Eukaryotic cells are compartmentalized by membrane-bound organelles, which communicate with each other through the transport of cargo vesicles that culminates in membrane fusion. We use vacuolar lysosomes isolated from the yeast Saccharomyces cerevisiae to examine membrane fusion. Vacuolar fusion requires a group of regulatory lipids that includes phosphoinositides, ergosterol, phosphatidic acid and diacylglycerol. Fusion occurs at membrane microdomains where regulatory lipids interdependently assemble with the proteins that catalyze fusion including SNAREs, the Rab GTPase Ypt7 the tethering complex HOPS, and actin. Because the lipid bilayer undergoes constant remodeling through lipid modification, we hypothesize that the metabolic interconversion of lipid species is integrally coupled to membrane fusion. In this proposal we examine the relationship between the lipid composition of vacuolar membranes and the function of SNAREs as well as the Rab7/Ypt7 nucleotide exchange factor Mon1. We plan to elucidate the role of lipid composition and the regulation of vacuole fusion by pursuing the following aims: 1 - Examine the function of SNARE trans-membrane domains during fusion and how these domains are controlled by the local lipid environment;2 - Determine the role of lipid composition on kinetics of affinities of SNARE complex formation;3 - Examine the relationship between the lipid phosphatidylinositol 3-phosphate and the recruitment and activation of the Rab7/Ypt7 nucleotide exchange factor Mon1. PUBLIC HEALTH RELAVANCE: Membrane fusion is essential for cell viability, neurotransmission, the secretion of antibodies and hormones, blood clotting, and the destruction of microbes. Dysregulation of lipid metabolism/modification is tied t many diseases including Charcot- Marie-Tooth disease, obesity, type-2 diabetes, and heart disease. In each case, membrane trafficking and fusion is deleteriously affected, yet the mechanisms that link lipid modification and the regulation of the fusion machinery remain unknown. The proposed study will define how the composition of the vacuolar membrane regulates SNARE and Mon1 function and will give us clues about the relationship between lipid modification and human health and disease.

Public Health Relevance

Dysfunctional membrane fusion can have severe consequences to human health, and defects are found in many genetic and infectious diseases that are too numerous to list here. However, suffice it to say that proper membrane fusion is essential for cell viability and cellular functions that include the release of neurotransmitters, hormones and antibodies. Therefore, understanding the fundamental mechanisms of membrane fusion is critical if we are to understand the diseases that target this machinery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM101132-03
Application #
8651501
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Chin, Jean
Project Start
2012-05-01
Project End
2017-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Champaign
State
IL
Country
United States
Zip Code
61820
Miner, Gregory E; Starr, Matthew L; Hurst, Logan R et al. (2017) Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity. Traffic 18:315-329
Starr, Matthew L; Hurst, Logan R; Fratti, Rutilio A (2016) Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming. Traffic 17:1091-109
Sparks, Robert P; Jenkins, Jermaine L; Miner, Gregory E et al. (2016) Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding. Biochem Biophys Res Commun 479:551-556
Miner, Gregory E; Starr, Matthew L; Hurst, Logan R et al. (2016) The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain. J Biol Chem 291:17651-63
Sparks, Robert P; Guida, Wayne C; Sowden, Mark P et al. (2016) Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells. Biochem Biophys Res Commun 478:546-52
Lawrence, Gus; Brown, Christopher C; Flood, Blake A et al. (2014) Dynamic association of the PI3P-interacting Mon1-Ccz1 GEF with vacuoles is controlled through its phosphorylation by the type 1 casein kinase Yck3. Mol Biol Cell 25:1608-19
Sasser, Terry L; Fratti, Rutilio A (2014) Class C ABC transporters and Saccharomyces cerevisiae vacuole fusion. Cell Logist 4:e943588
Sasser, Terry L; Lawrence, Gus; Karunakaran, Surya et al. (2013) The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion. J Biol Chem 288:18300-10
Karunakaran, Surya; Fratti, Rutilio A (2013) The lipid composition and physical properties of the yeast vacuole affect the hemifusion-fusion transition. Traffic 14:650-62
Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya et al. (2012) Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion. J Biol Chem 287:2221-36

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