ATP- and Cytosol-dependent Release of Adaptor Proteins from Clathrin-coated Vesicles: A Dual Role for Hsc70
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Abstract:
Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP- and cytosol- dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release.Keywords:
Clathrin adaptor proteins
Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
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Clathrin-mediated endocytosis (CME) plays a central role in fundamental processes such as synaptic vesicle recycling, receptor recycling, signalling and development. CME begins with clathrin assembly on the plasma membrane, facilitated by adaptor proteins. This process forms an endocytic vesicle that allows transport of cargo into the cell, and is followed by clathrin disassembly through the action of different adaptor/accessory proteins. A large number of different adaptor and accessory proteins are recruited during CME, in a spatially and temporally ordered manner. Although our understanding is growing as to the roles of individual adaptor proteins, we still do not understand the way in which some adaptors interact with clathrin or the molecular details of their interactions with one another in the presence of clathrin. Clathrin adaptor proteins contain short, linear clathrin-binding motifs, which form the basis of their interaction with the four distinct sites on the clathrin N-terminal domain (TD). An adaptor protein with tighter binding or more numerous clathrin binding sequences could displace one with weaker or fewer binding elements. This raises the question of whether adaptor proteins compete for binding to clathrin or whether they can bind simultaneously.
Using certain biochemical and biophysical techniques in vitro and purified WT and mutant adaptor proteins, I have shown the complex ‘multiple TD linking effect’ of epsin 1 via the cooperative action of its two clathrin box motifs and unstructured region. Using the newly developed SPR/IAC (2-injection) method, I explored competition between five purified structurally and functionally diverse adaptor proteins when simultaneously binding to clathrin TD. I have shown how the complex structure of epsin 1 causes competition with β-arrestin 1 for clathrin TD binding. Such competition is observed between espin 1 and auxilin 1 as well, which reveals information about the mechanism of disassembly. However, β2-adaptin and auxilin 1 demonstrate no such competition.
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ABSTRACT During the last decade the term ‘endocytosis’ has become virtually synonymous with the activity of clathrin-coated vesicles. These vesicles, which are derived from cell surface clathrin-coated pits, are transport vehicles responsible for the transfer of plasma membrane receptors and their ligands, between the first two stations of the endocytic pathway: namely, the plasma membrane and early endosomes (Goldstein et al., 1985; van Deurs et al., 1989; Griffiths and Gruenberg, 1991). Despite the irrefutable evidence that clathrin-coated vesicles mediate endocytosis, their contribution to the total endocytic activity of the cell and the composition of the membrane they internalise remains controversial. Here we discuss: (1) the evidence that non-clathrin-mediated endocytic mechanisms operate alongside the clathrin-mediated pathway; (2) the evidence that endocytosis occurs for surface molecules that are not enriched in clathrin-coated pits and; (3) the sorting activities of cell surface clathrincoated pits and the notion that plasma membrane proteins that show particularly slow rates of uptake are actively excluded from the endocytic pathway.
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Abstract Clathrin coats drive transport vesicle formation from the plasma membrane and in pathways between the trans-Golgi network (TGN) and endosomes. Clathrin adaptors play central roles orchestrating assembly of clathrin coats. The yeast clathrin adaptor-interacting protein Irc6 is an orthologue of human p34, which is mutated in the inherited skin disorder punctate palmoplantar keratoderma type I. Irc6 and p34 bind to clathrin adaptor complexes AP-1 and AP-2 and are members of a conserved family characterized by a two-domain architecture. Irc6 is required for AP-1-dependent transport between the TGN and endosomes in yeast. Here we present evidence that the C-terminal two amino acids of Irc6 are required for AP-1 binding and transport function. Additionally, like the C-terminal domain, the N-terminal domain when overexpressed partially restores AP-1-mediated transport in cells lacking full-length Irc6. These findings support a functional role for Irc6 binding to AP-1. Negative genetic interactions with irc6∆ are enriched for genes related to membrane traffic and nuclear processes, consistent with diverse cellular roles for Irc6.
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Vesicular Transport Proteins
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Recent evidence has proved that in addition to the well-documented clathrin-mediated endocytic route (vesicles of 100-150 nm), at least three distinct non-clathrin-coated endocytic pathways function at the surface of mammalian cells. Endocytosis via these pathways is initiated by caveolae (50-80 nm), macropinosomes (500-2000 nm) and micropinosomes (95-100 nm). The current state of knowledge about these non-clathrin coated endocytic routes is presented and evidence that endocytic routes other than via clathrin-coated vesicles are utilised by viruses is discussed. The recent advances in these areas have provided us with tools to investigate the entry of those viruses which appear to enter cells via endocytosis into non-clathrin-coated vesicles. Data indicate that these four endocytic pathways differ in the absence, presence and/or type of coat on the vesicles, the size of the vesicles, their sensitivity to a variety of inhibitors, and in the ligands endocytosed. A historical perspective of the discovery of these non-clathrin-coated endocytic pathways is provided and recent information is summarised and discussed. The entry of viruses via non-clathrin-coated pits is destined to be an exciting new area of viral-cell entry, as has been indicated recently by the finding that entry of simian virus type 40 into cells occurs via caveolae. Copyright 1997 by John Wiley & Sons, Ltd.
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CDK5 inhibits constitutive internalization of the nociceptor TRPV1 and contributes to inflammatory hyperalgesia.
Internalization
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Cyclin-dependent kinase 5
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Internalization
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Lipid microdomain
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Linker
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