The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Lois E. Greene - One of the best experts on this subject based on the ideXlab platform.
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endocytosis and clathrin uncoating defects at synapses of Auxilin knockout mice
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Yangin Yim, Pietro De Camilli, Evan Eisenberg, Tao Sun, Andrea Raimondi, Lois E. GreeneAbstract:Neuronally expressed Auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an Auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of Auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.
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essential role of cyclin g associated kinase Auxilin 2 in developing and mature mice
Molecular Biology of the Cell, 2008Co-Authors: Dong Won Lee, Evan Eisenberg, Xiaohong Zhao, Yangin Yim, Lois E. GreeneAbstract:Hsc70 with its cochaperone, either Auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.
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multiple roles of Auxilin and hsc70 in clathrin mediated endocytosis
Traffic, 2007Co-Authors: Evan Eisenberg, Lois E. GreeneAbstract:The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either Auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific Auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of Auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and Auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and Auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and Auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, Auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and Auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.
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recruitment dynamics of gak and Auxilin to clathrin coated pits during endocytosis
Journal of Cell Science, 2006Co-Authors: Dong Won Lee, Evan Eisenberg, Lois E. GreeneAbstract:Cyclin G-associated kinase (GAK), the ubiquitous form of the neuronal-specific protein Auxilin 1, is an essential cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Total internal reflectance microscopy was used to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits is inhibited by actin depolymerization, there is repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating.
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depletion of gak Auxilin 2 inhibits receptor mediated endocytosis and recruitment of both clathrin and clathrin adaptors
Journal of Cell Science, 2005Co-Authors: Dong Won Lee, Evan Eisenberg, Xiaohong Zhao, Fang Zhang, Lois E. GreeneAbstract:Cyclin G-associated kinase (GAK/Auxilin 2), the ubiquitous form of the neuronal-specific protein Auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.
Evan Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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endocytosis and clathrin uncoating defects at synapses of Auxilin knockout mice
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Yangin Yim, Pietro De Camilli, Evan Eisenberg, Tao Sun, Andrea Raimondi, Lois E. GreeneAbstract:Neuronally expressed Auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an Auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of Auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.
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essential role of cyclin g associated kinase Auxilin 2 in developing and mature mice
Molecular Biology of the Cell, 2008Co-Authors: Dong Won Lee, Evan Eisenberg, Xiaohong Zhao, Yangin Yim, Lois E. GreeneAbstract:Hsc70 with its cochaperone, either Auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.
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multiple roles of Auxilin and hsc70 in clathrin mediated endocytosis
Traffic, 2007Co-Authors: Evan Eisenberg, Lois E. GreeneAbstract:The ATP-dependent dissociation of clathrin from clathrin-coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J-domain cofactor proteins, either Auxilin or cyclin-G-associated kinase (GAK). Both the nerve-specific Auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of Auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and Auxilin are involved in several other key processes that occur during clathrin-mediated endocytosis. First, Hsc70 and Auxilin are required for the clathrin exchange that occurs during coated-pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin-coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and Auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, Auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and Auxilin provide the energy for this curvature by inducing ATP-dependent clathrin exchange and rearrangement during endocytosis and ATP-dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.
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recruitment dynamics of gak and Auxilin to clathrin coated pits during endocytosis
Journal of Cell Science, 2006Co-Authors: Dong Won Lee, Evan Eisenberg, Lois E. GreeneAbstract:Cyclin G-associated kinase (GAK), the ubiquitous form of the neuronal-specific protein Auxilin 1, is an essential cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Total internal reflectance microscopy was used to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits is inhibited by actin depolymerization, there is repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating.
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depletion of gak Auxilin 2 inhibits receptor mediated endocytosis and recruitment of both clathrin and clathrin adaptors
Journal of Cell Science, 2005Co-Authors: Dong Won Lee, Evan Eisenberg, Xiaohong Zhao, Fang Zhang, Lois E. GreeneAbstract:Cyclin G-associated kinase (GAK/Auxilin 2), the ubiquitous form of the neuronal-specific protein Auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.
Tsvika Greener - One of the best experts on this subject based on the ideXlab platform.
