Cell fusion is essential for human fertilization and development, and represents a new frontier for studies on the mechanism of membrane fusion. The cell fusion event of yeast mating provides a genetically accessible system to study this fundamental process. In yeast, cell fusion mutants accumulate prezygotes containing two haploid cells that have bound to each other but have not fused.

We monitor cell fusion by following the diffusion of fluorescent proteins such as GFP across the fusion pore Play Movie. Calculations from this data reveal that a typical fusion pore opens with a burst and then gradually expands. In fus1 mutant mating pairs, the fusion pores are 10-fold smaller than normal, and they expand more slowly. Fus1 is a transmembrane protein that is expressed only during mating and is targeted to the plasma membrane at contact sites between the two cells of a mating pair. The extracellular domain of Fus1 is not essential for its mating functions, so we are currently investigating proteins that physically and genetically interact with the cytoplasmic domain.

A screen for new cell fusion mutants revealed that ergosterol promotes both pheromone signaling and plasma membrane fusion. For signaling, ergosterol interacts with sphingolipids to facilitate recruitment of a signaling protein complex to polarized sites on the plasma membrane. In contrast, normal membrane fusion requires ergosterol, but not sphingolipids. We would like to learn whether ergosterol activates a membrane protein or simply alters the biophysical properties of the membrane to facilitate fusion.

The multispanning membrane protein Prm1 regulates the initiation of membrane fusion. A mixed phenotype is observed in prm1 mutant matings. Some mating pairs fuse, some arrest prior to fusion, and in some mating pairs, the two cells simultaneously lyse as soon as their plasma membranes come into contact Play Movie 2. Since Prm1 is not absolutely essential for cell fusion, we hypothesize that it regulates fusion proteins: without Prm1, uncoordinated action of these fusion proteins can lead to lysis. Interestingly, lysis can be prevented by adding high concentrations of calcium to the medium. Calcium enters the cytoplasm shortly before fusion in prm1 mating pairs by a pathway that does not require any known calcium channels. We would like to elucidate the pathway for calcium entry and identify intracellular targets that regulate the fidelity of membrane fusion.

Like Fus1, Prm1 is expressed only in mating cells and is targeted to contact sites in mating pairs. However, if Prm1 is expressed in non-mating cells, it is targeted to the vacuole and rapidly degraded. We are investigating how Prm1 transport is regulated and the physiological consequences of prematurely targeting Prm1 to the plasma membrane.

Biochemistry and molecular biology, cell-cell fusion, fusion of a sperm with an egg.

Jin, H., McCaffery, J.M. and Grote, E. (2008) Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast. J. Cell. Biol. 180: In press.

Chen, E., Grote, E., Mohler, W. and Vignery, A. (2007) Cell-Cell Fusion. FEBS Letters 581:2181-2193.

Nolan, S., Cowan, A.E., Koppel, D., Jin, H., and Grote, E. (2006) Fus1 regulates the opening and expansion of fusion pores between mating yeast. Mol. Biol. Cell 17: 2439-50.

Jin, H., Carlile, C., Nolan, S., and Grote, E. (2004). Prm1 prevents contact-dependent lysis of yeast mating pairs. Eukaryotic Cell 3: 1664-73.

Grote, E., Vlacich, G. Pypaert, M., and Novick, P. (2000). A SNC endocytosis mutant: phenotypic analysis and suppression by overproduction of dihydrosphingosine lyase. Mol. Biol. Cell 11: 4051-65.

Grote, E., Baba, M., Ohsumi, Y. and Novick, P. (2000). Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion. J. Cell Biol. 151: 453-465.

Grote, E., Carr, C.M., and Novick, P. (2000). Ordering the final events in yeast exocytosis. J. Cell Biol. 151: 439-451.

Grote, E. and Novick, P. (1999). Promiscuity in Rab/SNARE interactions. Mol. Biol. Cell 10: 4149-4161.

Carr, C.M., Grote, E. and Novick, P. (1999). Sec1p binds to SNARE complexes and concentrates at sites of secretion. J. Cell Biol. 146:333-344.

Abeliovich, H., Grote, E. Novick, P. and Ferro-Novick S. (1998). Tlg2p, a yeast syntaxin homolog that resides on the Golgi and endocytic structures. J. Biol. Chem. 273:11719-27.

Grote, E., Kelly, R.B. (1996). Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal. J. Cell Biol. 132:537-547.

Grote, E., Hao, J.C., Bennett, M.K., Kelly, R.B. (1995). A targeting signal in VAMP regulating transport to synaptic vesicles. Cell 81:581-589.