GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules invol ved in the docking and fusion of synaptic vesicles with the presynaptic mem brane. We report here the molecular, cell biological, and biochemical chara cterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequ ence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 3 2-kDa protein present in both cytosol and membrane fractions. The membrane- bound form of this protein is revealed to be primarily localized to the Gol gi apparatus by indirect immunofluorescence microscopy, a finding that is f urther established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi appara tus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in th e Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustain s the preferential interaction of GS32 with Golgi syntaxin 6.