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 ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and gamma-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as beta-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that approximately 70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and approximately 75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.
Yamaji
R
R
Pulmonary-Critical Care Medicine Branch and Pathology Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. yamajir@fido.nhlbi.nih.gov
Adamik
R
R
Takeda
K
K
Togawa
A
A
Pacheco-Rodriguez
G
G
Ferrans
V J
VJ
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors / Q000737:chemistry*; D015153:Blotting, Western; D020126:Brefeldin A / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008861:Microsomes / Q000378:metabolism; D010455:Peptides; D011233:Precipitin Tests; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
C119087:ARFGEF1 protein, human; C119088:ARFGEF2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors