Rutilio Fratti

Protein-Lipid interactions; Membrane Fusion; Lipid Metabolism

Research in the Fratti lab focuses on how the chemical and physical properties of membrane bilayers control the function of proteins. The connection between lipids and protein function are important throughout biology and dysregulation is linked to a wide array of diseases including cancer, diabetes, and Alzheimer’s disease. Therefore, it is important that we understand the fundamentals of protein-lipid interactions so that we can better address their disruption in diseases.

We use lysosomes/vacuoles from the yeast Saccharomyces cerevisiae to reveal how specific lipids inhibit or promote different mechanisms in the process of membrane fusion catalyzed by SNARE proteins. Fusion events are often carried out in highly organized membrane platforms, collectively known as microdomains that are enriched in regulatory lipids that partition from bulk lipids (e.g., phosphatidylcholine) that make up a vesicle. Although low in concentration, regulatory lipids are critical in cellular signaling and the formation of membrane microdomains. The lipids that drive microdomain assembly include phosphatidic acid (PA), diacylglycerol (DAG), phosphoinositides (PI), cholesterol, and sphingolipids. The stoichiometry of these lipids is constantly changing through the action of lipid phosphatases, kinases, and lipases. Lipid modification and remodeling of membranes dramatically changes how lipids interact with proteins and can affect local physical properties such as curvature, fluidity, and bilayer stability. Thus, protein function can be regulated by changes by the lipids in a membrane. Using yeast vacuoles as well as synthetic vesicles we will determine how lipid modification regulates proteins on vesicles as they move through the different stages of the fusion pathway and how dysregulation of these events affects specific genetic and infectious diseases.

Regulatory Lipids and Protein Function

Eukaryotic cells are compartmentalized by membrane-bound organelles that communicate through the trafficking of transport vesicles. The transfer of vesicular cargo between organelles is finalized by the fusing of two membrane bilayers into a continuous membrane. Membrane fusion is catalyzed by proteins called SNAREs that are present on both donor and acceptor membranes. Although seemingly straight forward, membrane fusion is highly orchestrated and tightly controlled to prevent unchecked fusion when unwanted, or missorting cargo to the wrong organelle. Thus, it is essential to regulate membrane trafficking and fusion to maintain homeostasis. Many proteins control these pathways, yet the role of the membrane itself remains underappreciated.

Membranes contain a small, yet critical group of “Regulatory Lipids” that aid in signal transduction, recruit proteins to their site of action, or alter membrane curvature and fluidity to modulate protein function. In the context of membrane fusion, regulatory lipids are essential for the fusion of synaptic vesicles to the plasma membrane, autophagosomes-to-lysosome fusion, phagosome-endosome fusion, et cetera. Regulatory lipids are essential throughout the different stages of membrane fusion and their effects can be altered though modification. Phosphoinositides, for example, can be differentially phosphorylated and dephosphorylated by specific kinase and phosphatases, or hydrolyzed by various lipases.

My lab focuses on how lipid modification affects protein function in the fusion pathway. It is clear that dysregulation of lipid modification has severe deleterious effects on fusion and overall cellular upkeep. In many cases pathologies have been mapped to lipid modifying enzymes, however, their effects on membrane function and cellular homeostasis is often unknown. Our long term goal is to bridge the gap between mapping diseases to genes and understanding the underlying mechanisms of the corresponding pathologies.

Current projects include:

I. Phosphatidic acid and Diacylglycerol Interconversion • Regulation of SNARE activation • Endolysosomal maturation • Autophagy

II. PI3P binding by Vam7 • Autoregulation of PI3P binding by Vam7 • Role of PI3P-Vam7 interaction in pathogenic yeast

III. PI(3,5)P2 production during fusion and fission • Reciprocal modulation of Ca2+ during fusion and fission • Role in regulating vacuole pH

IV. ABC transporters and vacuole fusion • Regulation of Ca2+ efflux • Interplay with PI(4,5)P2 production