Preferential association of syntaxin 8 with the early endosome

Members of the syntaxin family play a fundamental role in vesicle docking a nd fusion of diverse transport events. We have molecularly characterized sy ntaxin 8, a novel member of the syntaxin family. The nucleotide sequence of cloned rat cDNA predicts a polypeptide of 236 residues with a carboxyl-ter minal 18-residue hydrophobic domain that may function as a membrane anchor. Characteristic of syntaxins, syntaxin 8 also contain regions that have the potential to form coiled-coil structures. Among the known syntaxins, synta xin 8 is most homologous to syntaxin 6 which is predominantly associated wi th the trans-Golgi network (TGN), The syntaxin 8 transcript is detected in all rat tissues examined by northern blot. Antibodies against recombinant s yntaxin 8 recognize a 27 kDa protein that is enriched in membrane fractions containing the Golgi apparatus and the endosomal/lysosomal compartments. S yntaxin 8 in membrane extract could be incorporated into a 20S protein comp lex in a way that is dependent on the soluble N-ethylmaleimide-sensitive fa ctor (NSF) and soluble NSF attachment protein (a-SNAP), suggesting that syn taxin 8 is indeed a SNAP receptor (SNARE), Indirect immunofluorescence micr oscopy reveals that the majority of syntaxin 8 is localized to the early en dosome marked by Rab5, This is corroborated by immunogold labeling experime nts showing enrichment of syntaxin 8 in the early endosome and its co-label ing with Rab5.