title: Pericentriolar targeting of GDP-dissociation inhibitor isoform 2.
abstract: Cellular mechanisms for regulating membrane movements appear to involve small GTPases of the Rab subfamily. Binding of GDP-bound Rab proteins to donor membranes and their release from target membranes appear to be regulated by GDP-dissociation inhibitor (GDI) protein isoforms. Previous work showed strikingly higher levels of GDI-2 than GDI-1 fractionate with total membranes of cultured cells and are visualized in the perinuclear region in 3T3-L1 adipocytes. Here we report that GDI-2-containing structural elements are concentrated predominantly in the pericentriolar area in interphase CHO-T cells and differentiated 3T3-L1 adipocytes based on colocalization of GDI-2 and the centrosomal marker pericentrin. This finding is documented by both immunofluorescence and immunoelectron microscopy. Expressed c-Myc-tagged GDI-2 in transfected COS-7 cells targets to the same region. During mitotic resolution of the centrosome into two identifiable foci in CHO-T cells, GDI-2 containing structures remain intact and also resolve into two regions surrounding the centrosome. Dissociation of pericentriolar GDI-2 from the Golgi markers beta-COP and lectin receptors was apparent upon brefeldin A treatment of 3T3-L1 adipocytes or CHO-T cells. The integrity of pericentriolar GDI-2-binding elements was not disrupted by either brief Triton X-100 extraction or microtubule cytoskeletal disassembly, achieved with nocodazole. These data demonstrate the presence of highly ordered, detergent-resistant GDI-2-specific structural elements around the centrosome and indicate functional differences for the GDI-1 and GDI-2 protein isoforms. The results suggest the presence of selective GDI-2 acceptors in this region and a possible role of pericentriolar GDI-2 in membrane recycling.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
authors.forename: A
authors.initials: A
authors.lastname: Shisheva
authors.forename: S J
authors.initials: SJ
authors.lastname: Doxsey
authors.forename: J M
authors.initials: JM
authors.lastname: Buxton
authors.forename: M P
authors.initials: MP
authors.lastname: Czech
mesh_terms: D016475:3T3 Cells / Q000378:metabolism / Q000648:ultrastructure; D000273:Adipose Tissue; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000378:metabolism; D001692:Biological Transport; D020126:Brefeldin A; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D002502:Centrioles; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D003517:Cyclopentanes / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D020730:Guanine Nucleotide Dissociation Inhibitors; D007399:Interphase; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020692:rab3 GTP-Binding Proteins
chemical_list: D000941:Antigens; D003517:Cyclopentanes; D003902:Detergents; C098085:Gdi2 protein, mouse; D020730:Guanine Nucleotide Dissociation Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C086178:pericentrin; D020126:Brefeldin A; D017830:Octoxynol; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D015739:Nocodazole