Regulation of the AAA+ protein Sec18 through membrane lipid modification

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Eukaryotic cells are compartmentalized into organelles that communicate through the trafficking of membranous transport vesicles. Cargo transfer is finalized through the fusion of these compartments. Many proteins control fusion, yet the role of the membrane itself remains under-examined. Membranes contain specialized lipids that are essential for fusion and the PI's lab examines how these lipids affect protein function during fusion. Altering lipid content negatively affects fusion, however the mechanisms are unknown. The long-term goal of this project is to elucidate how lipid modification affects membrane fusion. The project will allow the investigator to continue his training of scientists at all levels, including female and other underrepresented students. This research will benefit society by spreading scientific discovery and education broadly to students from all walks of life. Eukaryotic cellular homeostasis is maintained in part through membrane fusion, which is catalyzed by SNARE proteins. Prior to fusion, inactive SNARE complexes are disassembled into individual proteins by Sec18/NSF. The PI's lab discovered that phosphatidic acid (PA) binds Sec18 to sequester it from SNAREs. The PA phosphatase Pah1/Lipin1 converts PA to diacylglycerol to release Sec18 and allows it to engage SNAREs. The objective of this research is to undertake a detailed analysis of the interactions between PA and Sec18. First, they will use biochemical, biophysical and computational approaches to define the PA binding sites on Sec18. Second, they will elucidate how PA-binding induces Sec18 conformational changes. Third, they will test conservation of Sec18 regulation by PA in other organelles. The completion of this project will define the how Sec18 is regulated by the membrane to activate SNAREs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.