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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 targeting of the exocyst to mitochondria and peroxisomes. (A) The Tom20-mCherry and Tom20-mCherry-Sec3p fusions. See Materials and Methods for the construction of the plasmids. (B) Colocalization of Tom20-mCherry and Tom20-mCherry-Sec3p with Cit1-GFP, a marker protein for yeast mitochondria. (C) Sec8-GFP is recruited to mitochondria in cells expressing Tom20-mCherry–tagged exocyst subunits Sec3p, Sec5p, Sec6p, Sec10p, Sec15p, and Exo84p but not Exo70p. (D) Summary of Sec6-GFP and Sec8-GFP localization in cells expressing Tom20-mCherry–tagged exocyst subunits. +, located to mitochondria; –, not located to mitochondria; N/A, not applicable. (E) The exocyst complex is assembled at mitochondria in cells expressing Tom20-2xFLAG-Sec3p. Cells expressing Tom20-2xFLAG was used as a negative control. The exocyst complex was immunoprecipitated using the anti-FLAG antibody and the exocyst subunits (Sec6p, Sec10p, Sec15p, and Exo84p) coimmunoprecipitated with Tom20-2xFLAG-Sec3p. Molecular weights (in kilodaltons) are indicated to the left. (F) The exocyst can be ectopically targeted to peroxisomes. Sec6-GFP and Sec8-GFP were targeted to peroxisomes in cells expressing Pex3N-mCherry-Sec3p but not Pex3N-mCherry.
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Figure 1: Ectopic targeting of the exocyst to mitochondria and peroxisomes. (A) The Tom20-mCherry and Tom20-mCherry-Sec3p fusions. See Materials and Methods for the construction of the plasmids. (B) Colocalization of Tom20-mCherry and Tom20-mCherry-Sec3p with Cit1-GFP, a marker protein for yeast mitochondria. (C) Sec8-GFP is recruited to mitochondria in cells expressing Tom20-mCherry–tagged exocyst subunits Sec3p, Sec5p, Sec6p, Sec10p, Sec15p, and Exo84p but not Exo70p. (D) Summary of Sec6-GFP and Sec8-GFP localization in cells expressing Tom20-mCherry–tagged exocyst subunits. +, located to mitochondria; –, not located to mitochondria; N/A, not applicable. (E) The exocyst complex is assembled at mitochondria in cells expressing Tom20-2xFLAG-Sec3p. Cells expressing Tom20-2xFLAG was used as a negative control. The exocyst complex was immunoprecipitated using the anti-FLAG antibody and the exocyst subunits (Sec6p, Sec10p, Sec15p, and Exo84p) coimmunoprecipitated with Tom20-2xFLAG-Sec3p. Molecular weights (in kilodaltons) are indicated to the left. (F) The exocyst can be ectopically targeted to peroxisomes. Sec6-GFP and Sec8-GFP were targeted to peroxisomes in cells expressing Pex3N-mCherry-Sec3p but not Pex3N-mCherry.

Mentions: The mitochondrial protein Tom20p associates with the outer membrane of mitochondria through its N-terminal transmembrane segment and exposes its C-terminus to the cytosol (Yamamoto et al., 2011). We engineered a Tom20-mCherry fusion in yeast expression vectors (Figure 1A). The Tom20-mCherry fusion protein was correctly targeted to mitochondria as indicated by its colocalization with a GFP-tagged mitochondria marker protein, Cit1p (Figure 1B). Using this vector, we cloned each of the eight exocyst subunits 3′ to the Tom20-mCherry fusion. When expressed in yeast, all of the exocyst fusion proteins were ectopically targeted to mitochondria, as shown by their colocalization with Cit1-GFP (Figure 1B and Supplemental Figure S1). We noticed that mitochondria tended to cluster in cells expressing Tom20-mCherry-exocyst subunits, which will be discussed later.


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 and peroxisomes. (A) The Tom20-mCherry and Tom20-mCherry-Sec3p fusions. See Materials and Methods for the construction of the plasmids. (B) Colocalization of Tom20-mCherry and Tom20-mCherry-Sec3p with Cit1-GFP, a marker protein for yeast mitochondria. (C) Sec8-GFP is recruited to mitochondria in cells expressing Tom20-mCherry–tagged exocyst subunits Sec3p, Sec5p, Sec6p, Sec10p, Sec15p, and Exo84p but not Exo70p. (D) Summary of Sec6-GFP and Sec8-GFP localization in cells expressing Tom20-mCherry–tagged exocyst subunits. +, located to mitochondria; –, not located to mitochondria; N/A, not applicable. (E) The exocyst complex is assembled at mitochondria in cells expressing Tom20-2xFLAG-Sec3p. Cells expressing Tom20-2xFLAG was used as a negative control. The exocyst complex was immunoprecipitated using the anti-FLAG antibody and the exocyst subunits (Sec6p, Sec10p, Sec15p, and Exo84p) coimmunoprecipitated with Tom20-2xFLAG-Sec3p. Molecular weights (in kilodaltons) are indicated to the left. (F) The exocyst can be ectopically targeted to peroxisomes. Sec6-GFP and Sec8-GFP were targeted to peroxisomes in cells expressing Pex3N-mCherry-Sec3p but not Pex3N-mCherry.
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Figure 1: Ectopic targeting of the exocyst to mitochondria and peroxisomes. (A) The Tom20-mCherry and Tom20-mCherry-Sec3p fusions. See Materials and Methods for the construction of the plasmids. (B) Colocalization of Tom20-mCherry and Tom20-mCherry-Sec3p with Cit1-GFP, a marker protein for yeast mitochondria. (C) Sec8-GFP is recruited to mitochondria in cells expressing Tom20-mCherry–tagged exocyst subunits Sec3p, Sec5p, Sec6p, Sec10p, Sec15p, and Exo84p but not Exo70p. (D) Summary of Sec6-GFP and Sec8-GFP localization in cells expressing Tom20-mCherry–tagged exocyst subunits. +, located to mitochondria; –, not located to mitochondria; N/A, not applicable. (E) The exocyst complex is assembled at mitochondria in cells expressing Tom20-2xFLAG-Sec3p. Cells expressing Tom20-2xFLAG was used as a negative control. The exocyst complex was immunoprecipitated using the anti-FLAG antibody and the exocyst subunits (Sec6p, Sec10p, Sec15p, and Exo84p) coimmunoprecipitated with Tom20-2xFLAG-Sec3p. Molecular weights (in kilodaltons) are indicated to the left. (F) The exocyst can be ectopically targeted to peroxisomes. Sec6-GFP and Sec8-GFP were targeted to peroxisomes in cells expressing Pex3N-mCherry-Sec3p but not Pex3N-mCherry.
Mentions: The mitochondrial protein Tom20p associates with the outer membrane of mitochondria through its N-terminal transmembrane segment and exposes its C-terminus to the cytosol (Yamamoto et al., 2011). We engineered a Tom20-mCherry fusion in yeast expression vectors (Figure 1A). The Tom20-mCherry fusion protein was correctly targeted to mitochondria as indicated by its colocalization with a GFP-tagged mitochondria marker protein, Cit1p (Figure 1B). Using this vector, we cloned each of the eight exocyst subunits 3′ to the Tom20-mCherry fusion. When expressed in yeast, all of the exocyst fusion proteins were ectopically targeted to mitochondria, as shown by their colocalization with Cit1-GFP (Figure 1B and Supplemental Figure S1). We noticed that mitochondria tended to cluster in cells expressing Tom20-mCherry-exocyst subunits, which will be discussed later.

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