The role of Sec3p in secretory vesicle targeting and exocyst complex assembly.
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.
Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.Show MeSH
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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).
Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.