated membrane potential. We observed that reconstituted Vesicles containing the 17/19-kDa subunits carried out passive proton transport, with the addition of the 38- and 100-kDa subunits increasing proton conductance. Reconstituted Vesicles containing the 38- and/or 100-kDa subunits showed no proton transport. Partial separation of the 17- and 19-kDa subunits revealed that the 17-kDa subunit alone carried out proton transport, with increased conductance on the addition of the 19-kDa subunit. These results indicate that the V0 domain possesses the information necessary to form a dicyclohexylcarbodiimide-inhibitable passive proton channel. Bafilomycin binding by native and reassembled V0 complexes was also measured by their ability to protect V-ATPase activity against bafilomycin inhibition. The native V0 domain, the isolated 100-kDa subunit and the 100/38-kDa subunits were able to protect against inhibition by bafilomycin, suggesting that the binding site for bafilomycin resides on the 100-kDa subunit.

  • characterization of the v0 domain of the Coated Vesicle h atpase
    Journal of Biological Chemistry, 1992
    Co-Authors: Jimin Zhang, Melanie Myers, Michael Forgac
    Abstract:

    Abstract 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.