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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 in cells following transient expression of PKD-K618N. NRK-52E and MDCK cells were transiently transfected with plasmid encoding GFP-PKD-K618N according to Materials and methods. MDCK cells were subsequently incubated at 19°C for 2 h. Cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bar, 5 μm.
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fig9: Immunofluorescent staining of Sec6 in cells following transient expression of PKD-K618N. NRK-52E and MDCK cells were transiently transfected with plasmid encoding GFP-PKD-K618N according to Materials and methods. MDCK cells were subsequently incubated at 19°C for 2 h. Cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bar, 5 μm.

Mentions: PKD/PKCμ was recently shown to be present in exocytic compartments of the TGN and expression of a kinase-inactive mutant (PKD-K618N) caused tubulation of the Golgi complex and arrested post-Golgi trafficking of VSVG in HeLa cells (Liljedahl et al., 2001). We examined effects of PKD-K618N expression on Sec6/8 complex distribution in NRK-52E cells and MDCK cells. Expression of PKD-K618N caused extensive tubulation in HeLa cells (unpublished data), similar to results reported previously (Liljedahl et al., 2001). When GFP-PKD-K618N was transiently expressed in NRK-52E cells, the mutant kinase colocalized with Sec6 in a perinuclear region, strengthening our conclusion that Sec6/8 complex is associated with a TGN compartment involved in exocytic protein trafficking (Fig. 9). A small amount of GFP-PKD-K618N was also present in peripheral vesicular structures that did not contain Sec6. Importantly, expression of GFP-PKD-K618N in these cells caused an expansion of the TGN and led to a significant increase in the level of Sec6 associated with this organelle compared with nontransfected neighboring cells (Fig. 9). Significantly, transient overexpression of GFP-PKD-K618N, combined with incubation of cells at 19°C for 2 h, resulted in accumulation of a pool of Sec6 in the TGN of MDCK cells (Fig. 9). Note that the low temperature incubation augmented TGN accumulation of Sec6 compared with transfected cells incubated at 37°C (unpublished data), but low temperature incubation alone was not sufficient to cause Sec6 accumulation on the TGN (Fig. 9, nontransfected cells). We conclude that although incubation of cells at 19°C slows exocytic trafficking in MDCK cells, association of Sec6/8 complex with the TGN is dependent on a process regulated by PKD, and that inactivation of this kinase is required for stable TGN binding of Sec6/8 complex in these cells.


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 in cells following transient expression of PKD-K618N. NRK-52E and MDCK cells were transiently transfected with plasmid encoding GFP-PKD-K618N according to Materials and methods. MDCK cells were subsequently incubated at 19°C for 2 h. Cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bar, 5 μm.
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fig9: Immunofluorescent staining of Sec6 in cells following transient expression of PKD-K618N. NRK-52E and MDCK cells were transiently transfected with plasmid encoding GFP-PKD-K618N according to Materials and methods. MDCK cells were subsequently incubated at 19°C for 2 h. Cells were fixed, permeabilized, and stained for Sec6 (mAb 9H5). Bar, 5 μm.
Mentions: PKD/PKCμ was recently shown to be present in exocytic compartments of the TGN and expression of a kinase-inactive mutant (PKD-K618N) caused tubulation of the Golgi complex and arrested post-Golgi trafficking of VSVG in HeLa cells (Liljedahl et al., 2001). We examined effects of PKD-K618N expression on Sec6/8 complex distribution in NRK-52E cells and MDCK cells. Expression of PKD-K618N caused extensive tubulation in HeLa cells (unpublished data), similar to results reported previously (Liljedahl et al., 2001). When GFP-PKD-K618N was transiently expressed in NRK-52E cells, the mutant kinase colocalized with Sec6 in a perinuclear region, strengthening our conclusion that Sec6/8 complex is associated with a TGN compartment involved in exocytic protein trafficking (Fig. 9). A small amount of GFP-PKD-K618N was also present in peripheral vesicular structures that did not contain Sec6. Importantly, expression of GFP-PKD-K618N in these cells caused an expansion of the TGN and led to a significant increase in the level of Sec6 associated with this organelle compared with nontransfected neighboring cells (Fig. 9). Significantly, transient overexpression of GFP-PKD-K618N, combined with incubation of cells at 19°C for 2 h, resulted in accumulation of a pool of Sec6 in the TGN of MDCK cells (Fig. 9). Note that the low temperature incubation augmented TGN accumulation of Sec6 compared with transfected cells incubated at 37°C (unpublished data), but low temperature incubation alone was not sufficient to cause Sec6 accumulation on the TGN (Fig. 9, nontransfected cells). We conclude that although incubation of cells at 19°C slows exocytic trafficking in MDCK cells, association of Sec6/8 complex with the TGN is dependent on a process regulated by PKD, and that inactivation of this kinase is required for stable TGN binding of Sec6/8 complex in these cells.

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