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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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Sec6/8 complex is associated with membranes in fibroblastic and epithelial-like NRK cells. (A) Immunofluorescent staining of NRK-49F and NRK-52E cells with antibodies to E/P-cadherin, ZO-1, or occludin. Cells were fixed with 4% paraformaldehyde then extracted with 1% Triton X-100. (B) Immunodetection of Sec6, Sec8, and Exo70 in detergent lysates (50 μg/lane) from NRK-49F (F) and NRK-52E (E) cells. Protein standards indicated are myosin (205 kD), β-galactosidase (116 kD), phosphorylase b (97 kD), bovine serum albumin (66 kD), and egg albumin (45 kD). (C) Separation of membrane-bound and cytosolic Sec6/8 complexes from NRK-49F and NRK-52E cells in 30% (wt/vol) iodixanol gradients. Bar, 20 μm.
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fig1: Sec6/8 complex is associated with membranes in fibroblastic and epithelial-like NRK cells. (A) Immunofluorescent staining of NRK-49F and NRK-52E cells with antibodies to E/P-cadherin, ZO-1, or occludin. Cells were fixed with 4% paraformaldehyde then extracted with 1% Triton X-100. (B) Immunodetection of Sec6, Sec8, and Exo70 in detergent lysates (50 μg/lane) from NRK-49F (F) and NRK-52E (E) cells. Protein standards indicated are myosin (205 kD), β-galactosidase (116 kD), phosphorylase b (97 kD), bovine serum albumin (66 kD), and egg albumin (45 kD). (C) Separation of membrane-bound and cytosolic Sec6/8 complexes from NRK-49F and NRK-52E cells in 30% (wt/vol) iodixanol gradients. Bar, 20 μm.

Mentions: Western blot analysis of detergent extracts of fibroblastic normal rat kidney (NRK)*-49F and epithelioid NRK-52E cells (Fig. 1 A) revealed that Sec6, Sec8, and Exo70 are expressed at similar levels in both cell lines (Fig. 1 B) and that most of the Sec6/8 complex was recovered in low-density membrane fractions at the top of iodixanol gradients (Fig. 1 C). Immunofluorescence microscopy of NRK-49F cells fixed before extraction revealed Sec6 staining diffusely distributed throughout the cell, but discontinuous dot-like concentrations of staining were observed along the edges of the plasma membrane between contacting cells (Fig. 2 A). The Sec6-positive dots at the cell periphery were also stained by antibodies to ZO-1, and decorated the ends of parallel bundles of actin filaments stained with rhodamine-phalloidin (Fig. 2 A). In cells that were extracted before fixation, detergent-insoluble protein was localized to spots at membranes between adjacent cells (Fig. 2 A). Bundles of actin filaments appear to terminate in these spots. Note that P-cadherin, α-catenin, ZO-1, and ZO-2 coaccumulated in these Sec6-positive spots (Fig. 2 A).


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)

Sec6/8 complex is associated with membranes in fibroblastic and epithelial-like NRK cells. (A) Immunofluorescent staining of NRK-49F and NRK-52E cells with antibodies to E/P-cadherin, ZO-1, or occludin. Cells were fixed with 4% paraformaldehyde then extracted with 1% Triton X-100. (B) Immunodetection of Sec6, Sec8, and Exo70 in detergent lysates (50 μg/lane) from NRK-49F (F) and NRK-52E (E) cells. Protein standards indicated are myosin (205 kD), β-galactosidase (116 kD), phosphorylase b (97 kD), bovine serum albumin (66 kD), and egg albumin (45 kD). (C) Separation of membrane-bound and cytosolic Sec6/8 complexes from NRK-49F and NRK-52E cells in 30% (wt/vol) iodixanol gradients. Bar, 20 μm.
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Related In: Results  -  Collection

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

fig1: Sec6/8 complex is associated with membranes in fibroblastic and epithelial-like NRK cells. (A) Immunofluorescent staining of NRK-49F and NRK-52E cells with antibodies to E/P-cadherin, ZO-1, or occludin. Cells were fixed with 4% paraformaldehyde then extracted with 1% Triton X-100. (B) Immunodetection of Sec6, Sec8, and Exo70 in detergent lysates (50 μg/lane) from NRK-49F (F) and NRK-52E (E) cells. Protein standards indicated are myosin (205 kD), β-galactosidase (116 kD), phosphorylase b (97 kD), bovine serum albumin (66 kD), and egg albumin (45 kD). (C) Separation of membrane-bound and cytosolic Sec6/8 complexes from NRK-49F and NRK-52E cells in 30% (wt/vol) iodixanol gradients. Bar, 20 μm.
Mentions: Western blot analysis of detergent extracts of fibroblastic normal rat kidney (NRK)*-49F and epithelioid NRK-52E cells (Fig. 1 A) revealed that Sec6, Sec8, and Exo70 are expressed at similar levels in both cell lines (Fig. 1 B) and that most of the Sec6/8 complex was recovered in low-density membrane fractions at the top of iodixanol gradients (Fig. 1 C). Immunofluorescence microscopy of NRK-49F cells fixed before extraction revealed Sec6 staining diffusely distributed throughout the cell, but discontinuous dot-like concentrations of staining were observed along the edges of the plasma membrane between contacting cells (Fig. 2 A). The Sec6-positive dots at the cell periphery were also stained by antibodies to ZO-1, and decorated the ends of parallel bundles of actin filaments stained with rhodamine-phalloidin (Fig. 2 A). In cells that were extracted before fixation, detergent-insoluble protein was localized to spots at membranes between adjacent cells (Fig. 2 A). Bundles of actin filaments appear to terminate in these spots. Note that P-cadherin, α-catenin, ZO-1, and ZO-2 coaccumulated in these Sec6-positive spots (Fig. 2 A).

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