Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex

Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for AD P-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. Wh en observed by immunofluorescence in Hela S3 and HepG2 cells, endogenous BI G1 and coexpressed BIG2 were distributed in a punctate pattern throughout t he cytosol, and also concentrated in the perinuclear region, where endogeno us BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and gamma-adaptin. On Western blot analysis, both BIG1 and BIG2 were clear ly more abundant in the cytosol than in the microsomal fractions. After den sity gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were r ecovered in the same fraction as beta-CDP, a marker for Golgi membranes. Wh en cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 an d BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa, Western blotting using anti-peptide antibodies specific for B IG1 or BIG2 demonstrated that approximate to 70% of BIG2 was immunoprecipit ated along with 100% of BIG1 by the anti-BIG1 IgG, and approximate to 75% o f BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG, All obser vations were consistent with the conclusion that significant fractions of B IG1 and BIG2 exist as components of the same macromolecular complexes in bo vine brain cytosol and are similarly localized in cultured cells.