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. 2000 Mar 14;97(6):2567-72.
doi: 10.1073/pnas.97.6.2567.

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

R Yamaji  1 , R Adamik, K Takeda, A Togawa, G Pacheco-Rodriguez, V J Ferrans, J Moss, M Vaughan
Affiliations

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

R Yamaji et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Western blot analysis of BIG1 and BIG2 in the cytosolic and microsomal fractions from HeLa and HepG2 cells. (A) Samples of cytosol (C, 100 μg of proteins) or microsomes (M, 10 μg from HeLa, 15 μg from HepG2 cells) were subjected to SDS/PAGE and transferred to PVDF membrane, followed by reaction with anti-BIG1 (Left) or anti-BIG2 (Right) IgG. (B) The indicated amounts of proteins from M or C fractions of HepG2 cells were subjected to SDS/PAGE in 6% gel and transferred to PVDF membrane, followed by Western blotting using anti-BIG1 or anti-BIG2 IgG. Immunoreactive proteins were quantified by densitometry. On standard curves for cytosolic BIG1 and BIG2, positions of the M samples (15 μg) are indicated. Total C and M protein was, respectively, 3.9 and 0.39 mg for HeLa, and 16 and 2.4 mg for HepG2 cells. Similar data were obtained in four independent experiments.
Figure 2
Figure 2
Subcellular fractionation of microsomes from HepG2 cells. Microsomal proteins (1.7 mg) from HepG2 cells were fractionated on a discontinuous sucrose density gradient. Proteins from the 0.6/0.25, 0.85/0.6, and 1.15/0.85 interfaces were precipitated with trichloroacetic acid and separated by SDS/PAGE, followed by Western blotting using anti-BIG1 or anti-BIG2 IgG or anti-β-COP antibodies, as indicated. Observations were replicated three times.
Figure 3
Figure 3
Golgi localization of BIG1 and BIG2. HepG2 cells were reacted with rabbit anti-BIG1 or anti-BIG2 IgG and mouse anti-58K or -AP-1 antibodies, as indicated, followed by FITC-labeled anti-rabbit IgG and Texas Red-labeled anti-mouse IgG. In the third panel of each row, images of the preceding two panels are superimposed. Observations were replicated with other samples of cells, five times for BIG1 and three times for BIG2.
Figure 4
Figure 4
Colocalization of endogenous BIG1 and overexpressed BIG2. HeLa and HepG2 cells were transfected with pcDNA3.1/BIG2-Myc encoding a fusion protein of BIG2 with a C-terminal myc tag. After 24 hr, cells were fixed, permeabilized, and incubated with rabbit anti-BIG1 IgG and mouse anti-myc antibodies (to detect BIG2), as indicated, followed by FITC-labeled anti-rabbit IgG and Texas Red-labeled anti-mouse IgG. In the third panel of each row, images of the preceding two panels are superimposed. Observations were replicated twice.
Figure 5
Figure 5
Fractionation of BIG1 and BIG2 on Sepharose CL-6B. Cytosol (16 mg protein) from HeLa cells was fractionated on Sepharose CL-6B. Samples of fractions were subjected to SDS/PAGE, followed by Western blotting using anti-BIG1 and anti-BIG2 IgGs. Immunoreactive bands were quantified by densitometry; amounts of BIG1 and BIG2 are recorded as percentage of the total of each recovered. Elution positions of standard proteins are indicated. Th, thyroglobulin (669 kDa); Fe, ferritin (440 kDa); Ca, catalase (232 kDa); Al, aldolase (158 kDa). Data from a replicate experiment were similar.
Figure 6
Figure 6
Immunoprecipitation of BIG1 and BIG2 complexes from HepG2 cells. Samples of proteins, cytosol (C) 2.6 mg, or microsomes (M) 0.4 mg, were immunoprecipitated (IP) by anti-BIG1 or anti-BIG2 IgG with or without the peptide used for immunization, as indicated. Immunoprecipitated proteins were separated by SDS/PAGE, and reacted on Western blots (WB) with anti-BIG1 or anti-BIG2 IgG. Data were similar in three experiments.
Figure 7
Figure 7
Immunoprecipitation of protein complexes containing BIG1 and BIG2. (A) Proteins immunoprecipitated from samples of cytosolic proteins (30 μg) from HepG2 cells with the indicated amounts of anti-BIG1 or anti-BIG2 IgG were analyzed by Western blotting using anti-BIG1 or anti-BIG2 IgG, followed by quantification of immunoreactive protein by densitometry. (B) Samples of HepG2 cell cytosolic proteins (30 μg) were incubated first without or with anti-BIG1 or anti-BIG2 IgG (3.75 μg) and protein A beads. After centrifugation, the supernatant was incubated a second time with anti-BIG1 or anti-BIG2 IgG and protein A beads. Immunoprecipitated proteins were analyzed by SDS/PAGE and Western blotting (WB) using anti-BIG1 or anti-BIG2 IgG, and quantified by densitometry. Data were similar in three experiments.
Figure 8
Figure 8
Effect of BFA on intracellular distribution of BIG1 and BIG2. HepG2 cells were incubated without or with BFA (10 μg/ml) for 0, 1, or 10 min, fixed, permeabilized, and incubated with anti-BIG1 or anti-BIG2 IgG, or rabbit anti-β-COP or mouse anti-58K antibodies, as indicated, followed by FITC-labeled anti-rabbit IgG or Texas Red-labeled anti-mouse IgG. Data were similar in three experiments.
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