Rutilio "Rudy" A. 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. 

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Figure 1. The Vacuole Fusion Cycle.

Depicted are the stages of vacuole fusion. 1) Isolated vacuoles contain inactive cis-SNARE complexes composed of the 3Q-SNAREs (Vam7, Vam3 and Vti1, shades of green) and 1R-SNARE (Nyv1, red). Also pictured is a PA-bound monomeric Sec18 (mSec18, purple wedge) associated with the membrane. Pah1 activity promotes moving to the next stage while excess PA blocks the progression. 2) Hexameric Sec18 (hSec18) associates with cis-SNARE through the use of Sec17 (orange trapezoids). This is promoted by ergosterol and PI(4,5)P₂. 3) Sec18 hydrolyzes ATP to use Sec17 as a molecular wedge to separate the cis-SNARE bundle into individual proteins. Sec17 are and Vam7 released from the membrane. Individual SNAREs are shown to partially secondary structure in their SNARE motifs depicted as elongated lines extending from the ovals. Sec18 is thought to re-associate with the membrane as a PA-bound protomers. 4) During vacuole docking SNAREs interact in trans and partially zipper. This is promoted by PI3P, PI4P and PI(4,5)P₂. 5) trans-SNARE complexes continue to fully zipper leading to hemifusion. The outer leaflets of both compartments merge (purple), while the inner leaflets remain separated to prevent content mixing. This is promoted by DAG, while it is inhibited by PI(3,5)P₂, LPC and PA. 6) Inner leaflets fusion to fully merge the compartments into a continuous membrane and mixing luminal content (purple). The post-fusion SNAREs are shown now in cis and the vacuole returns to step 1 of the cycle. (See Starr & Fratti TIBS 2019)