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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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Related in: MedlinePlus

Sec2p and Sec4p control exocyst assembly at the mitochondria in cells expressing Tom20-mCherry-Sec3p. Localization of Sec8-GFP was examined in wild-type, sec1-1, sec2-41, sec4-8, and sec9-4 cells expressing Tom20-mCherry or Tom20-mCherry-Sec3p. In mutant cells expressing Tom20-mCherry, Sec8-GFP was located to the bud at the permissive temperature (25°C) and became diffused in the cytoplasm at the restrictive temperature (37°C). In cells expressing Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria at 25°C. At 37°C, Sec8-GFP remained associated with mitochondria in wild-type, sec1-1, and sec9-4 cells but diffused in sec2-41 and sec4-8 cells.
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Figure 5: Sec2p and Sec4p control exocyst assembly at the mitochondria in cells expressing Tom20-mCherry-Sec3p. Localization of Sec8-GFP was examined in wild-type, sec1-1, sec2-41, sec4-8, and sec9-4 cells expressing Tom20-mCherry or Tom20-mCherry-Sec3p. In mutant cells expressing Tom20-mCherry, Sec8-GFP was located to the bud at the permissive temperature (25°C) and became diffused in the cytoplasm at the restrictive temperature (37°C). In cells expressing Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria at 25°C. At 37°C, Sec8-GFP remained associated with mitochondria in wild-type, sec1-1, and sec9-4 cells but diffused in sec2-41 and sec4-8 cells.

Mentions: Using the ectopic targeting assay, we examined exocyst assembly to Sec3p on mitochondria in a number of post-Golgi secretory pathway mutants. Sec8-GFP was expressed in wild-type, sec2-41, sec4-8, sec1-1, and sec9-4 cells carrying either Tom20-mCherry (as a vector control) or Tom20-mCherry-Sec3p. As shown in Figure 5, in cells carrying Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria in all strains at the permissive temperature (25°C). At the restrictive temperature (37°C), Sec8-GFP failed to be recruited to mitochondria in the sec2-41 and sec4-8 cells. Because Sec2p is the guanine nucleotide exchange factor of Sec4p, this result further supports the role of Sec4p in regulating exocyst assembly and targeting. The mitochondria targeting of the Sec8-GFP in sec1-1 and sec9-4 strains expressing Tom20-mCherry-Sec3p was unaffected, which is consistent with the idea that Sec1p and SNARE proteins function downstream of the exocyst (Grote et al., 2000).


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

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

Sec2p and Sec4p control exocyst assembly at the mitochondria in cells expressing Tom20-mCherry-Sec3p. Localization of Sec8-GFP was examined in wild-type, sec1-1, sec2-41, sec4-8, and sec9-4 cells expressing Tom20-mCherry or Tom20-mCherry-Sec3p. In mutant cells expressing Tom20-mCherry, Sec8-GFP was located to the bud at the permissive temperature (25°C) and became diffused in the cytoplasm at the restrictive temperature (37°C). In cells expressing Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria at 25°C. At 37°C, Sec8-GFP remained associated with mitochondria in wild-type, sec1-1, and sec9-4 cells but diffused in sec2-41 and sec4-8 cells.
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Related In: Results  -  Collection

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Figure 5: Sec2p and Sec4p control exocyst assembly at the mitochondria in cells expressing Tom20-mCherry-Sec3p. Localization of Sec8-GFP was examined in wild-type, sec1-1, sec2-41, sec4-8, and sec9-4 cells expressing Tom20-mCherry or Tom20-mCherry-Sec3p. In mutant cells expressing Tom20-mCherry, Sec8-GFP was located to the bud at the permissive temperature (25°C) and became diffused in the cytoplasm at the restrictive temperature (37°C). In cells expressing Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria at 25°C. At 37°C, Sec8-GFP remained associated with mitochondria in wild-type, sec1-1, and sec9-4 cells but diffused in sec2-41 and sec4-8 cells.
Mentions: Using the ectopic targeting assay, we examined exocyst assembly to Sec3p on mitochondria in a number of post-Golgi secretory pathway mutants. Sec8-GFP was expressed in wild-type, sec2-41, sec4-8, sec1-1, and sec9-4 cells carrying either Tom20-mCherry (as a vector control) or Tom20-mCherry-Sec3p. As shown in Figure 5, in cells carrying Tom20-mCherry-Sec3p, Sec8-GFP was recruited to mitochondria in all strains at the permissive temperature (25°C). At the restrictive temperature (37°C), Sec8-GFP failed to be recruited to mitochondria in the sec2-41 and sec4-8 cells. Because Sec2p is the guanine nucleotide exchange factor of Sec4p, this result further supports the role of Sec4p in regulating exocyst assembly and targeting. The mitochondria targeting of the Sec8-GFP in sec1-1 and sec9-4 strains expressing Tom20-mCherry-Sec3p was unaffected, which is consistent with the idea that Sec1p and SNARE proteins function downstream of the exocyst (Grote et al., 2000).

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