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

  • 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.

  • sh3 domain containing proteins function at distinct steps in clathrin coated Vesicle formation
    Nature Cell Biology, 1999
    Co-Authors: Fiona Simpson, Natasha K Hussain, Britta Qualmann, Regis B Kelly, Peter S Mcpherson, Sandra L. Schmid
    Abstract:

    Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of Clathrin-Coated Vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-Vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-Vesicle formation.

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.

  • structure and properties of the clathrin coated Vesicle and yeast vacuolar v atpases
    Journal of Bioenergetics and Biomembranes, 1999
    Co-Authors: Michael Forgac
    Abstract:

    The V-ATPases are a family of ATP-dependent proton pumps responsible foracidification of intracellular compartments in eukaryotic cells. This reviewfocuses on the the V-ATPases from Clathrin-Coated Vesicles and yeastvacuoles. The V-ATPase of Clathrin-Coated Vesicles is a precursor to thatfound in endosomes and synaptic Vesicles, which function in receptorrecycling, intracellular membrane traffic, and neurotransmitter uptake. Theyeast vacuolar ATPase functions to acidify the central vacuole and to drivevarious coupled transport processes across the vacuolar membrane. TheV-ATPases are composed of two functional domains. The V1 domain isa 570-kDa peripheral complex composed of eight subunits of molecular weight70—14 kDa (subunits A—H) that is responsible for ATP hydrolysis.The V0 domain is a 260-kDa integral complex composed of fivesubunits of molecular weight 100—17 kDa (subunits a, d, c, c8 and c9)that is responsible for proton translocation. Using chemical modification andsite-directed mutagenesis, we have begun to identify residues that play arole in ATP hydrolysis and proton transport by the V-ATPases. A centralquestion in the V-ATPase field is the mechanism by which cells regulatevacuolar acidification. Several mechanisms are described that may play a rolein controlling vacuolar acidification in vivo. One mechanisminvolves disulfide bond formation between cysteine residues located at thecatalytic nucleotide binding site on the 70-kDa A subunit, leading toreversible inhibition of V-ATPase activity. Other mechanisms includereversible assembly and dissociation of V1 and V0domains, changes in coupling efficiency of proton transport and ATPhydrolysis, and regulation of the activity of intracellular chloride channelsrequired for vacuolar acidification.

  • interaction of the clathrin coated Vesicle v atpase with adp and sodium azide
    Journal of Biological Chemistry, 1998
    Co-Authors: Elena Vasilyeva, Michael Forgac
    Abstract:

    Abstract The kinetics of adenosine triphosphate (ATP)-dependent proton transport into Clathrin-Coated Vesicles from bovine brain have been studied. We observe that the vacuolar proton-translocating ATPase (V-ATPase) from Clathrin-Coated Vesicles is subject to two different types of inhibition by ADP. The first is competitive inhibition with respect to ATP, with aK i for ADP of 11 μm. The second type of inhibition occurs after preincubation of the V-ATPase in the presence of ADP and Mg2+, which results in inhibition of the initial rate of proton transport followed by reactivation over the course of several minutes. The second effect is observed at ADP concentrations as low as 0.1–0.2 μm, indicating that a high affinity inhibitory complex is formed between ADP and the V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F1-F0-ATP synthase (F-ATPase). We observed that azide inhibited ATP-dependent proton transport by the purified, reconstituted V-ATPase with aK 0.5 of 0.2–0.4 mm but had no effect on ATP hydrolysis. Azide was shown not to increase the passive proton permeability of reconstituted Vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis. Thus, azide does not appear to act as a simple uncoupler of proton transport and ATP hydrolysis. Rather, azide may have some more direct effect on V-ATPase activity. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.

  • 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.

Tomas Kirchhausen - One of the best experts on this subject based on the ideXlab platform.

