HIGH-AFFINITY BINDING OF THE YEAST CIS-GOLGI T-SNARE, SED5P, TO WILD-TYPE AND MUTANT SLY1P, A MODULATOR OF TRANSPORT VESICLE DOCKING

Docking of ER-derived vesicles to the cis-Golgi compartment in yeast r equires vesicle and target membrane receptors (v-SNAREs and t-SNAREs) and the GTPase Ypt1p. The t-SNARE Sed5p is complexed with Sly1p in viv o. The mutant form Sly1-20p rescues Ypt1p-lacking cells from lethality , suggesting an inhibitory function of Sly1p in V-SNARE/t-SNARE intera ction. Using surface plasmon resonance spectroscopy, we found that Sed 5p binds Sly1p and Sly1-20p with equally high affinity (K-D = 5.13 x 1 0(-9) M and 4.74 x 10(-9) M, respectively). Deletion studies show that the N-terminal half of Sly1p rather than the C-terminus (harbouring t he E532K substitution in Sly1-20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibi tory role of Sly1p in vesicle docking. (C) 1997 Federation of European Biochemical Societies.