Browsing by Subject "Vesicular Transport Proteins"
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Item Characterization of Class D VPS Proteins(2003-03-11) Friedberg, Andrew Seth; Sternweis, Paul C.The vacuole of the yeast Saccharomyces cerevisiae is functionally similar to the mammalian lysosome. The components of the VPS (vacuolar protein sorting) system are responsible for proper delivery of vacuolar biosythetic enzymes. Efforts to dissect the genetics of this system have revealed several classes of mutants, each defective in one transport step in the VPS pathway. The Class D VPS proteins are thought to control anterograde traffic between the late Golgi and late endosome. Although most of these proteins have homologues of known function in other systems, two exceptions are the Vps3p and Vps8p proteins. Analysis of Vps3p reveals that it is associated with a highdensity structure, possibly a coated vesicle or a large protein complex. The Vps8p protein contains a C-terminal H2 RING finger motif, a domain often associated with E3 ubiquitin ligase activity. In vitro analysis reveals that a Vps8p fragment containing this domain has this activity. Deletion of the RING finger reveals that the endocytic marker Ste3p accumulates in an abnormally large late-endosome-derived structure, but that sorting of the soluble vacuolar cargo CPY is relatively unaffected. These results suggest a division of function within the Vps8p molecule.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.Item The Sec6/8 (a.k.a. Exocyst) Complex Supports DNA Repair Fidelity(2014-04-14) Torres, Michael Jason 1982-; Brekken, Rolf A.; Cobb, Melanie H.; Burma, SandeepThe exocyst complex, first described in yeast, is a heterooctomeric complex that serves as a signaling platform to mediate cellular responses to diverse spatial and temporal cues. Evidence suggests that the exocyst might contribute to oncogenesis, potentially by disrupting spatial and temporal regulation of pathways critical to determining cell survival vs. apoptosis. Our work investigated how cancer cells subvert the exocyst to upregulate the AKT (v-akt murine thymoma viral oncogene) pro-survival pathway through the innate immune protein TBK1 (TANK-binding kinase 1). siRNA-mediated depletion of TBK1 in pancreatic and breast cancer cell lines results in apoptosis, which is mediated through the AKT pathway. Pharmacological inhibition of TBK1 recapitulates the apoptotic phenotype in mouse orthotopic models. Additionally, my work uncovered exocyst participation in the regulation of DNA repair. The isolation of multiple components of the DNA damage response (DDR) within the human exocyst protein-protein interaction network, together with the identification of Sec8 as a suppressor of the p53 response, prompted an investigation of functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in a radioresistance phenotype to ionizing radiation (IR) that was associated with accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Exocyst-dependent modulation of the DDR is, at least in part, through restraint of the associated chromatin modifiers ATF2 and RNF20. Exocyst perturbation resulted in aberrant accumulation of ATF2 and RNF20; the promiscuous accumulation of DDR-associated chromatin marks; and IR-induced increased Rad51 repairosomes. Thus, the exocyst indirectly supports DNA repair fidelity by limiting formation of repair chromatin in the absence of a DNA damage signal. This newly revealed regulation of DNA repair by the exocyst may provide additional insight into the emerging observations of DNA damage protein involvement in pathways not canonically associated DNA repair, such as the host cytokinesis, host defense response, and maintenance of cilia. This work further substantiates the importance of the exocyst in normal cell biology and gives insight into how disruption of exocyst function can result in disease.Item Structural Studies of Complexin/SNARE Interactions(2008-09-18) Lee, Daeho; Rizo-Rey, JoséVesicular neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. The Ca2+-triggered release process is extremely fast, lasts less than half a millisecond, and is tightly regulated by Ca2+. Action potentials cause Ca2+ influx through voltage-gated Ca2+ channels, which in turn triggers synaptic vesicle fusion. The typical sub-millisecond latency between an action potential and neurotransmitter release and the precise timing of the release process are crucial for information processing in the nervous system. To achieve this exquisite regulation, many proteins are involved. The goal of our investigations was to delineate interactions between complexin and SNARE components that lead to the formation of a primed minimal fusion machinery. We have generated and used new constructs of short forms of synaptobrevin and complexin, as well as constructs of SNAP-25 and syntaxin that have previously been shown to be part of the minimal fusion machinery. With NMR spectroscopy, the use of the short synaptobrevin constructs has led to experimental results suggesting at least two key intermediates during the docking/priming process that are independent of complexin. We found evidence for a modular assembly of the full SNARE complex. In the absence of Syb2-CT, the N-terminal half of SNARE complex forms a four-helix bundle, while the C-terminal half, starting just after the polar layer, is disordered. In the presence of the Syb2-CT, however, both halves of the SNARE complex form a four-helix bundle. It is interesting to note that Syb2 residues 29-84 are sufficient for formation of the fully assembled SNARE complex. This evidence strongly suggests the existence of at least two intermediates during the docking priming reaction. Furthermore, with NMR spectroscopy, we have found new evidence that complexin can bind to the t-SNARE complex, in contrast to earlier evidence suggesting that complexin regulates the fully assembled SNARE complex. We demonstrated that Cpx-FL binds the t-SNARE complex SN1/SN3/Syx1a(188-259) in solution, as wa suggested for membrane-bound t-SNAREs. Note, however, that the t-SNARE complex does not contain the large complexin-binding interface provided by Syb2. Furthermore, we found that Cpx-FL also binds t-SNARE sub-complexes formed by SN1/SN3, and SN1/Syx1a(188-259), while very little binding was observed between Cpx-FL and Syx1a(188-259) alone. This finding is particularly interesting, because the cryst structure of the fully assembled SNARE complex does not suggest any binding between Cpx26-83 and either SN1 or SN3, whereas the only common component in all of the above experiments was SN1 domain.