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.