Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth.

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis.

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.

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Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions.

The synaptotagmin family of membrane proteins has been implicated in both exocytosis and endocytosis. Synaptotagmin I, a protein containing two tandem C2 domains (the C2A and the C2B) in its cytoplasmic tail, is involved in regulated exocytosis of synaptic vesicles as well as compensatory endocytosis. A related family member, synaptotagmin VII, is involved in multiple forms of regulated exocytosis of lysosomes and secretory granules. In this study we show that the cytoplasmic C2 domains in synaptotagmin VII contain unique internalization signals and regulators of these signals. The C-terminal portion of the C2B is internalized in much the same way as the corresponding region of synaptotagmin I. This signal is tryptophan-based and dynamin and eps15 dependent. In contrast, the C2A contains an unusual internalization signal that is not seen in the C2A of synaptotagmin I. This signal is not based on the homologous tryptophan in its C-terminus. Moreover, internalization of the C2A domain is both dynamin and eps15 independent. Finally, the C2B domain of synaptotagmin VII contains an inhibitory motif that prevents internalization. Endocytic trafficking of synaptotagmin VII is thus governed by these two latent internalization signals, which are concealed by intramolecular inhibition. We propose that endocytosis of synaptotagmin VII is regulated in this way to allow it to couple the processes of regulated exocytosis and compensatory endocytosis.

Endocytosis of synaptotagmin 1 is mediated by a novel, tryptophan-containing motif.

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.