Eileen M. Lafer, Ph.D.

Professor

Department of Biochemistry

Location:	MED, Room 415B
Phone:	(210) 567-3764
Fax:	(210) 567-6595
E-mail:	lafer@biochem.uthscsa.edu

Research Interests:

Neurotransmission is a cycle of exo- and endocytosis. Following depolarization and calcium influx synaptic vesicles fuse with the presynaptic plasma membrane and release their neurotransmitter cargo into the synaptic cleft. The neurotransmitter molecules diffuse across the cleft and bind to receptors on the post-synaptic plasma membrane, thereby propagating the nerve impulse. The synaptic vesicle membrane proteins are then recovered by the presynaptic terminal, and reutilized for the local biogenesis of new synaptic vesicles. We have demonstrated that the clathrin pathway is essential for synaptic vesicle endocytosis in vivo (Morgan et al, 1999, 2000, 2001). We have reconstituted the assembly and uncoating reactions from purified components in vitro (Ye and Lafer, 1995a; Hao et al., 1999; Morgan et al, 2001), and have characterized the protein-protein (Ye and Lafer, 1995a,b; Hao et al., 1999; Morgan et al, 2000, 2001) and protein-lipid (Ye et al., 1995; Hao et al., 1997) interactions underlying clathrin coated vesicle assembly and disassembly. We are particularly interested in understanding how coated vesicle dynamics are regulated. We have evidence that both protein binding (Morgan et al., 2003), protein phosphorylation (Hao et al., 1999), and lipid phosphorylation (Hao et al., 1997) contribute to this process (Morgan et al., 2001b). Recently, we have started to incorporate structural approaches into our mechanistic studies of the endocytic machinery. Auxilin and Hsc70 are proteins critical for coated vesicle uncoating (Morgan et al., 2001). Together with faculty colleagues Rui Sousa and John Hart, we solved the structure of the auxilin J-domain by X-ray crystallography (Jiang et al., 2003). Together with Rui Sousa, we recently solved the structure of Hsc70. This is extremely significant, since while there had been structures of isolated domains available for 10-15 years, a structure of an Hsc70 containing both an ATPase domain and substrate binding domain had been elusive. Our structural study provides novel insights into the mechanism of interdomain communication for this important chaperone protein (Jiang et al., 2005).

Unique Technical and Clinical Research Capabilities/Instrumentation:

surface plasmon resonance (Biacore), analytical ultracentrifugation, protein expression and purification, directed mutagenesis, microinjection

Publications (selected):

Ye, W. and Lafer, E.M. Bacterially expressed F1-20/AP-3 assembles clathrin into cages with a narrow size distribution: Implications for the regulation of quantal size during neurotransmission. J. Neurosci. Res. 41:15-26 (1995a).

Ye, W. and Lafer, E.M. Clathrin binding and assembly activities of expressed domains of the synapse-specific clathrin assembly protein AP-3. J. Biol. Chem. 270:10933-10939, (1995b).

Ye, W., Ali, N., Bembenek, M., Shears, S.B. and Lafer, E.M. Inhibition of clathrin assembly by high- affinity binding of specific inositol polyphosphates to the synapse-specific clathrin assembly protein AP-3. J. Biol. Chem. 270:1564-1568, (1995).

Hao, W, Tan, T, Prasad, K., Reddy, K.K., Chen, J., Prestwich, G.D., Falck, J.R., Shears, S.B. and Lafer, E.M.. Regulation of AP-3 Function by Inositides: Identification of Phosphatidylinositol 3,4,5-trisphosphate as a Potent Ligand. J. Biol. Chem. 272: 6393-6398 (1997).

Hao, W. , Luo,Z., Zheng, L. , Prasad, K., and Lafer, E.M. AP180 and AP-2 Interact Directly in a Complex that Cooperatively Assembles Clathrin. J. Biol. Chem. 274: 22785-22795 (1999).

Morgan, J.R., Xiao, X., Womack, M., Prasad, K., Augustine, G.J., and Lafer, E.M. A Role for the Clathrin Assembly Domain of AP180 in Synaptic Vesicle Endocytosis. J. Neurosci. 19: 10201-10212 (1999).

Morgan, J.R., Prasad, K., Hao, W., Augustine, G.J., and Lafer, E.M. A Conserved Clathrin Assembly Motif Essential for Synaptic Vesicle Endocytosis. J. Neurosci. 20: 8667-8676 (2000).

Morgan, J.R., Prasad, K., Jin, S., Augustine, G.J. and Lafer, E.M. Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc70 and Auxilin. Neuron 32: 289-300 (2001).

Lafer, E.M.. Clathrin-Protein Interactions. Traffic 3: 513-520 (2002).

Morgan, J.R., Augustine, G.J. and Lafer, E.M. Synaptic Vesicle Endocytosis: The Races, Places, and Molecular Faces. NeuroMolecular Medicine 2: 101-114 (2002).

Jiang, J., Taylor, A.B., Prasad, K., Ishikawa-Brush, Y., Hart, P.J., Lafer, E.M., and Sousa, R. Structure-Function Analysis of the Auxilin J-Domain Reveals an Extended Hsc70 Interaction Interface. Biochemistry 42: 5748-5753 (2003).

Morgan, J.R., Prasad, K., Jin, S., Augustine, G.J., and Lafer, E.M. Eps15 Homology Domain ≠ NPF Motif Interactions Regulates Clathrin Coat Assembly During Synaptic Vesicle Recycling. J. Biol. Chem. 278: 33583-92 (2003).

Yu, S. Yin, X., Lafer, E.M., and Jiang, J.X. Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C-terminus of Major Intrinsic Protein (Aquaporin-0). J. Biol. Chem. 280:22081-90 (2005).

Jiang, J., Prasad, K., Lafer, E.M., and Sousa, R. Structural Basis of Interdomain Communication in the Hsc70 Chaperone. Molecular Cell 20:513-524 (2005).

Augustine, G.J., Morgan, J.R., Villalba-Galea, C., Jin, S., Prasad, K., and Lafer, E.M. Clathrin and Synaptic Vesicle Endocytosis: Studies at the squid giant synapse. Biochemical Society Transactions, in press (2006).

Jin, A.J., Prasad, K., Smith, P.D, Lafer, E.M, and Nossal, R.J. Measuring the elasticity of clathrin coated vesicles via atomic force microscopy. Biophysical Journal, in press (2006).

Jiang, J., Lafer, E.M., and Sousa, R. Crystallization of a Functionally Intact Bovine Hsc70.  ACTA F, in press (2006)

Key Words:

neurotransmission, synaptic vesicles, coated vesicles, organelle biogenesis, synapse, endocytosis, clathrin