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Linton M. Traub - One of the best experts on this subject based on the ideXlab platform.
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endocytic adaptor molecules reveal an endosomal population of Clathrin by total internal reflection fluorescence microscopy
Journal of Biological Chemistry, 2004Co-Authors: Peter A Keyel, Simon C Watkins, Linton M. TraubAbstract:Abstract Most eukaryotes utilize a single pool of Clathrin to assemble Clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble Clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow Clathrin dynamics close to the cell surface, we find that the majority of labeled Clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the α subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 β2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile Clathrin puncta may be distinct from plasma membrane-associated Clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than Clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, Clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile Clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of Clathrin structures.
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two distinct interaction motifs in amphiphysin bind two independent sites on the Clathrin terminal domain beta propeller
Nature Structural & Molecular Biology, 2004Co-Authors: Linton M. Traub, Adriana E Miele, Peter J Watson, Philip R Evans, David J OwenAbstract:During the assembly of Clathrin-coated vesicles, many peripheral membrane proteins, including the amphiphysins, use LLDLD-type Clathrin-box motifs to interact with the N-terminal beta-propeller domain (TD) of Clathrin. The 2.3 A-resolution structure of the Clathrin TD in complex with a TLPWDLWTT peptide from amphiphysin 1 delineates a second Clathrin-binding motif, PWXXW (the W box), that binds at a site on the TD remote from the Clathrin box-binding site. The presence of both sequence motifs within the unstructured region of the amphiphysins allows them to bind more tightly to free TDs than do other endocytic proteins that contain only Clathrin-box motifs. This property, along with the propensity of the N-terminal BAR domain to bind curved membranes, will preferentially localize amphiphysin and its partner, dynamin, to the periphery of invaginated Clathrin lattices.
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sorting it out ap 2 and alternate Clathrin adaptors in endocytic cargo selection
Journal of Cell Biology, 2003Co-Authors: Linton M. TraubAbstract:The AP-2 adaptor complex is widely viewed as a linchpin molecule in Clathrin-mediated endocytosis, simultaneously binding both Clathrin and receptors. This dual interaction couples cargo capture with Clathrin coat assembly, but it has now been discovered that the association with cargo is tightly regulated. Remarkably, AP-2 is not obligatory for all Clathrin-mediated uptake, and several alternate adaptors appear to perform similar sorting and assembly functions at the Clathrin bud site.
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Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins.
The Journal of biological chemistry, 2001Co-Authors: Sanjay K. Mishra, Nicole R. Agostinelli, Tom J. Brett, Ikuko F. Mizukami, Theodora S. Ross, Linton M. TraubAbstract:Abstract Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, Clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain Clathrin-coated vesicles and associates directly with both AP-2 and Clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminalhomology (ENTH) domain, HIP1 associates with AP-2 to provide coincident Clathrin-binding sites that together efficiently recruit Clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated Clathrin lattice biogenesis.
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epsin binds to Clathrin by associating directly with the Clathrin terminal domain evidence for cooperative binding through two discrete sites
Journal of Biological Chemistry, 2000Co-Authors: Matthew T Drake, Maureen A Downs, Linton M. TraubAbstract:Abstract Epsin is a recently identified protein that appears to play an important role in Clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the α subunit. We have found that this central portion of epsin 1 also associates with Clathrin. The interaction with Clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that Clathrin binding depends on the sequence 257LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known Clathrin-binding sequences in the adaptor β subunits, amphiphysin, and β-arrestin, facilitates the association of epsin 1 with the terminal domain of the Clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of Clathrin in parallel with AP-2. Because the β subunit of the AP-2 complex also contains a Clathrin-binding site, optimal association with soluble Clathrin appears to depend on the presence of at least two distinct Clathrin-binding sites, and we show that a second Clathrin-binding sequence 480LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with Clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in Clathrin recruitment assays, suggesting that they bind to different sites on the Clathrin-terminal domain. The evolutionary conservation of similar Clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing Clathrin-coated bud is an important aspect of epsin function.
