Although the membrane-trafficking functions of most SNAREs are conserved from yeast to humans, some mammalian SNAREs have evolved specialized functions unique to multicellular life. The mammalian homolog of the prenylated yeast SNARE Ykt6p might be one such example, because rat Ykt6 is highly expressed only in brain neurons. Furthermore, neuronal Ykt6 displayed a remarkably specialized, punctate localization that did not overlap appreciably with conventional compartments of the endomembrane system, suggesting that Ykt6 might be involved in a pathway unique to or specifically modified for neuronal function. Targeting of Ykt6 to its unique subcellular location was directed by its profilin-like longin domain. We have taken advantage of high-resolution structural data available for the yeast Ykt6p longin domain to examine mechanisms by which the mammalian longin domain controls Ykt6 conformation and subcellular targeting. We found that the overall tertiary structure of the longin domain, not sequence-specific surface features, drives direct targeting to the Ykt6 punctate structures. However, several sequence-specific surface features of the longin domain indirectly regulate Ykt6 localization through intramolecular interactions that mask otherwise-dominant targeting signals on the SNARE motif and lipid groups. Specifically, two hydrophobic binding pockets, one on each face of the longin domain, and one mixed hydrophobic/charged surface, participate in protein-protein interactions with the SNARE motif and protein-lipid interactions with the lipid group(s) at the molecule's C-terminus. One of the hydrophobic pockets suppresses protein-palmitoylation-dependent mislocalization of Ykt6 to the plasma membrane. The Ykt6 intramolecular interactions would be predicted to create a compact, closed conformation of the SNARE that prevents promiscuous targeting interactions and premature insertion into membranes. Interestingly, both protein-protein and protein-lipid interactions are required for a tightly closed conformation and normal targeting.