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structure of the functional fragment of Auxilin required for catalytic uncoating of clathrin coated vesicles
Biochemistry, 2004Co-Authors: James M Gruschus, Tsvika Greener, Lois E. Greene, Chae J Han, James A Ferretti, Evan EisenbergAbstract:The three-dimensional structure of the C-terminal 20 kDa portion of Auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa Auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to Auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the Auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of Auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as Auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.
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identification of domain required for catalytic activity of Auxilin in supporting clathrin uncoating by hsc70
Journal of Biological Chemistry, 2002Co-Authors: Tsvika Greener, Lois E. Greene, Michael E Pacold, S Kaushal, Evan EisenbergAbstract:During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein Auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for Auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of Auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike Auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the Auxilin clathrin-binding domain, it seemed possible that this subdomain is required for Auxilin to act catalytically, and indeed its deletion caused Auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened Auxilin-clathrin binding and prevented Auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of Auxilin is required for Auxilin to act catalytically, whereas the N-terminal subdomain strengthens Auxilin-clathrin binding.
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Caenorhabditis elegans Auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo
Nature Cell Biology, 2001Co-Authors: Tsvika Greener, Lois E. Greene, Barth Grant, Yinhua Zhang, David Hirsh, Evan EisenbergAbstract:The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro , the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as Auxilin. However, there is little evidence that either Hsc70 or Auxilin is essential in vivo . Here we show that C. elegans has a single Auxilin homologue that is identical to mammalian Auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit Auxilin expression in C. elegans , oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP–clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that Auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans .
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Expression of Auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin.
Journal of Cell Science, 2001Co-Authors: Xiaohong Zhao, Evan Eisenberg, Tsvika Greener, Hadi Al-hasani, Samuel W. Cushman, Lois E. GreeneAbstract:Although uncoating of clathrin-coated vesicles is a key event in clathrin-mediated endocytosis it is unclear what prevents uncoating of clathrin-coated pits before they pinch off to become clathrin-coated vesicles. We have shown that the J-domain proteins Auxilin and GAK are required for uncoating by Hsc70 in vitro. In the present study, we expressed Auxilin in cultured cells to determine if this would block endocytosis by causing premature uncoating of clathrin-coated pits. We found that expression of Auxilin indeed inhibited endocytosis. However, expression of Auxilin with its J-domain mutated so that it no longer interacted with Hsc70 also inhibited endocytosis as did expression of the clathrin-assembly protein, AP180, or its clathrin-binding domain. Accompanying this inhibition, we observed a marked decrease in clathrin associated with the plasma membrane and the trans-Golgi network, which provided us with an opportunity to determine whether the absence of clathrin from clathrin-coated pits affected the distribution of the clathrin assembly proteins AP1 and AP2. Surprisingly we found almost no change in the association of AP2 and AP1 with the plasma membrane and the trans-Golgi network, respectively. This was particularly obvious when Auxilin or GAK was expressed with functional J-domains since, in these cases, almost all of the clathrin was sequestered in granules that also contained Hsc70 and Auxilin or GAK. We conclude that expression of clathrin-binding proteins inhibits clathrin-mediated endocytosis by sequestering clathrin so that it is no longer available to bind to nascent pits but that assembly proteins bind to these pits independently of clathrin.
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Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells
Journal of Biological Chemistry, 2000Co-Authors: Tsvika Greener, Evan Eisenberg, Xiaohong Zhao, Hiroshi Nojima, Lois E. GreeneAbstract:Abstract Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an Auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to Auxilin; like Auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not Auxilin, are uncoated by Hsc70, suggesting that GAK acts as an Auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.
Ernst Ungewickell - One of the best experts on this subject based on the ideXlab platform.
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molecular and functional characterization of clathrin and ap 2 binding determinants within a disordered domain of Auxilin
Journal of Biological Chemistry, 2003Co-Authors: Urte Scheele, Christoph Kalthoff, Jurgen Alves, Ronald Frank, Michael Duwel, Ernst UngewickellAbstract:Uncoating of clathrin-coated vesicles requires the J-domain protein Auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of Auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547–813). CD spectroscopy of Auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of Auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and Auxilin compete for binding to the α-ear domain of AP-2. Like AP180, Auxilin also directly interacts with the ear domain of β-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.