Lois E. Greene - One of the best experts on this subject based on the ideXlab platform.
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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.
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adaptor and Clathrin exchange at the plasma membrane and trans golgi network
Molecular Biology of the Cell, 2003Co-Authors: Xiaohong Zhao, Juan S Bonifacino, Evan Eisenberg, Rosa Puertollano, Lois E. GreeneAbstract:We previously demonstrated, using fluorescence recovery after photobleaching, that Clathrin in Clathrin-coated pits at the plasma membrane exchanges with free Clathrin in the cytosol, suggesting that Clathrin-coated pits are dynamic structures. We now investigated whether Clathrin at the trans-Golgi network as well as the Clathrin adaptors AP2 and AP1 in Clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of Clathrin-coated vesicle is blocked without significantly affecting the structure of Clathrin-coated pits, both Clathrin and AP2 at the plasma membrane and Clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of Clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of Clathrin-coated pits, Clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that Clathrin-coated pits are dynamic structures with rapid exchange of both Clathrin and adaptors and that adaptors are able to exchange independently of Clathrin when Clathrin exchange is blocked.
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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.
Peter S Mcpherson - One of the best experts on this subject based on the ideXlab platform.
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huntingtin interacting protein 1 hip1 regulates Clathrin assembly through direct binding to the regulatory region of the Clathrin light chain
Journal of Biological Chemistry, 2005Co-Authors: Valerie Legendreguillemin, Martina Metzler, Lu Gan, Jacynthe Philie, Michael R Hayden, Jeanfrancois Lemaire, Peter S McphersonAbstract:Huntingtin interacting protein 1 (HIP1) is a component of Clathrin coats. We previously demonstrated that HIP1 promotes Clathrin assembly through its central helical domain, which binds directly to Clathrin light chains (CLCs). To better understand the relationship between CLC binding and Clathrin assembly we sought to dissect this interaction. Using C-terminal deletion constructs of the HIP1 helical domain, we identified a region between residues 450 and 456 that is required for CLC binding. Within this region, point mutations showed the importance of residues Leu-451, Leu-452, and Arg-453. Mutants that fail to bind CLC are unable to promote Clathrin assembly in vitro but still mediate HIP1 homodimerization and heterodimerization with the family member HIP12/HIP1R. Moreover, HIP1 binding to CLC is necessary for HIP1 targeting to Clathrin-coated pits and Clathrin-coated vesicles. Interestingly, HIP1 binds to a highly conserved region of CLC previously demonstrated to regulate Clathrin assembly. These results suggest a role for HIP1/CLC interactions in the regulation of Clathrin assembly.
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hip1 and hip12 display differential binding to f actin ap2 and Clathrin identification of a novel interaction with Clathrin light chain
Journal of Biological Chemistry, 2002Co-Authors: Valerie Legendreguillemin, Martina Metzler, Martine Charbonneau, Lu Gan, Vikramjit Chopra, Jacynthe Philie, Michael R Hayden, Peter S McphersonAbstract:Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the Clathrin coat that interact but differ in their ability to bind Clathrin and the Clathrin adaptor AP2. HIP1 contains a Clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the Clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity Clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate Clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the Clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in Clathrin-mediated endocytosis.
Frances M Brodsky - One of the best experts on this subject based on the ideXlab platform.
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Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Lisa Redlingshofer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M BrodskyAbstract:Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterized the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more effective in membrane deformation than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin activity in neurons, which requires their functional balance.
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Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo
bioRxiv, 2020Co-Authors: Lisa Redlingshofer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M BrodskyAbstract:Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterised the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more effective in membrane deformation than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin activity in neurons, which requires their functional balance. SIGNIFICANCE STATEMENT This study reveals that diversity of Clathrin light chain (CLC) subunits alters Clathrin properties and demonstrates that the two neuronal CLC subunits work together for optimal Clathrin function in synaptic vesicle formation. Our findings establish a role for CLC diversity in synaptic transmission and illustrate how CLC variability expands the complexity of Clathrin to serve tissue-specific functions. ### Competing Interest Statement The authors have declared no competing interest.
