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The role of Sec3p in secretory vesicle targeting and exocyst complex assembly.

Luo G, Zhang J, Guo W - Mol. Biol. Cell (2014)

Bottom Line: We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria.We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells.In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.

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Ectopic recruitment of secretory vesicles to mitochondria. (A) Sec4p was examined by immunofluorescence microscopy using a rabbit polyclonal antibody. In the presence of DMSO, Sec4p was partially recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p, with the rest localized to the bud. After latrunculin B treatment, however, Sec4p signal in the bud diminished. Concomitantly, significantly more Sec4p associated with mitochondria. As a control, in cells expressing Tom20-mCherry, Sec4p was localized to the bud in the presence of DMSO. After treatment with latrunculin B, Sec4p dispersed in the cytosol. (B) Bgl2p was detected by immunofluorescence microscopy using a rabbit polyclonal antibody. In cells expressing Tom20-mCherry, there was very little Blg2p accumulation in the presence of DMSO. After latrunculin B treatment, Bgl2p fluorescence became more detectable and was dispersed in the cytoplasm rather than associated with mitochondria. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in the presence of DMSO. After latrunculin B treatment, brighter Bgl2p signal was detected at mitochondria. (C) Chs3-GFP was recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry. (D) Thin-section electron microscopy analysis of cells expressing Tom20-mCherry, Tom20-mCherry-Sec3p, or Tom20-mCherry-Sec6p. Secretory vesicles (shown in dark staining) associated with mitochondria clustered in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry or Tom20-mCherry-Sec6p. The selected areas are magnified and shown at the bottom. There was no vesicle accumulation in cells expressing Tom20-mCherry. Cells were fixed with permanganate, and the vesicles are shown in dark staining. Scale bar, 1 μm.
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Figure 4: Ectopic recruitment of secretory vesicles to mitochondria. (A) Sec4p was examined by immunofluorescence microscopy using a rabbit polyclonal antibody. In the presence of DMSO, Sec4p was partially recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p, with the rest localized to the bud. After latrunculin B treatment, however, Sec4p signal in the bud diminished. Concomitantly, significantly more Sec4p associated with mitochondria. As a control, in cells expressing Tom20-mCherry, Sec4p was localized to the bud in the presence of DMSO. After treatment with latrunculin B, Sec4p dispersed in the cytosol. (B) Bgl2p was detected by immunofluorescence microscopy using a rabbit polyclonal antibody. In cells expressing Tom20-mCherry, there was very little Blg2p accumulation in the presence of DMSO. After latrunculin B treatment, Bgl2p fluorescence became more detectable and was dispersed in the cytoplasm rather than associated with mitochondria. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in the presence of DMSO. After latrunculin B treatment, brighter Bgl2p signal was detected at mitochondria. (C) Chs3-GFP was recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry. (D) Thin-section electron microscopy analysis of cells expressing Tom20-mCherry, Tom20-mCherry-Sec3p, or Tom20-mCherry-Sec6p. Secretory vesicles (shown in dark staining) associated with mitochondria clustered in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry or Tom20-mCherry-Sec6p. The selected areas are magnified and shown at the bottom. There was no vesicle accumulation in cells expressing Tom20-mCherry. Cells were fixed with permanganate, and the vesicles are shown in dark staining. Scale bar, 1 μm.

Mentions: Using the ectopic targeting assay, we examined whether secretory vesicles were recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p. We first examined vesicles by immunofluorescence using the Rab protein Sec4p as a marker. In the Tom20-mCherry-Sec3p cells, while some Sec4p remained detectable at the bud, a portion of Sec4p colocalized with Tom20-mCherry-Sec3p at the mitochondria in 35.1% of the cells (n = 105; Figure 4A, left). We noted that not all of the Sec4p was recruited to mitochondria, which will be discussed later. As a control, all Sec4p proteins were localized to the bud in cells expressing Tom20-mCherry (>95% of the cells, n = 100). The secretory vesicles were also examined by following the cargo protein, Bgl2p, using immunofluorescence microscopy (Figure 4B, left). There was very little Blg2p detected inside the cells expressing Tom20-mCherry, consistent with the observation that Tom20-mCherry alone does not block secretion in cells shown earlier. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in 88.5% of the cells (n = 139). We also examined the localization of chitin synthase III (Chs3p), a protein required for cell wall remodeling at the mother–daughter junction. It was previously shown that Chs3-GFP was localized to mother–bud junction and some endosomal compartments (Chuang and Schekman, 1996), and the functional exocyst complex is required for its proper localization (Zanolari et al., 2011). As shown in Figure 4C, portions of Chs3-GFP were recruited to mitochondria in 89.7% of the cells (n = 108) expressing Tom20-mCherry-Sec3p, suggesting that Sec3p mediates the targeting of secretory vesicles. The fluorescence microscopy result was further supported by biochemistry experiments in which Sec4p, Bgl2p, and Chs3p were found to associate with mitochondria purified from yeast cells expressing Tom20-mCherry-Sec3p but not cells expressing Tom20-mCherry (Supplemental Figure S3).


The role of Sec3p in secretory vesicle targeting and exocyst complex assembly.

