Browsing by Subject "Multiprotein Complexes"
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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 Insights into the Metabolic Regulation by GATOR1 in Response to Amino Acid Signaling(2017-07-27) Chen, Jun; Liu, Yi; Tu, Benjamin; Phillips, Margaret A.; Goodman, Joel M.The GATOR1/SEACIT complex consisting of Iml1-Npr2-Npr3 inhibits Target of Rapamycin Complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, yeast mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite hallmarks of increased TORC1 signaling. Such mutants perceive they are amino acid-replete and thus repress metabolic activities that are important for achieving this state. I find that npr2∆ mutants have defective mitochondrial TCA cycle activity and retrograde response. Supplementation of glutamine, and especially aspartate, which are nitrogen-containing forms of TCA cycle intermediates, rescue growth of npr2∆ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings reveal that negative regulators of TORC1 such as GATOR1/SEACIT regulate the cataplerotic synthesis of these amino acids from the TCA cycle in tune with the amino acid and nitrogen status of cells.Item Recruitment of Enzyme Cascade to Phase-Separated Biomolecular Condensates Accelerates Reactions via Concentration-Dependent and Concentration-Independent Mechanisms(August 2021) Peeples, William Benjamin; De Martino, George; Phillips, Margaret A.; Kohler, Jennifer J.; Rosen, Michael K.Biomolecular condensates are ubiquitous throughout biology, but their functions remain largely poorly understood. Biomolecular condensates concentrate biomolecules relative to the surrounding medium. For biomolecular condensates that concentrate enzymes and their substrates, classic enzyme kinetics predicts an acceleration of the reaction rate within the condensates, but the effect of condensates on enzymatic activity both within and outside condensates has not been widely investigated. In order to understand these effects in more detail, we developed an in vitro model system consisting of multivalent protein scaffolds and a minimal enzyme system-the SUMOylation cascade. By inducibly recruiting various combinations of components of the SUMOylation cascade to condensates, we are able to uncouple the contributions of individual components and phases to enzymatic activity. We find that the reaction is accelerated when all SUMOylation components are recruited to condensates, and this acceleration requires recruitment of both enzyme and substrate. This is despite condensates representing only 1 % of total solution volume. This enhancement is limited to substrates whose KM is well above total substrate concentration. This selective enhancement is further demonstrated with simple modeling to show that substrate concentration relative to KM is a key factor in understanding the degree to which different substrates are likely to be influenced through condensate recruitment. Recruitment accelerates not only the reaction within the condensate but also the reaction outside the condensates. To understand what fraction of this increased activity within condensates is attributable to increased concentration of enzyme and substrate, we measured activity at identical concentrations of enzyme and substrate but lacking the scaffolds. We find that condensate activity exceeds the concentration-matched reaction, suggesting there is concentration-independent activity enhancement. Further investigation found that this excess enhancement is likely due to a scaffold-induced reduction in apparent KM. These results suggest that condensates can accelerate enzymatic activity through multiple mechanisms, including concentration and molecular organization of enzyme and substrate. Condensates selectively accelerate substrates whose total concentration is low relative to KM. Together these effects demonstrate the capacity of condensates to impart activity enhancement, specificity, and potentially sequestration through regulated enzyme and substrate recruitment.Item [Southwestern News](1999-05-25) Steeves, Susan A.