Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat.
The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially 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 terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with 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 small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C. These findings suggest that p137 is the mammalian orthologue of Sec31p.
Shugrue
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Gastroenterology Section, Dept of Medicine, Yale University School of Medicine, New Haven, CT, USA.
Kolen
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Peters
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Czernik
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Kaiser
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Matovcik
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Hubbard
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Gorelick
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022181:COP-Coated Vesicles; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005656:Fungal Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D010750:Phosphoproteins; C079620:SEC13 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; C106113:SEC31 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins