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: 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.
Affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.