Intracellular membrane docking and fusion are fundamental processes in cell biology. They are essential for the operation of the secretory and endocytic pathways and for neurotransmission, hormone secretion, lipid metabolism and immunity. Fusion events are usually catalyzed by SNARE proteins that, on native membranes, act together with an array of chaperones and regulatory proteins including small G proteins and multisubunit tethering complexes. The yeast vacuole is the most technically advanced system for understanding the SNARE-mediated fusion of intracellular organelles. It offers superb in vivo tools, an unsurpassed cell- free assay of fusion, and a fully reconstituted system that allows Rab-regulated fusion. In the previous funding cycle we extensively characterized HOPS, a 640 kDa tethering complex required for vacuole fusion, we delineated new mechanisms that control the activity of the vacuolar Rab protein Ypt7, we studied interactions between HOPS and a coat complex, AP-3, and we developed methods that for the first time allow the capture and study of unambiguous trans-SNARE holocomplexes. We now propose to combine these advances with innovative new technologies as well as classical approaches, to obtain an integrated view of the complex processes leading to pre-fusion complex assembly, and the mechanisms through which these complexes initiate and regulate fusion.
In Specific Aim 1 we use biochemical and genetic approaches to dissect a newly discovered mechanism of SNARE complex quality control that operates in vivo, and we explore the mechanism by which the universal chaperone Secl7 restores fusion activity to certain defective trans-SNARE complexes.
In Aim 2 we use newly developed optical assays of Rab and SNARE function to probe the dynamics of docking and fusion.
In Aim 3 we use trans-SNARE capture and a new AP-3 mutant to dissect the heterotypic delivery of Golgi-derived AP-3 vesicles to the lysosomal vacuole. Box 357350 1959 NE Pacific St Seattle, WA 93195 206.543.1660 fax 206.685.1792 bioc@u, http. :deptsyashIngtonedu biowww; Modified Specific Aims Our goal is to understand how the complex events of membrane tethering, docking and fusion are executed and regulated on native organelles. Membrane fusion is one of the most fundamental processes in cell biology. Fusion and the docking reactions preceding it are essential for the operation of the secretory and endocytic pathways, lipid metabolism, neurotransmission, nutrient homeostasis, and immunity. We build on biological and technical advances achieved during the previous funding cycle to further explore universal mechanisms of SNARE-mediated docking and to obtain a coherent understanding of the specific machinery that directs traffic into lysosomal organelles. Because the requested funding period for this Project was reduced from 5 years to 4, and because the requested budget over years 1-4 was cut by an average of 31% per year, we are reluctantly compelled to scale back the Specific Aims. We now omit the original Aim 1 (mass spectrometry of trans-SNARE complexes) due to its expense and technical complexity, and we eliminate sub-Aim 3C (electrical recordings from isolated organelles), again for reasons of technical complexity.
Both Aims were identified by the Study Section as high-risk and, relative to the other Aims, lacking in preliminary data and clear end-points. The Modified Aims are to: 1. Identify mechanisms of SNARE complex quality control that operate in living cells. We have obtained evidence that SNARE complex assembly is monitored by a quality control system in vivo. Biochemical and genetic strategies will be used to understand the mechanisms through which this quality control system operates. We have also discovered that, through an apparently separate mechanism, the universal SNARE chaperone Secl7 (a-SNAP) can rescue certain defective trans-SNARE complexes. Mutational analyses and biochemical assays will be used to clarify the underlying mechanism of this novel and unexpected activity. 2. Use optical methods to probe the dynamics of docking, SNARE-cofactor interaction, and fusion. We have developed new optical assays and reporters to probe docking and fusion. A noninvasive optical assay of Rab activity allows us to follow Rab activation status in real time during docking and fusion. We have prepared fluorescent SNAREs that will allow us to simultaneously capture trans complex assembly intermediates and probe their organization. 3. Discover the molecular requirements for AP-3 vesicle transport to the lysosomal vacuole. In Saccharomyces, direct traffic from the Golgi to the lysosomal vacuole requires the AP-3 cargo adaptor complex. Despite enormous efforts in several labs, only a few of the components specific to this pathway are known. In vivo SNARE capture, and a new AP-3 mutant that is stuck at the Golgi, will be used to identify additional components of the AP-3 pathway and to understand the mechanisms through which they operate.

Public Health Relevance

[No modifications] Membrane docking and fusion are among the most fundamental processes in cell biology, essential for embryonic development, lipid homeostasis, neuronal and endocrine function, and immunity. We have dissected the domains and interactions of a crucial lysosomal docking complex, HOPS, and we have developed methods to arrest the SNARE-zippering process that initiates organelle fusion, to capture and study bona fide pre-fusion intermediates. Together these advances will illuminate how diverse cargoes are targeted to lysosomes and how membrane docking and fusion occur with tremendous precision and reliability at the correct times and locations in the cell. Please let me know if this is sufficient information. Regards, Alexey Jarrell erz J2 Associate Professor of Biochemistry University of Washington or if you will need some additional Concurrence / Office ofSponsored Programs Acting for Lysme Chronig

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
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University of Washington
Schools of Medicine
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