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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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Immunofluorescent staining of Sec6 compared with endocytic and exocytic cargo. (A) NRK-49F cells were labeled with Texas red transferrin as described in Materials and methods. Following internalization for 5 or 30 min, cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). (B) NRK-49F cells were transiently transfected with plasmid encoding ts-G-GFP, and incubated under conditions to arrest this protein in either the ER or TGN, as described in Materials and methods. Cells were then fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bars, (A) 8 μm; (B) 5 μm.
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fig6: Immunofluorescent staining of Sec6 compared with endocytic and exocytic cargo. (A) NRK-49F cells were labeled with Texas red transferrin as described in Materials and methods. Following internalization for 5 or 30 min, cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). (B) NRK-49F cells were transiently transfected with plasmid encoding ts-G-GFP, and incubated under conditions to arrest this protein in either the ER or TGN, as described in Materials and methods. Cells were then fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bars, (A) 8 μm; (B) 5 μm.

Mentions: Additional evidence that Sec6/8 complex is primarily associated with TGN and not endosomes was obtained by determining whether Sec6-positive compartments were accessible to endocytic (Texas red transferrin; Fig. 6 A) or exocytic (green fluorescent protein [GFP]–vesicular stomatitis virus G protein [VSVG] protein; Fig. 6 B) cargo. Internalization of Texas red transferrin for 5 min labeled a population of vesicular structures in the cell periphery that were negative for Sec6 (Fig. 6 A). Following a 30 min (or 60 min; unpublished data) uptake, transferrin labeled a perinuclear tubulovesicular compartment that was in the same general vicinity as membranes containing Sec6/8 complex. However, there was no overlap in the distributions of Sec6 and transferrin.


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)

Immunofluorescent staining of Sec6 compared with endocytic and exocytic cargo. (A) NRK-49F cells were labeled with Texas red transferrin as described in Materials and methods. Following internalization for 5 or 30 min, cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). (B) NRK-49F cells were transiently transfected with plasmid encoding ts-G-GFP, and incubated under conditions to arrest this protein in either the ER or TGN, as described in Materials and methods. Cells were then fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bars, (A) 8 μm; (B) 5 μm.
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Related In: Results  -  Collection

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fig6: Immunofluorescent staining of Sec6 compared with endocytic and exocytic cargo. (A) NRK-49F cells were labeled with Texas red transferrin as described in Materials and methods. Following internalization for 5 or 30 min, cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). (B) NRK-49F cells were transiently transfected with plasmid encoding ts-G-GFP, and incubated under conditions to arrest this protein in either the ER or TGN, as described in Materials and methods. Cells were then fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bars, (A) 8 μm; (B) 5 μm.
Mentions: Additional evidence that Sec6/8 complex is primarily associated with TGN and not endosomes was obtained by determining whether Sec6-positive compartments were accessible to endocytic (Texas red transferrin; Fig. 6 A) or exocytic (green fluorescent protein [GFP]–vesicular stomatitis virus G protein [VSVG] protein; Fig. 6 B) cargo. Internalization of Texas red transferrin for 5 min labeled a population of vesicular structures in the cell periphery that were negative for Sec6 (Fig. 6 A). Following a 30 min (or 60 min; unpublished data) uptake, transferrin labeled a perinuclear tubulovesicular compartment that was in the same general vicinity as membranes containing Sec6/8 complex. However, there was no overlap in the distributions of Sec6 and transferrin.

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