ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase-activating proteins (GAPs). Two general types of GEPs have been recognized,a family of ~200-kDa proteins that are inhibited by BFA (a drug that interferes with protein secretion and causes reversible disintegration of Golgi cisternae) and smaller ~55-kDa GEPs that are BFA-resistant. All GEPs have so-called Sec7 domains of ~200 amino acids that are responsible for the GEP activity, as well as its BFA sensitivity. This group had earlier purified, from bovine brain cytosol, two BFA- inhibited GEPs (BIG1 and BIG2) that behaved on gel filtration as molecules of ~670 kDa. To determine whether they were parts of the same or different protein complexes of similar size and to define their intracellular localization, specific anti-peptide antibodies were prepared and affinity purified. Immunofluorescence microscopy revealed a punctate distribution of both throughout cells with concentration in the perinuclear region, partially colocalized with Golgi-specific p58 protein. All observations, including results of sequential immunoprecipitation of proteins from cytosol with BIG1- and BIG2- specific antibodies were consistent with the conclusion that significant fractions of both proteins exist in the same macromolecular complexes in cytosol and are similarly localized in cultured cells. Identification of other proteins present in the complexes or interacting with the GEPs in cells is ongoing.

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National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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Puxeddu, Ermanno; Uhart, Marina; Li, Chun-Chun et al. (2009) Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity. Proc Natl Acad Sci U S A 106:6158-63
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