  • cellular mechanisms for transferrin receptor endocytosis vital for terminal erythroid differentiation
    Blood, 2013
    Co-Authors: Yuichi Ishikawa, Francois Aguet, Tomas Kirchhausen, Silvia K Tachevagrigorova, Manami Maeda, Maria Ericsson, Takahiro Maeda
    Abstract:

    Clathrin-mediated endocytosis (CME) is a key endocytic portal that regulates transferrin receptor (TfR) endocytosis. Although CME is among the most-studied cellular systems in cell biology, little is known regarding how this key system physiologically functions in vivo.The CALM gene (for Clathrin Assembly Lymphoid Myeloid, also known as PICALM ), located on human chromosome 11q14, encodes a 652 aa protein containing multiple domains functioning in Clathrin-Coated Vesicle formation. Since CALM is predominantly expressed in erythroblasts, and hematopoietic-specific Calm conditional knockout mice, which we developed, exhibit a severe iron-deficient anemia, we hypothesized that CALM functions to increase efficiency of TfR endocytosis in erythroblasts so that iron uptake is not rate-limiting. To test this hypothesis, we performed FACS-based endocytosis assay in primary erythroblasts, in vitro erythroid differentiation assay, live-cell imaging and CALM add-back rescue experiments. TfR endocytosis was severely attenuated in Calm -deficient basophilic- and polychromatophilic erythroblasts: efficiency of TfR endocytosis was less than 25% of that of control, as revealed by FACS-based endocytosis assay. Unexpectedly, TfR endocytosis was not active in orthochromatophilic erythroblasts regardless of genotype, while they express TfR at high levels.For live cell imaging, we established immortalized mouse embryonic fibroblasts (MEFs), in which the Calm gene can be deleted in an inducible manner (CalmF/F ERT2-Cre+ MEFs). The MEFs were then stably transduced with the retrovirus encoding an EGFP-tagged AP2-bound adaptin σ2. The dynamics of Clathrin-Coated Vesicle formation was determined using fast live cell fluorescence spinning disk confocal microscopy. Clathrin-coat dynamics was barely affected in the absence of Calm under regular culture condition; however, it was completely stalled under high membrane tension (e.g. hypo-osmotic medium, jasplakinolide treatment). To determine functional domain(s) of CALM, a series of CALM mutants were generated, “added-back” to Calm -deficient MEFs and coat dynamics examined by live cell imaging. Mutant that does not bind to phosphatidylinositol 4,5-bisphosphate of the plasma membrane (PIP2-mutant CALM) failed to rescue TfR endocytosis in Calm -deficient MEFs, suggesting that CALM binding to PIP2, but not to v-SNAREs or EPS15, is essential for CALM-mediated TfR endocytosis. Splenic erythroblasts were significantly expanded in Calm conditional knockout mice. In contrast, Calm -deficient hematopoietic progenitors cannot give rise to immature erythroblasts in vitro, suggesting that Calm -deficiency is partially compensated in vivo via non-cell autonomous mechanisms (e.g. central macrophages). Iron supplement treatment could significantly rescue erythroid development of Calm -deficient progenitors in vitro, indicating that iron-deficiency is a primary cause of developmental defects seen in Calm -deficient erythroblasts. As expected, retrovirus-mediated expression of WT-Calm, but not PIP2-mutant, completely rescued erythroid development in Calm -deficient erythroblasts in vitro.Our data indicate that CALM plays a key role in clathrin-mediated endocytosis in a context-dependent (high membrane tension) and cell-type-specific (erythroblasts) manner. We propose that CALM is essential for transferrin uptake in erythroblasts by functioning as an erythroid-specific clathrin adaptor. Disclosures: No relevant conflicts of interest to declare.

