Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy. T he sorting of receptors and other cell-surface proteins from the plasma membrane via clathrin-mediated endocytosis is the basis for a range of essential cellular processes, including the uptake of nutrient and signaling receptors, the control of cell and serum homeostasis through the internalization of plasma membrane pumps, and a contribution to learning and memory through the regulation of surface expression of neurotransmitter receptors (1). Until recently, it was thought that clathrin assembly into progressively curved lattices provided the driving force for the formation of clathrin-coated pits (CCPs) and vesicles (CCVs), and that the adaptor protein 2 (AP-2) complex was solely responsible for recruiting clathrin to the membrane and for binding to endocytic cargo, concentrating the cargo in CCPs (1, 2). However, clathrin assembly may not be sufficient to drive membrane curvature (3), and the previously accepted obligatory role for AP-2 in coat assembly and cargo recruitment has been recently questioned (4-6).In neuronal tissues, CCVs are postulated to be responsible for the recycling of synaptic vesicles (SVs) during neurotransmission (7). As such, CCVs retrieve SV membranes from the plasma membrane after SV collapse, concomitant with neurotransmitter release. Many of the components of the endocytic machinery are concentrated in the presynaptic compartment (8), and disruption of these proteins affects neurotransmission (9). Moreover, a number of SV proteins have been identified as components of isolated CCVs (10, 11). Synaptic transmission involving intermittent fusion of SVs without complete collapse (12, 13) has also been demonstrated. The prevalence of such a ''kiss-and-run'' mechanism with the alternative model of full fusion is uncertain (14). Even in the membrane retrieval model via CCVs, it remains unclear whether SVs are generated directly from CCVs (15, 16) or whether they require an additional sorting step through endosomal membranes localized in the presynaptic compartment (7, 17). Here, using ...

Summary The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn DENN domain functions as a guanine nucleotide exchange factor for Rab35 to regulate endosomal trafficking. Loss of Rab35 activity causes an enlargement of early endosomes, inhibits MHCI recycling, and prevents the early endosomal recruitment of EHD1, a common component recycling tubules on endosomes. Our data are the first to reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

SummaryCells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to downregulate an Arf6-dependent recycling pathway for b1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes of decreased cell adhesion and increased cell migration observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the ␣-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three ␣-earbinding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.

We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Vesicle budding and trafficking via clathrin-coated pits (CCPs) 1 and vesicles (CCVs) provides a major route by which proteins are transported out of the trans-Golgi network (TGN) and by which receptors, transporters, and nutrients are endocytosed at the plasma membrane (1-3). Many clathrin-dependent trafficking events mediate cargo transport that is needed in all cell types. These "housekeeping" forms of clathrin trafficking include the turnover of plasma membrane proteins and lipids, endocytic uptake of nutrients such as ironsaturated transferrin and low density lipoproteins, and endocytosis of a diverse range of activated growth factor receptors (1-3). Moreover all cells have housekeeping trafficking at the TGN. An important example is the delivery of mannose 6-phosphate-tagged lysosomal hydrolases from the TGN to endosomes/lysosomes via the mannose 6-phosphate receptor (MPR) (4).In addition to these housekeeping activities of CCVs, some tissues have specialized trafficking needs. For example, in secretory cells, clathrin coats are involved in the formation of secretory granules at the TGN (5), and polarized cells utilize CCVs for the trafficking of certain receptors from the TGN to the basolateral membrane necessary for the maintenance of polarity (2). At the plasma membrane, intestinal epithelial cells in rat or placental cells in humans use CCVs for the uptake of maternal immunoglobulins, a necessary aspect of maternal derived immunity (6). A striking example of specialized CCV function is seen in neurons, which communicate by releasing neurotransmitters through fusion of synaptic vesicles with the plasma membrane following transient increases in Ca 2ϩ concentration (7). These vesicles are then retrieved through CCVs (8 -10). Thus, neurons need CCVs not only for housekeeping forms of clathrin-mediated endocytosis but also to retrieve synaptic vesicle membranes. It has been unclear whether or not the mechanisms mediating these two related but distinct events taking place at the plasma membrane could be distinguished. Moreover the relative amount of brain CCVs specialized for synaptic function has never been assessed.The presence of clathr...

AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 a-ear, is the recruitment of accessory proteins. NECAPs are a-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 a-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the a-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrinmediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.

Endocytosis of nutrients, signaling receptors, and other cell surface regulatory proteins via clathrin-coated vesicles (CCVs) is essential for normal cellular function. CCVs are also involved in the transport of proteins, such as lysosomal hydrolases, from the trans-Golgi network to the endosomal/lysosomal system. CCVs are relatively uniform in size and, at 50 to 100 nm in diameter, they are among the smallest membranous organelles. Moreover, they are encased in a dense, proteinaceous coat, which contributes to the formation of the vesicular structure. Protocols for isolating CCVs take advantage of the small size and high density of these organelles by using differential centrifugation coupled with velocity and equilibrium gradients to separate CCVs from contaminating membranes.The first reliable protocol for isolating CCVs was described by Pearse (1975), working with pig brain. CCVs were enriched in a microsomal fraction prepared by differential centrifugation. They were subsequently separated from larger and less dense membranes using velocity and equilibrium sedimentation, respectively, on linear sucrose gradients. Pearse (1982) later introduced a protocol (for purification of CCVs from human placenta) in which Ficoll and D 2 O were substituted for sucrose. Most protocols currently in use are derived from these original protocols.In Basic Protocol 1, the authors describe a procedure for isolating CCVs from adult rat brain. This procedure, which is based on the protocol of Maycox et al. (1992), uses differential centrifugation coupled with Ficoll and D 2 O-sucrose density gradient centrifugation. The application of an additional step involving velocity sedimentation in linear sucrose gradients, as originally described by Wasiak et al. (2002), is also outlined. In Alternate Protocols 1 and 2, the authors describe how the same basic approach can be applied to the isolation of CCVs from developing rat brain and cell lines, respectively.When applied to other tissues, such as rat liver, the steps outlined in Basic Protocol 1 yield CCV preparations that exhibit only modest enrichment and purity. In Basic Protocol 2, therefore, the authors describe a fractionation procedure that has been used to purify CCVs from rat liver. This protocol, based on that of Pilch et al. (1983), involves differential centrifugation coupled with velocity and equilibrium centrifugation using discontinuous sucrose gradients.Following fractionation, it is necessary to characterize CCV enrichment and purity. One important method for doing so is the analysis of equal protein aliquots of the various subcellular fractions by SDS-PAGE (UNIT 6.1) using Coomassie blue staining and/or immunoblotting (UNIT 6.2) with antibodies against specific CCV proteins (e.g., clathrin). Thus, for each protocol, we indicate fractions from which aliquots should be retained for analysis. The purity of CCVs is best assessed by electron microscopy (EM). In the Support Protocol, we describe an EM procedure, based on that of Baudhuin et al. (1967), that involves filtrati...

Highlights d Casein kinase 1 d (CK1d) stabilizes nervous system architecture after axon outgrowth d CK1d phosphorylates and inhibits SSUP-72, an RNA polymerase II CTD phosphatase d CK1d inhibits transcription termination to promote giant Ankyrin expression d Expression of giant Ankyrin in CK1d mutants rescues axon maturation defects