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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 expression of Sec3p is required for exocyst targeting and assembly at mitochondria. (A) Sec6-GFP and Sec8-GFP can be recruited to mitochondria in cells expressing Tom20-mCherry tagged Sec3C (amino acids [aa] 321–1336) but not Sec3N (aa 1–320). (B) Cells expressing Tom20-mCherry-Sec3p or -Sec3C grew more slowly than cells expressing Tom20-mCherry-Sec3N on a synthetic complete plate at 25°C. (C) Cells expressing Tom20-mCherry-Sec3 or -Sec3C showed accumulation of Bgl2p inside the cells as detected by Western blotting. Adh1p was used as a loading control. Molecular weights (in kilodaltons) are indicated to the left.
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Figure 3: Ectopic expression of Sec3p is required for exocyst targeting and assembly at mitochondria. (A) Sec6-GFP and Sec8-GFP can be recruited to mitochondria in cells expressing Tom20-mCherry tagged Sec3C (amino acids [aa] 321–1336) but not Sec3N (aa 1–320). (B) Cells expressing Tom20-mCherry-Sec3p or -Sec3C grew more slowly than cells expressing Tom20-mCherry-Sec3N on a synthetic complete plate at 25°C. (C) Cells expressing Tom20-mCherry-Sec3 or -Sec3C showed accumulation of Bgl2p inside the cells as detected by Western blotting. Adh1p was used as a loading control. Molecular weights (in kilodaltons) are indicated to the left.

Mentions: Sec3p contains two functional domains. Its N-terminus (amino acids 1–320) interacts with the Rho family of GTPases and PI(4,5)P2, which mediates the polarized localization of Sec3p at the plasma membrane (Guo et al., 2001; Zhang et al., 2001, 2008; Baek et al., 2010). The C-terminal region (amino acids 321–1336) of Sec3p binds to the exocyst subunit Sec5p (Guo et al., 2001). We fused the two domains of Sec3p to Tom20-mCherry (Tom20-mCherry-Sec3N and Tom20-mCherry-Sec3C) and expressed them in cells. Sec6-GFP and Sec8-GFP were recruited to mitochondria in cells carrying Tom20-mCherry-Sec3C but not in cells carrying Tom20-mCherry-Sec3N (Figure 3A). Cells expressing Tom20-mCherry-Sec3N grew similarly to those containing the control plasmid, whereas cells carrying Tom20-mCherry-Sec3C were defective in growth, similar to cells expressing Tom20-mCherry-Sec3p (Figure 3B). Consistent with the growth phenotype, Bgl2p secretion was also defective in these cells (Figure 3C). This result suggests that Sec3 C-terminal region recruits the exocyst subunits to mitochondria for exocyst assembly, which is consistent with the previous Sec3p domain mapping results (Guo et al., 2001; Zhang et al., 2008).


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

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

Ectopic expression of Sec3p is required for exocyst targeting and assembly at mitochondria. (A) Sec6-GFP and Sec8-GFP can be recruited to mitochondria in cells expressing Tom20-mCherry tagged Sec3C (amino acids [aa] 321–1336) but not Sec3N (aa 1–320). (B) Cells expressing Tom20-mCherry-Sec3p or -Sec3C grew more slowly than cells expressing Tom20-mCherry-Sec3N on a synthetic complete plate at 25°C. (C) Cells expressing Tom20-mCherry-Sec3 or -Sec3C showed accumulation of Bgl2p inside the cells as detected by Western blotting. Adh1p was used as a loading control. Molecular weights (in kilodaltons) are indicated to the left.
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Related In: Results  -  Collection

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Figure 3: Ectopic expression of Sec3p is required for exocyst targeting and assembly at mitochondria. (A) Sec6-GFP and Sec8-GFP can be recruited to mitochondria in cells expressing Tom20-mCherry tagged Sec3C (amino acids [aa] 321–1336) but not Sec3N (aa 1–320). (B) Cells expressing Tom20-mCherry-Sec3p or -Sec3C grew more slowly than cells expressing Tom20-mCherry-Sec3N on a synthetic complete plate at 25°C. (C) Cells expressing Tom20-mCherry-Sec3 or -Sec3C showed accumulation of Bgl2p inside the cells as detected by Western blotting. Adh1p was used as a loading control. Molecular weights (in kilodaltons) are indicated to the left.
Mentions: Sec3p contains two functional domains. Its N-terminus (amino acids 1–320) interacts with the Rho family of GTPases and PI(4,5)P2, which mediates the polarized localization of Sec3p at the plasma membrane (Guo et al., 2001; Zhang et al., 2001, 2008; Baek et al., 2010). The C-terminal region (amino acids 321–1336) of Sec3p binds to the exocyst subunit Sec5p (Guo et al., 2001). We fused the two domains of Sec3p to Tom20-mCherry (Tom20-mCherry-Sec3N and Tom20-mCherry-Sec3C) and expressed them in cells. Sec6-GFP and Sec8-GFP were recruited to mitochondria in cells carrying Tom20-mCherry-Sec3C but not in cells carrying Tom20-mCherry-Sec3N (Figure 3A). Cells expressing Tom20-mCherry-Sec3N grew similarly to those containing the control plasmid, whereas cells carrying Tom20-mCherry-Sec3C were defective in growth, similar to cells expressing Tom20-mCherry-Sec3p (Figure 3B). Consistent with the growth phenotype, Bgl2p secretion was also defective in these cells (Figure 3C). This result suggests that Sec3 C-terminal region recruits the exocyst subunits to mitochondria for exocyst assembly, which is consistent with the previous Sec3p domain mapping results (Guo et al., 2001; Zhang et al., 2008).

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