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multiple interactions of Auxilin 1 with clathrin and the ap 2 adaptor complex
Journal of Biological Chemistry, 2001Co-Authors: Urte Scheele, Christoph Kalthoff, Ernst UngewickellAbstract:The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the Auxilin protein family to clathrin lattices. In vitro,this function of Auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of Auxilin (Auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the Auxilin 1 carboxyl-terminal segment 547–910. The overlapping Auxilin 1 fragments 547–714 and 619–738 bind the clathrin terminal domain with high affinity, whereas Auxilin 1-(715–901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the α-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that Auxilin 1-(715–901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of Auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.
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identification of the universal cofactor Auxilin 2 in clathrin coat dissociation
European Journal of Cell Biology, 2000Co-Authors: Akiko Umeda, Anika Meyerholz, Ernst UngewickellAbstract:Summary Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor Auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal Auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with Auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the γ- and α-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/Auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.
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primary structure of the neuronal clathrin associated protein Auxilin and its expression in bacteria
FEBS Journal, 1995Co-Authors: Stephan Schroder, Stephen A Morris, Ruth Knorr, Uwe Plessmann, Klaus Weber, Nguyen G Vinh, Ernst UngewickellAbstract:The protein Auxilin is a coat component of brain clathrin-coated vesicles. It interacts directly with the heavy chain of clathrin and supports its assembly into regular cages [Ahle, S. & Ungewickell, E. (1990) J. Cell Biol. 111, 19–29]. The combined open reading frames of three cow brain cDNA clones with a total of 4531 nucleotides predict a molecular mass of 99 504 Da for Auxilin. The coding region is followed by a very long untranslated region of at least 1670 nucleotides. By Northern analysis, Auxilin transcripts are found only in brain tissue. Auxilin is not related to any of the previously sequenced clathrin-binding proteins, but the region of positions 50–350 is 29% identical (similarity 56%) to the corresponding region of the actin-binding protein tensin from chicken fibroblasts. Recombinant Auxilin expressed in and purified from bacteria by affinity chromatography is functional with respect to clathrin binding.
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Light-chain-independent binding of adaptors, AP180, and Auxilin to clathrin.
Biochemistry, 1991Co-Authors: Robert Lindner, Ernst UngewickellAbstract:Binding of coated vesicle assembly proteins to clathrin causes it to assemble into regular coat structures. The assembly protein fraction of bovine brain coated vesicles comprises AP180, Auxilin, and HA1 and HA2 adaptors. Clathrin heavy chains, separated from their light chains, polymerize with unimpaired efficiency when assembly proteins are added. The reassembled coats were purified by sucrose gradient centrifugation and examined for composition by SDS-PAGE and immunoblotting. We found that all four major coat proteins are incorporated in the presence and absence of light chains. Moreover, each of the purified coat proteins is able to associate directly with clathrin heavy chains in preassembled cages as efficiently as with intact clathrin. We conclude that light chains are not essential for the interaction of AP180, Auxilin, and HA1 and HA2 with clathrin.
Tomas Kirchhausen - One of the best experts on this subject based on the ideXlab platform.
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structure of the pten like region of Auxilin a detector of clathrin coated vesicle budding
Structure, 2010Co-Authors: Rong J Guan, Dai Han, Stephen C Harrison, Tomas KirchhausenAbstract: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.
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structure of clathrin coat with bound hsc70 and Auxilin mechanism of hsc70 facilitated disassembly
The EMBO Journal, 2010Co-Authors: Yi Xing, Till Bocking, Stephen C Harrison, Tomas Kirchhausen, Nikolaus Grigorieff, Matthias WolfAbstract: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.
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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, 2006Co-Authors: Ramiro Massol, Werner Boll, April M Griffin, Tomas KirchhausenAbstract: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.
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structure of an Auxilin bound clathrin coat and its implications for the mechanism of uncoating
Nature, 2004Co-Authors: Alexander Fotin, Stephen C Harrison, Yifan Cheng, Nikolaus Grigorieff, Thomas Walz, Tomas KirchhausenAbstract:Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule Auxilin. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-A-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of Auxilin that contains both the clathrin-binding region and the J domain. We have located the Auxilin fragment by computing differences between these structures and those lacking Auxilin (described in an accompanying paper). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two 'ankle' segments; it also contacts the terminal domain of yet another clathrin 'leg'. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.