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Clathrin light chain diversity regulates budding efficiency in vitro and synaptic vesicle formation in vivo
bioRxiv, 2020Co-Authors: Lisa Redlingshoefer, Faye Mcleod, Yu Chen, Marine D Camus, Jemima J Burden, Ernest Palomer, Kit Briant, Philip N Dannhauser, Patricia C Salinas, Frances M BrodskyAbstract:Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal Clathrin function, we characterised the biophysical properties of Clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced Clathrin knee conformation within assemblies, and Clathrin with neuronal CLC mixtures was more efficient in membrane budding than Clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to wild-type mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight functional differences between the CLC isoforms and show that isoform mixing influences tissue-specific Clathrin function in neurons.
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hsc70 induced changes in Clathrin auxilin cage structure suggest a role for Clathrin light chains in cage disassembly
Traffic, 2013Co-Authors: Anna Young, Frances M Brodsky, Helen M Kent, Barbara M F Pearse, Alice Rothnie, Svetla Stoilovamcphie, Yvonne Vallis, Phillip Harveysmith, Neil A Ranson, Alan M RosemanAbstract:The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of Clathrin during Clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of Clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to Clathrin and auxilin before Clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the Clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and Clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of Clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of Clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for Clathrin light chains in Clathrin disassembly in addition to their established role in regulating Clathrin assembly.
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diversity of Clathrin function new tricks for an old protein
Annual Review of Cell and Developmental Biology, 2012Co-Authors: Frances M BrodskyAbstract:Clathrin is considered the prototype vesicle coat protein whose self-assembly mediates sorting of membrane cargo and recruitment of lipid modifiers. Detailed knowledge of Clathrin biochemistry, structure, and interacting proteins has accumulated since the first observation, almost 50 years ago, of its role in receptor-mediated endocytosis of yolk protein. This review summarizes that knowledge, and focuses on properties of the Clathrin heavy and light chain subunits and interaction of the latter with Hip proteins, to address the diversity of Clathrin function beyond conventional receptor-mediated endocytosis. The distinct functions of the two human Clathrin isoforms (CHC17 and CHC22) are discussed, highlighting CHC22's specialized involvement in traffic of the GLUT4 glucose transporter and consequent role in human glucose metabolism. Analysis of Clathrin light chain function and interaction with the actin-binding Hip proteins during bacterial infection defines a novel actin-organizing function for CHC17 Clathrin. By considering these diverse Clathrin functions, along with intracellular sorting roles and influences on mitosis, further relevance of Clathrin function to human health and disease is established.
Evan Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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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.
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adaptor and Clathrin exchange at the plasma membrane and trans golgi network
Molecular Biology of the Cell, 2003Co-Authors: Xiaohong Zhao, Juan S Bonifacino, Evan Eisenberg, Rosa Puertollano, Lois E. GreeneAbstract:We previously demonstrated, using fluorescence recovery after photobleaching, that Clathrin in Clathrin-coated pits at the plasma membrane exchanges with free Clathrin in the cytosol, suggesting that Clathrin-coated pits are dynamic structures. We now investigated whether Clathrin at the trans-Golgi network as well as the Clathrin adaptors AP2 and AP1 in Clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of Clathrin-coated vesicle is blocked without significantly affecting the structure of Clathrin-coated pits, both Clathrin and AP2 at the plasma membrane and Clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of Clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of Clathrin-coated pits, Clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that Clathrin-coated pits are dynamic structures with rapid exchange of both Clathrin and adaptors and that adaptors are able to exchange independently of Clathrin when Clathrin exchange is blocked.
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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.