Identification of the putative mammalian orthologue of Sec31P, a componentof the COPII coat

The regulation of intracellular vesicular trafficking is mediated by specif ic families of proteins that are involved in vesicular budding, translocati on, and fusion with target membranes. We purified a vesicle-associated prot ein from hepatic microsomes using sequential column chromatography and part ially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137), The protein had an N term inus WD repeat moth with significant homology to Sec31p, a member of the ye ast COPII coat that complexes with Sec13p. We found that p137 interacted wi th mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to sm all punctate structures in the cytoplasm of multiple cultured cell lines, W hen Sec13p was transfected into these cells, it demonstrated considerable o verlap with p137, This overlap was maintained through several pharmacologic al manipulations. The p137 compartment also demonstrated partial overlap wi th ts045-VSVG protein when infected cells were incubated at 15 degrees C, T hese findings suggest that p137 is the mammalian orthologue of Sec31p.