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Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p.

Wiederkehr A, De Craene JO, Ferro-Novick S, Novick P - J. Cell Biol. (2004)

Bottom Line: Sec3p and Sec5p are more critical than Exo70p for ER inheritance.Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled.Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.

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Co-isolation of Sec1p and exocyst subunits with myc epitope–tagged Sec10p. The exocyst was isolated from a wild-type (lanes 6, 7, and 12), sec3Δ (lane 5), sec5Δ (lane 8), and exo70Δ (lane 13) mutant strain via a carboxy-terminal 13myc epitope–tagged Sec10p. Immunoprecipitations were performed as described in Fig. 7. Mutant genotypes (wt, sec3Δ, sec5Δ, or exo70Δ) and the presence of multi-copy plasmids (2μSEC1 or 2μSEC4) are indicated on the top of each lane. 1% of lysates (left lanes) are compared with 30% of the immunoprecipitate (IP, right lanes). The antigens detected by Western blot analysis are marked on the left. Nonspecific bands are marked with an asterisk.
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fig8: Co-isolation of Sec1p and exocyst subunits with myc epitope–tagged Sec10p. The exocyst was isolated from a wild-type (lanes 6, 7, and 12), sec3Δ (lane 5), sec5Δ (lane 8), and exo70Δ (lane 13) mutant strain via a carboxy-terminal 13myc epitope–tagged Sec10p. Immunoprecipitations were performed as described in Fig. 7. Mutant genotypes (wt, sec3Δ, sec5Δ, or exo70Δ) and the presence of multi-copy plasmids (2μSEC1 or 2μSEC4) are indicated on the top of each lane. 1% of lysates (left lanes) are compared with 30% of the immunoprecipitate (IP, right lanes). The antigens detected by Western blot analysis are marked on the left. Nonspecific bands are marked with an asterisk.

Mentions: The above results concerning exocyst assembly in different mutant backgrounds are consistent with two possible interpretations. Either in these mutants Sec8myc binds more weakly to an otherwise fully assembled exocyst, or the absence of Sec3p, Sec5p, or Exo70p has a more global effect on the binding of exocyst subunits to each other. To distinguish between these possibilities, we also isolated the exocyst using a myc tag on Sec10p, another subunit of the exocyst. In a wild-type background, isolation of the exocyst using Sec10myc was similarly efficient as with Sec8myc. A large fraction of Sec6p, Sec8p, and Sec15p was co-isolated with the Sec10myc subunit from a wild-type lysate. However, ∼10 times less Sec6p or Sec8p was co-isolated with Sec10myc from lysates of the different mutant strains (Fig. 8, lanes 5, 8, and 13). The results show that the exocyst is largely unassembled or less stably assembled in these mutant strains. Nonetheless, in all cases Sec10p still efficiently bound Sec15p. The amount of Sec15p in a Sec10myc immunoprecipitation was only slightly reduced in the sec3Δ, sec5Δ, and exo70Δ mutants relative to wild type (Fig. 8, lanes 5, 8, and 13). Therefore, Sec10p and Sec15p form a subcomplex that is little affected by the absence of Sec3p, Sec5p, or Exo70p from the complex. The abundance of Sec6p, Sec8p, Sec10p, and Sec15p in the lysate was not affected in the different mutants (Fig. 7 and Fig. 8). Therefore, the absence of Sec3p, Sec5p, or Exo70p does not result in proteolysis of these other exocyst subunits. The results show that the assembly or stability of the complex is affected in these mutants, although a Sec10p–Sec15p subcomplex and possibly other subcomplexes remain intact.


Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p.

Wiederkehr A, De Craene JO, Ferro-Novick S, Novick P - J. Cell Biol. (2004)

Co-isolation of Sec1p and exocyst subunits with myc epitope–tagged Sec10p. The exocyst was isolated from a wild-type (lanes 6, 7, and 12), sec3Δ (lane 5), sec5Δ (lane 8), and exo70Δ (lane 13) mutant strain via a carboxy-terminal 13myc epitope–tagged Sec10p. Immunoprecipitations were performed as described in Fig. 7. Mutant genotypes (wt, sec3Δ, sec5Δ, or exo70Δ) and the presence of multi-copy plasmids (2μSEC1 or 2μSEC4) are indicated on the top of each lane. 1% of lysates (left lanes) are compared with 30% of the immunoprecipitate (IP, right lanes). The antigens detected by Western blot analysis are marked on the left. Nonspecific bands are marked with an asterisk.
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Related In: Results  -  Collection

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fig8: Co-isolation of Sec1p and exocyst subunits with myc epitope–tagged Sec10p. The exocyst was isolated from a wild-type (lanes 6, 7, and 12), sec3Δ (lane 5), sec5Δ (lane 8), and exo70Δ (lane 13) mutant strain via a carboxy-terminal 13myc epitope–tagged Sec10p. Immunoprecipitations were performed as described in Fig. 7. Mutant genotypes (wt, sec3Δ, sec5Δ, or exo70Δ) and the presence of multi-copy plasmids (2μSEC1 or 2μSEC4) are indicated on the top of each lane. 1% of lysates (left lanes) are compared with 30% of the immunoprecipitate (IP, right lanes). The antigens detected by Western blot analysis are marked on the left. Nonspecific bands are marked with an asterisk.
Mentions: The above results concerning exocyst assembly in different mutant backgrounds are consistent with two possible interpretations. Either in these mutants Sec8myc binds more weakly to an otherwise fully assembled exocyst, or the absence of Sec3p, Sec5p, or Exo70p has a more global effect on the binding of exocyst subunits to each other. To distinguish between these possibilities, we also isolated the exocyst using a myc tag on Sec10p, another subunit of the exocyst. In a wild-type background, isolation of the exocyst using Sec10myc was similarly efficient as with Sec8myc. A large fraction of Sec6p, Sec8p, and Sec15p was co-isolated with the Sec10myc subunit from a wild-type lysate. However, ∼10 times less Sec6p or Sec8p was co-isolated with Sec10myc from lysates of the different mutant strains (Fig. 8, lanes 5, 8, and 13). The results show that the exocyst is largely unassembled or less stably assembled in these mutant strains. Nonetheless, in all cases Sec10p still efficiently bound Sec15p. The amount of Sec15p in a Sec10myc immunoprecipitation was only slightly reduced in the sec3Δ, sec5Δ, and exo70Δ mutants relative to wild type (Fig. 8, lanes 5, 8, and 13). Therefore, Sec10p and Sec15p form a subcomplex that is little affected by the absence of Sec3p, Sec5p, or Exo70p from the complex. The abundance of Sec6p, Sec8p, Sec10p, and Sec15p in the lysate was not affected in the different mutants (Fig. 7 and Fig. 8). Therefore, the absence of Sec3p, Sec5p, or Exo70p does not result in proteolysis of these other exocyst subunits. The results show that the assembly or stability of the complex is affected in these mutants, although a Sec10p–Sec15p subcomplex and possibly other subcomplexes remain intact.

Bottom Line: Sec3p and Sec5p are more critical than Exo70p for ER inheritance.Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled.Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.

Show MeSH
Related in: MedlinePlus