Rutilio "Rudy" A Fratti
Associate Head of Biochemistry
Associate Professor of Biochemistry
Associate Professor, Center for Biophysics and Quantitative Biology
Ion Channels, Membrane Biology, Protein Dynamics
B.S. 1992 California State University, Long Beach
Ph.D. 2002 University of Michigan
Postdoc. 2002-2006 Dartmouth Medical School
Membrane Microdomain Assembly and Membrane Fusion
Our lab is interested in the fundamental problem of how lipids and proteins cooperate to form functional microdomains at the site of membrane fusion. Membrane fusion at highly specialized microdomains is essential for many cellular processes including hormone secretion, neurotransmitter release, membrane repair and antigen presentation. Deciphering the regulation and pathways that lead to microdomain assembly has become central to the deeper understanding of membrane fusion and trafficking. We use a combination of genetics, cell-free biochemistry and microscopy to understand microdomain assembly and membrane fusion.
To study microdomain assembly and membrane fusion we use purified vacuoles (lysosomes) from the yeast Saccharomyces cerevisiae. Because the mechanisms and machinery of membrane fusion are conserved throughout eukaryotes, the vacuole offers an ideal system for our studies. Purified vacuoles bear a full complement of lipids and proteins necessary for in vitro fusion and can be easily prepared in large quantities, facilitating high throughput biochemical studies. Vacuoles are also large organelles, facilitating microscopic examination of lipid microdomains and visualization of vacuole docking and fusion. These characteristics, combined with the power of yeast genetics makes the vacuole a superb system for the study of membrane biology.
Vacuole fusion occurs at a specialized lipid microdomain called the vertex ring (See below). When vacuoles come into contact at a single point (tethering), they are drawn together and become tightly associated, forming two flattened discs of apposed membrane termed the boundary membrane (docking). The perimeter of the boundary membrane is the vertex ring which becomes enriched in the proteins and lipids that catalyze membrane fusion. These include SNAREs and their chaperones, the Rab Ypt7p and its effector complex HOPS, actin and regulatory lipids including sterols, diacylglycerol and phosphoinositides such as PI(3)P and PI(4,5)P2. Fusion only occurs if the vertex ring becomes properly enriched in protein and lipid fusion catalysts. The assembly of proteins and lipids at the vertex ring is highly complex and interdependent. The intricate regulation of these processes are only now beginning to be understood making this a very exciting time in the lab.
Lipids and proteins that mediate membrane fusion are co-enriched at identical vertices. (Top) A schematic view of vacuoles showing the three morphological features that are developed during docking and fusion. (Bottom) Epifluorescence images of purified vacuoles in an in vitro docking assay. Vacuoles isolated from yeast harboring Vps33p-GFP were incubated with fluorescent Cy3-labeled FYVE domain to specifically localize PI(3)P. Cy3-FYVE is shown in red. To view the entire cluster of membranes, phosphatidylserine (PS), which is evenly distributed on vacuoles, was labeled with the fluorescent probe PSS-380 and is shown in blue. Solid arrows show examples of vertex sites enriched in Vps33p and PI(3)P. Hollow arrows specify outer membrane microdomains. Bar, 2 µm.
Miner GE, Starr ML, Hurst LR, Fratti RA. 2017. Deleting the DAG Kinase Dgk1 Augments Yeast Vacuole Fusion Through Increased Ypt7 Activity and Altered Membrane Fluidity. Traffic. 18:315-329.
Sparks RP, Jenkins LJ, Miner GE, Wangm Y, Guida WC, Sparks CE, Fratti RA, Sparks JD. 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.
Starr ML, Hurst LR, Fratti RA. 2016. Phosphatidic acid sequesters Sec18p from cis-SNARE complexes to inhibit priming. Traffic. 17:1091-1109.
Sparks RP, Guida WC, Sowden MP, Jenkins JL, Starr ML, Fratti RA, Sparks CE, Sparks JD. 2016. Sortilin facilitates VLDL-B100 secretion by insulin sensitive McArdle RH7777 cells. Biochem. Biophys. Res. Commun. 478:546-552.
Miner G, Starr ML, Hurst LR, Sparks RP Padolina M, Fratti RA. 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. 291:17651-17663.
Sasser TL, Fratti RA. 2014. Class C ABC transporters and Saccharomyces cerevisiae vacuole fusion. Cell. Logist. 2014 Jul 3;4(3):e943588.
Lawrence G, Flood B, Brown C, Karunakaran S, Cabrera M, Nordmann M, Ungermann C, Fratti RA. 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. 25:1608-1619.
Sasser TL, Lawrence G, Karunakaran S, Brown C, Fratti RA. 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(25):18300-10.
Karunankaran S, Fratti RA. 2013. The Lipid Composition and Physical Properties of the Yeast Vacuole Affect the Hemifusion-Fusion Transition. Traffic. 14(6):650-62.
Sasser TL, Padolina M, Fratti RA. 2012. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation. Biochem. J. 448(3):365-72.
Karunakaran S, Sasser T, Rajalekshmi S, Fratti RA. 2012. SNAREs, HOPS and regulatory lipids control the dynamics of vacuolar actin during homotypic fusion in S. cerevisiae. J. Cell Sci. 125(Pt 7):1683-92.
Sasser T, Karunakaran S, Qiu Q, Padolina M, Reyes A, Flood B, Smith S, Fratti RA. 2012 The Yeast Lipin 1 Orthologue Pah1p Regulates Vacuole Homeostasis and Membrane Fusion. J. Biol. Chem. 287:2221-2236.
Qiu Q, Fratti RA. 2010. The Na+/H+ exchanger Nhx1p Regulates the Initiation of Saccharomyces cerevisiae Vacuole Fusion. J. Cell Science. 123:3266-3275.