The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

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..

Representative Publications

Karunankaran S, Fratti RF. 2012. Vacuolar SNARE Function is Regulated by the Lipid Composition, Curvature and Fluidity of the Membrane. (in submission at J Biol. Chem.).

Karunakaran S, Sasser T, Rajalekshmi S, Fratti RA. 2012. SNAREs, HOPS, and regulatory lipids control the lateral mobility of vacuolar actin. (in press J Cell Sci)

Sasser T, Karunakaran S, Qiu Q, Padolina M, Reyes A, Flood B, Smith S, Fratti RF. 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

Fratti RA, Collins K, Hickey C, Wickner W. 2007. Stringent 3Q:1R composition of the SNARE 0-layer can be bypassed for fusion by compensatory SNARE mutation or by lipid bilayer modification. J. Biol. Chem. 282:14861-14867.

Fratti RA, Wickner W. 2007. Distinct Targeting and Fusion Functions of the PX- and SNARE domains of Yeast Vacuolar Vam7p. J. Biol. Chem. 282:13133-13138.

Jun Y, Thorngren N, Starai V, Fratti RA, Collins K, Wickner W. 2006. Reversible, cooperative reactions of yeast vacuole docking. EMBO J. 25:5250-5259.

Stroupe, C.S., Collins, K.M., Fratti, R.A., and Wickner, W. (2006) "Purification of Active HOPS Complex Reveals Its Affinities for Phosphoinositides and the SNARE Vam7p " EMBO J. 25, 1579-1589. [Abstract]

Collins, K.M., Thorngren, N., Fratti, R.A., and Wickner, W. (2005) "Sec17p and HOPS, in Distinct SNARE Complexes Associate, Mediate SNARE Complex Disruption or Assembly for Fusion " EMBO J. 24, 1775-1786. [Abstract]

Starai, V.J., Thorngren, N., Fratti, R.A., and Wickner, W. (2005) "Ion Regulation of Homotypic Vacuole Fusion in Saccharomyces cerevisiae " J. Biol. Chem. 280, 16754-16762. [Abstract]

Fratti, R.A., Jun, Y., Merz, A.J., Margolis, N., and Wickner, W. (2004) "Interdependent Assembly of Specific 'Regulatory' Lipids and Membrane Fusion Proteins into the Vertex Ring Domain of Docked Vacuoles " J. Cell Biol. 167, 1087-1098. [Abstract]

Jun, Y., Fratti, R.A., and Wickner, W. (2004) "Diacylglycerol and Its Formation by Phospholipase C Regulate Rab- and SNARE-dependent Yeast Vacuole Fusion " J. Biol. Chem. 279, 53186-53195. [Abstract]

Thorngren, N., Collins, K., Fratti, R.A., Merz, A.J., and Wickner, W. (2004) "A Soluble SNARE Drives Rapid Docking, Bypassing ATP and Sec17/18p for Vacuole Fusion " EMBO J. 23, 2765-2776. [Abstract]

Complete Publications List