Luo G, Zhang J, Guo W - Mol. Biol. Cell (2014)

Ectopic recruitment of secretory vesicles to mitochondria. (A) Sec4p was examined by immunofluorescence microscopy using a rabbit polyclonal antibody. In the presence of DMSO, Sec4p was partially recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p, with the rest localized to the bud. After latrunculin B treatment, however, Sec4p signal in the bud diminished. Concomitantly, significantly more Sec4p associated with mitochondria. As a control, in cells expressing Tom20-mCherry, Sec4p was localized to the bud in the presence of DMSO. After treatment with latrunculin B, Sec4p dispersed in the cytosol. (B) Bgl2p was detected by immunofluorescence microscopy using a rabbit polyclonal antibody. In cells expressing Tom20-mCherry, there was very little Blg2p accumulation in the presence of DMSO. After latrunculin B treatment, Bgl2p fluorescence became more detectable and was dispersed in the cytoplasm rather than associated with mitochondria. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in the presence of DMSO. After latrunculin B treatment, brighter Bgl2p signal was detected at mitochondria. (C) Chs3-GFP was recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry. (D) Thin-section electron microscopy analysis of cells expressing Tom20-mCherry, Tom20-mCherry-Sec3p, or Tom20-mCherry-Sec6p. Secretory vesicles (shown in dark staining) associated with mitochondria clustered in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry or Tom20-mCherry-Sec6p. The selected areas are magnified and shown at the bottom. There was no vesicle accumulation in cells expressing Tom20-mCherry. Cells were fixed with permanganate, and the vesicles are shown in dark staining. Scale bar, 1 μm.
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Figure 4: Ectopic recruitment of secretory vesicles to mitochondria. (A) Sec4p was examined by immunofluorescence microscopy using a rabbit polyclonal antibody. In the presence of DMSO, Sec4p was partially recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p, with the rest localized to the bud. After latrunculin B treatment, however, Sec4p signal in the bud diminished. Concomitantly, significantly more Sec4p associated with mitochondria. As a control, in cells expressing Tom20-mCherry, Sec4p was localized to the bud in the presence of DMSO. After treatment with latrunculin B, Sec4p dispersed in the cytosol. (B) Bgl2p was detected by immunofluorescence microscopy using a rabbit polyclonal antibody. In cells expressing Tom20-mCherry, there was very little Blg2p accumulation in the presence of DMSO. After latrunculin B treatment, Bgl2p fluorescence became more detectable and was dispersed in the cytoplasm rather than associated with mitochondria. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in the presence of DMSO. After latrunculin B treatment, brighter Bgl2p signal was detected at mitochondria. (C) Chs3-GFP was recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry. (D) Thin-section electron microscopy analysis of cells expressing Tom20-mCherry, Tom20-mCherry-Sec3p, or Tom20-mCherry-Sec6p. Secretory vesicles (shown in dark staining) associated with mitochondria clustered in cells expressing Tom20-mCherry-Sec3p but not in cells expressing Tom20-mCherry or Tom20-mCherry-Sec6p. The selected areas are magnified and shown at the bottom. There was no vesicle accumulation in cells expressing Tom20-mCherry. Cells were fixed with permanganate, and the vesicles are shown in dark staining. Scale bar, 1 μm.
Mentions: Using the ectopic targeting assay, we examined whether secretory vesicles were recruited to mitochondria in cells expressing Tom20-mCherry-Sec3p. We first examined vesicles by immunofluorescence using the Rab protein Sec4p as a marker. In the Tom20-mCherry-Sec3p cells, while some Sec4p remained detectable at the bud, a portion of Sec4p colocalized with Tom20-mCherry-Sec3p at the mitochondria in 35.1% of the cells (n = 105; Figure 4A, left). We noted that not all of the Sec4p was recruited to mitochondria, which will be discussed later. As a control, all Sec4p proteins were localized to the bud in cells expressing Tom20-mCherry (>95% of the cells, n = 100). The secretory vesicles were also examined by following the cargo protein, Bgl2p, using immunofluorescence microscopy (Figure 4B, left). There was very little Blg2p detected inside the cells expressing Tom20-mCherry, consistent with the observation that Tom20-mCherry alone does not block secretion in cells shown earlier. In cells expressing Tom20-mCherry-Sec3p, however, Bgl2p was detectable at the mitochondria in 88.5% of the cells (n = 139). We also examined the localization of chitin synthase III (Chs3p), a protein required for cell wall remodeling at the mother–daughter junction. It was previously shown that Chs3-GFP was localized to mother–bud junction and some endosomal compartments (Chuang and Schekman, 1996), and the functional exocyst complex is required for its proper localization (Zanolari et al., 2011). As shown in Figure 4C, portions of Chs3-GFP were recruited to mitochondria in 89.7% of the cells (n = 108) expressing Tom20-mCherry-Sec3p, suggesting that Sec3p mediates the targeting of secretory vesicles. The fluorescence microscopy result was further supported by biochemistry experiments in which Sec4p, Bgl2p, and Chs3p were found to associate with mitochondria purified from yeast cells expressing Tom20-mCherry-Sec3p but not cells expressing Tom20-mCherry (Supplemental Figure S3).

Bottom Line: We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria.We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells.In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.

Show MeSH
Related in: MedlinePlus