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Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells.

Yeaman C, Grindstaff KK, Wright JR, Nelson WJ - J. Cell Biol. (2001)

Bottom Line: At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45.Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites.Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

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Requirement for plasma membrane- and TGN-bound Sec6/8 complex in exocytosis of ts-G-GFP in NRK-52E cells. NRK-52E cells were transiently transfected with plasmid encoding ts-G-GFP, incubated under conditions to arrest this protein in TGN, and then permeabilized with Digitonin as described in Materials and methods. Protein transport to the plasma membrane was reconstituted in the presence of nonspecific IgG (A) or anti-Sec6/8 antibodies directed against epitopes expressed at the plasma membrane (B) or TGN (C). Amounts of ts-G-GFP, ZO-1, and furin associated with perinuclear, cytoplasmic, or peripheral regions of ∼15 cells were quantified as described in Materials and methods. Representative analyses are shown above histograms of data from all analyses. (D) A gallery of representative images of semiintact NRK-52E cells in which TGN to plasma membrane transport of ts-G-GFP (green) was reconstituted in the presence of either control antibodies (left) or anti-PM Sec6/8 antibodies (right) and compared with the distribution of the plasma membrane-localized ZO-1 (red). Bars, 5 μm.
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fig10: Requirement for plasma membrane- and TGN-bound Sec6/8 complex in exocytosis of ts-G-GFP in NRK-52E cells. NRK-52E cells were transiently transfected with plasmid encoding ts-G-GFP, incubated under conditions to arrest this protein in TGN, and then permeabilized with Digitonin as described in Materials and methods. Protein transport to the plasma membrane was reconstituted in the presence of nonspecific IgG (A) or anti-Sec6/8 antibodies directed against epitopes expressed at the plasma membrane (B) or TGN (C). Amounts of ts-G-GFP, ZO-1, and furin associated with perinuclear, cytoplasmic, or peripheral regions of ∼15 cells were quantified as described in Materials and methods. Representative analyses are shown above histograms of data from all analyses. (D) A gallery of representative images of semiintact NRK-52E cells in which TGN to plasma membrane transport of ts-G-GFP (green) was reconstituted in the presence of either control antibodies (left) or anti-PM Sec6/8 antibodies (right) and compared with the distribution of the plasma membrane-localized ZO-1 (red). Bars, 5 μm.

Mentions: Sec6/8 complex associates with TGN and plasma membrane cell–cell contacts, and there appears to be a dynamic relationship between these complexes. What is the role of Sec6/8 complexes at each site? Because monoclonal antibodies specifically bind Sec6/8 complexes associated with either TGN or plasma membrane in NRK cells, we tested these different antibodies as inhibitors of post-Golgi trafficking of ts-G-GFP in digitonin-permeabilized NRK cells. Morphological transport assays were performed using NRK-52E cells, because NRK-49F cells rounded up following permeabilization, making analysis of data impossible. Distribution of ts-G-GFP in perinuclear or peripheral regions of cells, or the region in-between these (defined generally as “cytoplasm”), was determined by quantifying total pixel intensities within boundaries defined by distributions of plasma membrane (ZO-1) or TGN (furin) markers. Representative images of individual cells (from ∼15 analyses for each condition) are presented, together with histograms of data from all analyses (Fig. 10).


Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells.

Yeaman C, Grindstaff KK, Wright JR, Nelson WJ - J. Cell Biol. (2001)

Requirement for plasma membrane- and TGN-bound Sec6/8 complex in exocytosis of ts-G-GFP in NRK-52E cells. NRK-52E cells were transiently transfected with plasmid encoding ts-G-GFP, incubated under conditions to arrest this protein in TGN, and then permeabilized with Digitonin as described in Materials and methods. Protein transport to the plasma membrane was reconstituted in the presence of nonspecific IgG (A) or anti-Sec6/8 antibodies directed against epitopes expressed at the plasma membrane (B) or TGN (C). Amounts of ts-G-GFP, ZO-1, and furin associated with perinuclear, cytoplasmic, or peripheral regions of ∼15 cells were quantified as described in Materials and methods. Representative analyses are shown above histograms of data from all analyses. (D) A gallery of representative images of semiintact NRK-52E cells in which TGN to plasma membrane transport of ts-G-GFP (green) was reconstituted in the presence of either control antibodies (left) or anti-PM Sec6/8 antibodies (right) and compared with the distribution of the plasma membrane-localized ZO-1 (red). Bars, 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198873&req=5

fig10: Requirement for plasma membrane- and TGN-bound Sec6/8 complex in exocytosis of ts-G-GFP in NRK-52E cells. NRK-52E cells were transiently transfected with plasmid encoding ts-G-GFP, incubated under conditions to arrest this protein in TGN, and then permeabilized with Digitonin as described in Materials and methods. Protein transport to the plasma membrane was reconstituted in the presence of nonspecific IgG (A) or anti-Sec6/8 antibodies directed against epitopes expressed at the plasma membrane (B) or TGN (C). Amounts of ts-G-GFP, ZO-1, and furin associated with perinuclear, cytoplasmic, or peripheral regions of ∼15 cells were quantified as described in Materials and methods. Representative analyses are shown above histograms of data from all analyses. (D) A gallery of representative images of semiintact NRK-52E cells in which TGN to plasma membrane transport of ts-G-GFP (green) was reconstituted in the presence of either control antibodies (left) or anti-PM Sec6/8 antibodies (right) and compared with the distribution of the plasma membrane-localized ZO-1 (red). Bars, 5 μm.
Mentions: Sec6/8 complex associates with TGN and plasma membrane cell–cell contacts, and there appears to be a dynamic relationship between these complexes. What is the role of Sec6/8 complexes at each site? Because monoclonal antibodies specifically bind Sec6/8 complexes associated with either TGN or plasma membrane in NRK cells, we tested these different antibodies as inhibitors of post-Golgi trafficking of ts-G-GFP in digitonin-permeabilized NRK cells. Morphological transport assays were performed using NRK-52E cells, because NRK-49F cells rounded up following permeabilization, making analysis of data impossible. Distribution of ts-G-GFP in perinuclear or peripheral regions of cells, or the region in-between these (defined generally as “cytoplasm”), was determined by quantifying total pixel intensities within boundaries defined by distributions of plasma membrane (ZO-1) or TGN (furin) markers. Representative images of individual cells (from ∼15 analyses for each condition) are presented, together with histograms of data from all analyses (Fig. 10).

Bottom Line: At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45.Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites.Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

Show MeSH
Related in: MedlinePlus