Upon activation by Wnt, the Frizzled receptor is internalized in a process that requires the recruitment of Dishevelled. We describe a novel interaction between Dishevelled2 (Dvl2) and micro2-adaptin, a subunit of the clathrin adaptor AP-2; this interaction is required to engage activated Frizzled4 with the endocytic machinery and for its internalization. The interaction of Dvl2 with AP-2 requires simultaneous association of the DEP domain and a peptide YHEL motif within Dvl2 with the C terminus of micro2. Dvl2 mutants in the YHEL motif fail to associate with micro2 and AP-2, and prevent Frizzled4 internalization. Corresponding Xenopus Dishevelled mutants show compromised ability to interfere with gastrulation mediated by the planar cell polarity (PCP) pathway. Conversely, a Dvl2 mutant in its DEP domain impaired in PCP signaling exhibits defective AP-2 interaction and prevents the internalization of Frizzled4. We suggest that the direct interaction of Dvl2 with AP-2 is important for Frizzled internalization and Frizzled/PCP signaling.

Resealing after wounding, the process of repair following plasma membrane damage, requires exocytosis. Vacuolins are molecules that induce rapid formation of large, swollen structures derived from endosomes and lysosomes by homotypic fusion combined with uncontrolled fusion of the inner and limiting membranes of these organelles. Vacuolin-1, the most potent compound, blocks the Ca 2 þ -dependent exocytosis of lysosomes induced by ionomycin or plasma membrane wounding, without affecting the process of resealing. In contrast, other cell structures and membrane trafficking functions including exocytosis of enlargeosomes are unaffected. Because cells heal normally in the presence of vacuolin-1, we suggest that lysosomes are dispensable for resealing.

Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a diseaseassociated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregateassociated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases.proteostasis | chaperone-dependent processes | protein misfolding | HSP70

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP-and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70 -facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone. INTRODUCTIONClathrin-coated vesicles, the best known carrier of intracellular membrane traffic, transport proteins and lipids from the plasma membrane to endosomes and between endosomes and the trans-Golgi network. Polymerization of the principal coat protein clathrin into a lattice-like assembly by sequential addition of individual, cytosolic clathrin trimers to a growing shell shapes the budding coated pit (Ehrlich et al., 2004; for review, see Kirchhausen, 2000). Intermediary proteins, known as adaptors, form the interface between the outer clathrin coat and the incorporated membrane bilayer. These adaptors selectively recruit membrane-anchored proteins ("cargo"). The most prominent adaptors are the heterotetrameric AP-2 and its cousins AP-1, AP-3, and AP-4, but there are other, presumably more specialized, adaptors, such as ␤-arrestins, epsin, GGAs, and dishevelled (Yu et al., 2007; for review, see Owen et al., 2004;Robinson, 2004).Coats can assemble in vitro without incorporated membrane, either from clathrin alone ("cages", stable only at reduced pH) or from clathrin plus heteroteterameric APs ("coats", stable at neutral pH) (Keen et al., 1979;Kirchhausen and Harrison, 1981; Vigers et al., 1986a,b;Kirchhausen, 2000;Fotin et al., 2004b). Coat assembly in vitro can proceed to completion without intervention of other factors. In vivo, coat assembly occurs only on membrane surfaces, and fission (pinching off) of the incorporating bilayer requires the large GTPase, dynamin (Sever, 2002;Praefcke and McMahon, 2004;Kruchten and McNiven, 2006;Macia et al., 2006). Removal of the coat (uncoating), essential for subsequent vesicle f...