High-affinity binding of the yeast cis-Golgi t-SNARE, Sed5p, to wild-type and mutant Sly1p, a modulator of transport vesicle docking

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Docking of ER-derived vesicles to the cis-Golgi compartment in yeast requires vesicle and target membrane receptors (v-SNAREs and t-SNAREs) and the GTPase Ypt1p. The t-SNARE Sed5p is complexed with Sly1p in vivo. The mutant form Sly1-20p rescues Ypt1p-lacking cells from lethality, suggesting an inhibitory function of Sly1p in v-SNARE/t-SNARE interaction. Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1-20p with equally high affinity ( K D=5.13×10 −9 M and 4.74×10 −9 M, respectively). Deletion studies show that the N-terminal half of Sly1p rather than the C-terminus (harbouring the E532K substitution in Sly1-20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking.