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Vol. 17, Issue 7, 3176-3186, July 2006
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*Stahlman Cardiovascular Research Laboratories, Program for Developmental Biology, and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300; and Department of Surgery and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, and Nashville VAMC, Nashville, TN 37212-2175
Submitted December 13, 2005;
Revised April 17, 2006;
Accepted April 25, 2006
Monitoring Editor: Francis Barr
SNAP-25 is a component of the SNARE complex that is involved in membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between SNAP-25 and cytoplasmic Lek1 (cytLEK1), a protein previously demonstrated to associate with the microtubule network. The binding domains within each protein were defined by yeast two-hybrid, coimmunoprecipitation, and colocalization studies. Confocal analyses reveal a high degree of colocalization between the proteins. In addition, the endogenous proteins can be isolated as a complex by immunoprecipitation. Further analyses demonstrate that cytLEK1 and SNAP-25 colocalize and coprecipitate with Rab11a, myosin Vb, VAMP2, and syntaxin 4, components of the plasma membrane recycling pathway. Overexpression of the SNAP-25–binding domain of cytLEK1, and depletion of endogenous Lek1 alters transferrin trafficking, consistent with a function in vesicle recycling. Taken together, our studies indicate that cytLEK1 is a link between recycling vesicles and the microtubule network through its association with SNAP-25. This interaction may play a key role in the regulation of the recycling endosome pathway.
Address correspondence to: David M. Bader ( david.bader{at}vanderbilt.edu)
Abbreviations used: cytLEK1, cytoplasmic LEK1; 5'LCR, cytLEK coil region; 5'LSD, cytLEK SNAP-25 Binding Domain; nucLEK1, nuclear LEK1; QDO, Quadruple Dropout; Tf, transferrin, SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors; SNAP-25, synaptosomal-associated protein of 25 kD; VAMP, vesicle-associated membrane protein; Y2H, yeast two-hybrid
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