  • structure of the pten like region of auxilin a detector of clathrin coated Vesicle budding
    Structure, 2010
    Co-Authors: Rong J Guan, Dai Han, Stephen C Harrison, Tomas Kirchhausen
    Abstract:

    Auxilin, a J-domain containing protein, recruits the Hsc70 uncoating ATPase to newly budded Clathrin-Coated Vesicles. The timing of auxilin arrival determines that uncoating will commence only after the clathrin lattice has fully assembled and after membrane fission is complete. Auxilin has a region resembling PTEN, a PI3P phosphatase. We have determined the crystal structure of this region of bovine auxilin 1; it indeed resembles PTEN closely. A change in the structure of the P loop accounts for the lack of phosphatase activity. Inclusion of phosphatidylinositol phosphates substantially enhances liposome binding by wild-type auxilin, but not by various mutants bearing changes in loops of the C2 domain. Nearly all these mutations also prevent recruitment of auxilin to newly budded coated Vesicles. We propose a specific geometry for auxilin association with a membrane bilayer and discuss implications of this model for the mechanism by which auxilin detects separation of a Vesicle from its parent membrane.

  • structure of clathrin coat with bound hsc70 and auxilin mechanism of hsc70 facilitated disassembly
    The EMBO Journal, 2010
    Co-Authors: Yi Xing, Till Bocking, Stephen C Harrison, Tomas Kirchhausen, Nikolaus Grigorieff, Matthias Wolf
    Abstract:

    The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded Clathrin-Coated Vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

  • vesicular stomatitis virus enters cells through Vesicles incompletely coated with clathrin that depend upon actin for internalization
    PLOS Pathogens, 2009
    Co-Authors: David K Cureton, Tomas Kirchhausen, Ramiro Massol, Saveez Saffarian, Sean P J Whelan
    Abstract:

    Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical Clathrin-Coated Vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how Clathrin-Coated Vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially Clathrin-Coated Vesicles. We found that on average, virus-containing Vesicles contain more clathrin and clathrin adaptor molecules than conventional Vesicles, but this increase is insufficient to permit full coating of the Vesicle. We further show that virus-containing Vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing Vesicles, and chemical inhibition of actin polymerization trapped viral particles in Vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a Clathrin-Coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

  • a burst of auxilin recruitment determines the onset of clathrin coated Vesicle uncoating
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Ramiro Massol, Werner Boll, April M Griffin, Tomas Kirchhausen
    Abstract:

    Clathrin-Coated pits assemble on a membrane and pinch off as coated Vesicles. The released Vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and Vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after Vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Margaret S Robinson - One of the best experts on this subject based on the ideXlab platform.

  • a human genome wide screen for regulators of clathrin coated Vesicle formation reveals an unexpected role for the v atpase
    Nature Cell Biology, 2013
    Co-Authors: Patrycja Kozik, Nicola A Hodson, Daniela A Sahlender, Nikol Simecek, Christina Soromani, Lucy M Collinson, Margaret S Robinson
    Abstract:

    Robinson and colleagues perform a human-genome-wide siRNA screen to identify regulators of Clathrin-Coated Vesicle formation. The knockdown phenotype of one of their hits, V-ATPase, is rescued by exogenous cholesterol, revealing an intriguing link between cholesterol and Clathrin-Coated Vesicle formation.

  • epsinr is an adaptor for the snare protein vti1b
    Molecular Biology of the Cell, 2004
    Co-Authors: Jennifer Hirst, Gabriele Fischer Von Mollard, Sharon E Miller, Marcus J Taylor, Margaret S Robinson
    Abstract:

    EpsinR is a Clathrin-Coated Vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a approximately 50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

  • clathrin mediated endocytosis in ap 2 depleted cells
    Journal of Cell Biology, 2003
    Co-Authors: Alison Motley, Nicholas A Bright, Matthew N J Seaman, Margaret S Robinson
    Abstract:

    We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-Coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No Clathrin-Coated pits or Vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for Clathrin-Coated Vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

  • γ synergin an eh domain containing protein that interacts with γ adaptin
    Journal of Cell Biology, 1999
    Co-Authors: Lesley J Page, Penelope J. Sowerby, Winnie W.y. Lui, Margaret S Robinson
    Abstract:

    The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in Clathrin-Coated Vesicles. To identify other proteins that participate in the Clathrin-Coated Vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the γ-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both γ-adaptin and α-adaptin, and γ-synergin, an alternatively spliced protein with an apparent molecular mass of ∼110–190 kD, which only interacts with γ-adaptin. γ-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in Clathrin-Coated Vesicles. It binds directly to the ear domain of γ-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the γ-adaptin binding site. In cells expressing α-adaptin with the γ-adaptin ear, a construct that goes mainly to the plasma membrane, much of the γ-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that γ-synergin links the AP-1 complex to another protein or proteins.

  • recruitment of coat proteins onto golgi membranes in intact and permeabilized cells effects of brefeldin a and g protein activators
    Cell, 1992
    Co-Authors: Margaret S Robinson, Thomas E. Kreis
    Abstract:

    Abstract Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., γ-adaptin) associated with the Clathrin-Coated Vesicles that bud from the trans-Golgi network (TGN), while the Clathrin-Coated Vesicles that bud from the plasma membrane are unaffected. γ-Adaptin redistributes with the same kinetics as β-COP, a coat protein associated with the non-Clathrin-Coated Vesicles that bud from the Golgi complex. Upon removal of BFA, however, γ-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AIF 4 − . Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilized cell system and is increased by addition of GTPγS and blocked by addition of BFA. These results suggest a role for G proteins in the control of the Clathrin-Coated Vesicle cycle at the TGN and further extend the similarities between Clathrin-Coated Vesicles and non-Clathrin-Coated Vesicles.

Bill P Crider - One of the best experts on this subject based on the ideXlab platform.

  • 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, Sue Jean Tsai
    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.

  • reconstitution of the recombinant 70 kda subunit of the clathrin coated Vesicle h atpase
    Journal of Biological Chemistry, 1994
    Co-Authors: Sheng Bin Peng, Bill P Crider, Dennis K Stone, Ying Zhang, Allen E White, Victor A Fried, Xiao Song Xie
    Abstract:

    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.

  • role of a 50 57 kda polypeptide heterodimer in the function of the clathrin coated Vesicle proton pump
    Journal of Biological Chemistry, 1994
    Co-Authors: Xiao Song Xie, Bill P Crider, Yong Ming, Dennis K Stone
    Abstract:

    Abstract The vacuolar-type proton-translocating ATPase of Clathrin-Coated Vesicles is composed of an integral membrane proton channel (VB) and a peripheral catalytic sector (VC). Native enzyme can catalyze the hydrolysis of both MgATP and CaATP and support proton pumping when reconstituted into liposomes. In contrast, isolated VC catalyzes only Ca(2+)-activated ATP hydrolysis and cannot support proton pumping when reconstituted into liposomes (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We now report that solubilized isolated VC can be reassembled with purified VB to restore properties of native enzyme, including Mg(2+)-activated ATP hydrolysis and proton-pumping capability. Investigation of this reassembly revealed that a heterodimer, composed of polypeptides of 50 and 57 kDa, stimulates Ca(2+)-activated ATPase activity of isolated VC 2-fold and Mg(2+)-activated ATPase activity catalyzed by the reassembled pump 9-fold. Moreover, this heterodimer stimulated proton transport by the reassembled pump > 20-fold. When separated from the proton pump, the dimer has no detectable kinase activity. Maximal stimulation occurs at a molar ratio of heterodimer to reassembled pump of 3, implying a structural, nonenzymatic mechanism. These data indicate that the 50-kDa and/or the 57-kDa polypeptide likely plays an essential and potentially regulatory role in the function of the proton-translocating ATPase of Clathrin-Coated Vesicles.

  • isolation of a protein activator of the clathrin coated Vesicle proton pump
    Journal of Biological Chemistry, 1993
    Co-Authors: Bill P Crider
    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.