Browsing by Subject "Adaptor Proteins, Vesicular Transport"
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Item Dissecting Roles for the Macromolecular Machinery Involved in Neurotransmitter Release(2019-01-15) Prinslow, Eric Andrew; Rosenbaum, Daniel M.; Rizo-Rey, José; Rice, Luke M.; Yu, HongtaoNeurotransmitter release is a tightly regulated process that involves synaptic vesicle docking at presynaptic active zones, priming of the vesicles to a release-ready state, and calcium evoked fusion of the vesicle and plasma membranes. The probability of release is modulated by plastic changes that depend on synaptic activity; these changes shape the properties of neural networks and underlie multiple forms of information processing in the brain. Elucidating the mechanisms of neurotransmitter release and its regulation is thus critical for understanding brain function and establishing fundamental principles of neuronal communication. I have investigated the mechanism by which neurotransmitters send messages between neurons as a specific model system to study the general mechanism of intracellular membrane fusion. In one project, I investigated whether trans-SNARE complexes can be disassembled by NSF-αSNAP. I showed that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, and is facilitated by synaptotagmin-1. I proposed a model whereby Munc18-1 and Munc13-1 are critical for mediating vesicle priming as well as precluding de-priming by preventing trans-SNARE complex disassembly. Complexin-1 also impaired de-priming, while synaptotagmin-1 may have assisted in priming and hindered de-priming. Additionally, I used various biophysical approaches including ITC and NMR to shed light into how Complexin has dual roles in fusion. One of my projects investigated the inhibitory role of Complexin and solved a controversy over conflicting ITC data. Another project focused on the Complexin N-terminal and C-terminal domains to try and develop a complete model of how Complexin functions that incorporates all of its known interactions and activating/inhibiting properties. I observed cooperative interactions between Complexin, the SNARE complex, and lipids by forming SNARE complexes anchored on nanodiscs and liposomes. Such cooperative binding of Complexin to membranes and SNAREs may be critical for releasing the inhibition caused by the accessory helix, although the molecular mechanism of action has yet to be determined. Overall, these experiments highlight the importance of interactions between numerous accessory proteins and the trans-SNARE for proper regulation of SNARE complex formation, and therefore fusion.Item PI(4)P-Dependent Recruitment of Clathrin Adaptors to the Trans-Golgi Network(2005-04-29) Wang, Jing; Yin, Helen L.The Trans Golgi Network (TGN) is the cell's central sorting station, and the complex trafficking patterns are organized by many types of trafficking adaptors. These include the heterotetrameric adaptor protein complexes (APs) and the monomeric Golgi-localized, gamma-ear containing, Arf-binding proteins (GGAs). The fundamental question of how these adaptors are recruited to TGN membrane remains unclear. Previous studies have shown that adaptor recruitment to the TGN is absolutely dependent on the small GTPase ADP ribosylation factor 1 (Arf1), but paradoxically, Arf1 has a broader intracellular distribution than these adaptors. We found that the Golgi is particularly enriched in phosphatidylinositol 4 phosphate [PI(4)P] and that the clathrin adaptor AP-1 binds PI(4)P directly, suggesting that PI(4)P binding may specify the TGN-specific recruitment in conjunction with Arf1. My studies showed that another monomeric clathrin adaptor GGA also binds PI(4)P and Arf1 independently. The C-terminal "triple helix bundle" of the GGA GAT domain is a polyfunctional module that interacts with multiple partners including PI(4)P and ubiquitin, and ubiquitin may provide a recognition signal for GGAs to control protein sorting. We found that PI(4)P increases wild type GAT binding to ubiquitin-conjugated agarose beads, but has no effect on a mutant GAT that does not bind PI(4)P. Therefore, PI(4)P may be an allosteric regulator of GGAs which enhances ubiquitin binding to GGAs. Based on these results, we conclude: (1) PI(4)P defines the TGN organelle identity by recruiting TGN-targeted adaptors; (2) TGN-enriched adaptors are recruited to the Golgi by binding to both PI(4)P and Arf1, and neither alone is sufficient; (3) PI(4)P acts as a scaffold, and may also be an allosteric regulator for GGAs that modulates GGA function with other ligands. We propose that the integration of combinatorial inputs from PI(4)P, Arf1 and ubiquitin may coordinately specify clathrin adaptor TGN recruitment through multiple low-affinity interactions.