Limits...
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.

Show MeSH

Related in: MedlinePlus

Ectopic targeting of the exocyst to mitochondria led to secretion defects. (A) Growth of cells expressing Tom20-mCherry–tagged exocyst subunits. The same amount of yeast cells was serially diluted at 1:10 ratio and then spotted on SC plates at 25°C. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits grew more slowly than control cells expressing Tom20-mCherry. (B) The growth defect of Tom20-mCherry-Sec3 cells can be suppressed by high-copy 2μ plasmids expressing either SEC1 or SEC4 gene. (C) Bgl2p secretion in cells expressing Tom20-mCherry–tagged exocyst subunits. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits showed accumulation of Bgl2p in cells (Internal) detected by Western blotting. Molecular weights (in kilodaltons) are indicated to the left. Alcohol dehydrogenase (Adh1p) was used as a loading control. (D) Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits were defective in invertase secretion at 25°C. The percentage of external (secreted) relative to the total invertase is presented. Error bars represent SD (n = 3). *p < 0.01 as compared with cells expressing Tom20-mCherry.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230786&req=5

Figure 2: Ectopic targeting of the exocyst to mitochondria led to secretion defects. (A) Growth of cells expressing Tom20-mCherry–tagged exocyst subunits. The same amount of yeast cells was serially diluted at 1:10 ratio and then spotted on SC plates at 25°C. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits grew more slowly than control cells expressing Tom20-mCherry. (B) The growth defect of Tom20-mCherry-Sec3 cells can be suppressed by high-copy 2μ plasmids expressing either SEC1 or SEC4 gene. (C) Bgl2p secretion in cells expressing Tom20-mCherry–tagged exocyst subunits. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits showed accumulation of Bgl2p in cells (Internal) detected by Western blotting. Molecular weights (in kilodaltons) are indicated to the left. Alcohol dehydrogenase (Adh1p) was used as a loading control. (D) Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits were defective in invertase secretion at 25°C. The percentage of external (secreted) relative to the total invertase is presented. Error bars represent SD (n = 3). *p < 0.01 as compared with cells expressing Tom20-mCherry.

Mentions: The exocyst has been localized to the bud tip in yeast (TerBush and Novick, 1995; Finger et al., 1998; Guo et al., 1999a; Zajac et al., 2005). Using our newly developed assay, we tested whether mistargeting of the exocyst to mitochondria affected cell growth and exocytosis. First, growth of cells carrying different Tom20-mCherry-exocyst subunits was examined on plates using the standard yeast colony assay. As shown in Figure 2A, except for Exo70p and the Tom20-mCherry vector control, growth was inhibited in cells expressing the Tom20-mCherry-exocyst subunits. The growth defects of these cells could be rescued by the overexpression of SEC1 (encodes a SNARE regulator) and SEC4 (encodes the Rab small GTPase) using 2μ plasmids (Figure 2B). Because SEC1 and SEC4 have often been used to rescue secretion defects of the exocyst mutants, our results suggest that secretion in the exocyst mistargeted cells was affected. Next, we examined the secretion of 1,3-β-glucosyltransferase (Bgl2p), a cell wall–remodeling enzyme commonly used to assay for yeast exocytosis. As shown in Figure 2C, Bgl2p secretion was defective in cells carrying the Tom20-mCherry-exocyst plasmids (except for Exo70p). Similar to Bgl2p, secretion of another cargo protein, invertase, was also inhibited in these cells (Figure 2D).


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

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

Ectopic targeting of the exocyst to mitochondria led to secretion defects. (A) Growth of cells expressing Tom20-mCherry–tagged exocyst subunits. The same amount of yeast cells was serially diluted at 1:10 ratio and then spotted on SC plates at 25°C. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits grew more slowly than control cells expressing Tom20-mCherry. (B) The growth defect of Tom20-mCherry-Sec3 cells can be suppressed by high-copy 2μ plasmids expressing either SEC1 or SEC4 gene. (C) Bgl2p secretion in cells expressing Tom20-mCherry–tagged exocyst subunits. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits showed accumulation of Bgl2p in cells (Internal) detected by Western blotting. Molecular weights (in kilodaltons) are indicated to the left. Alcohol dehydrogenase (Adh1p) was used as a loading control. (D) Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits were defective in invertase secretion at 25°C. The percentage of external (secreted) relative to the total invertase is presented. Error bars represent SD (n = 3). *p < 0.01 as compared with cells expressing Tom20-mCherry.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230786&req=5

Figure 2: Ectopic targeting of the exocyst to mitochondria led to secretion defects. (A) Growth of cells expressing Tom20-mCherry–tagged exocyst subunits. The same amount of yeast cells was serially diluted at 1:10 ratio and then spotted on SC plates at 25°C. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits grew more slowly than control cells expressing Tom20-mCherry. (B) The growth defect of Tom20-mCherry-Sec3 cells can be suppressed by high-copy 2μ plasmids expressing either SEC1 or SEC4 gene. (C) Bgl2p secretion in cells expressing Tom20-mCherry–tagged exocyst subunits. Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits showed accumulation of Bgl2p in cells (Internal) detected by Western blotting. Molecular weights (in kilodaltons) are indicated to the left. Alcohol dehydrogenase (Adh1p) was used as a loading control. (D) Except for Tom20-mCherry-Exo70, yeast cells expressing Tom20-mCherry–tagged exocyst subunits were defective in invertase secretion at 25°C. The percentage of external (secreted) relative to the total invertase is presented. Error bars represent SD (n = 3). *p < 0.01 as compared with cells expressing Tom20-mCherry.
Mentions: The exocyst has been localized to the bud tip in yeast (TerBush and Novick, 1995; Finger et al., 1998; Guo et al., 1999a; Zajac et al., 2005). Using our newly developed assay, we tested whether mistargeting of the exocyst to mitochondria affected cell growth and exocytosis. First, growth of cells carrying different Tom20-mCherry-exocyst subunits was examined on plates using the standard yeast colony assay. As shown in Figure 2A, except for Exo70p and the Tom20-mCherry vector control, growth was inhibited in cells expressing the Tom20-mCherry-exocyst subunits. The growth defects of these cells could be rescued by the overexpression of SEC1 (encodes a SNARE regulator) and SEC4 (encodes the Rab small GTPase) using 2μ plasmids (Figure 2B). Because SEC1 and SEC4 have often been used to rescue secretion defects of the exocyst mutants, our results suggest that secretion in the exocyst mistargeted cells was affected. Next, we examined the secretion of 1,3-β-glucosyltransferase (Bgl2p), a cell wall–remodeling enzyme commonly used to assay for yeast exocytosis. As shown in Figure 2C, Bgl2p secretion was defective in cells carrying the Tom20-mCherry-exocyst plasmids (except for Exo70p). Similar to Bgl2p, secretion of another cargo protein, invertase, was also inhibited in these cells (Figure 2D).

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