Targeting of vesicles to the acceptor membrane in protein transport depends on membrane proteins called SNAREs. Saccharomyces cerevisiae Golgi t-SNARE Sed5 protein and its neural cognate syntaxin 1 have similar three alpha-helices which are predicted to form coiled coils. We dissected the helices of Sed5 and found several characteristics unexpectedly distinct from those of syntaxin 1. Most importantly, only the N-terminal helix is responsible for the binding of Sly1 protein while almost the entire molecule of syntaxin is necessary for the binding of the cognate, Munc-18. The N-terminal region of Sed5 protein also binds to the C-terminal helix and Sly1 protein interfered this binding.