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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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Sec1p overexpression increases SNARE complex levels. SNARE complexes were isolated from cleared yeast lysates by immunoprecipitation using the anti-Snc pAb as described in Materials and methods. The amount of Ssop bound to Sncp was compared by Western blot using a biotinylated polyclonal anti-Sso antibody followed by streptavidin coupled to HRP. The lysates were prepared from wt (A), sec3Δ (B), sec5Δ (C), or exo70Δ (D) strains carrying either a multi-copy 2μSEC1 or 2μSEC4 plasmid. In each panel a wild-type control without plasmid is included. The SNARE complex isolations grouped in each panel were performed in parallel. 30% of the immunoprecipitations (IP) and 1% lysate controls were run on 12% SDS-PAGE gels.
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fig9: Sec1p overexpression increases SNARE complex levels. SNARE complexes were isolated from cleared yeast lysates by immunoprecipitation using the anti-Snc pAb as described in Materials and methods. The amount of Ssop bound to Sncp was compared by Western blot using a biotinylated polyclonal anti-Sso antibody followed by streptavidin coupled to HRP. The lysates were prepared from wt (A), sec3Δ (B), sec5Δ (C), or exo70Δ (D) strains carrying either a multi-copy 2μSEC1 or 2μSEC4 plasmid. In each panel a wild-type control without plasmid is included. The SNARE complex isolations grouped in each panel were performed in parallel. 30% of the immunoprecipitations (IP) and 1% lysate controls were run on 12% SDS-PAGE gels.

Mentions: Members of the SM family bind to SNARE proteins and may regulate SNARE complex assembly, stability, or function (Kosodo et al., 2002; Peng and Gallwitz, 2002; Toonen and Verhage, 2003; Scott et al., 2004). Ongoing membrane traffic is essential for SNARE complex formation, and temperature-sensitive sec4 and exocyst mutants lead to the rapid loss of exocytic SNARE complexes after a shift to the restrictive temperature (Carr et al., 1999; Grote et al., 2000). Therefore, we tested how SNARE complex levels are affected by the absence of Sec3p, Sec5p, or Exo70p, as well as by Sec1p or Sec4p overproduction. For this analysis, steady-state levels of SNARE complexes were measured in the different mutants (Fig. 9). The SNARE complexes were isolated using an antibody against the v-SNARE Snc, and the relative amount of Sso in the immunoprecipitates was determined. Consistent with our earlier results, ∼1% of Sso was co-isolated with Snc from a wild-type strain (Grote et al., 2000). Upstream inhibition of membrane traffic and the concomitant slowed formation of SNARE complexes leads to a decrease in steady-state levels as Sec18p-mediated disassembly of SNARE complexes continues. As secretory function is only partially affected in the sec3Δ mutant, SNARE complex levels were only reduced in this strain (Fig. 9 B, lane 6). In a sec3Δ strain about half as much Sso was isolated in SNARE complexes together with Snc (40% ± 10; n = 5), compared with a wild-type strain. Consistent with its ability to restore secretion, Sec4p overproduction also restored the amount of SNARE complexes that can be isolated from a sec3Δ mutant background (Fig. 9 B, lane 8).


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)

Sec1p overexpression increases SNARE complex levels. SNARE complexes were isolated from cleared yeast lysates by immunoprecipitation using the anti-Snc pAb as described in Materials and methods. The amount of Ssop bound to Sncp was compared by Western blot using a biotinylated polyclonal anti-Sso antibody followed by streptavidin coupled to HRP. The lysates were prepared from wt (A), sec3Δ (B), sec5Δ (C), or exo70Δ (D) strains carrying either a multi-copy 2μSEC1 or 2μSEC4 plasmid. In each panel a wild-type control without plasmid is included. The SNARE complex isolations grouped in each panel were performed in parallel. 30% of the immunoprecipitations (IP) and 1% lysate controls were run on 12% SDS-PAGE gels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172455&req=5

fig9: Sec1p overexpression increases SNARE complex levels. SNARE complexes were isolated from cleared yeast lysates by immunoprecipitation using the anti-Snc pAb as described in Materials and methods. The amount of Ssop bound to Sncp was compared by Western blot using a biotinylated polyclonal anti-Sso antibody followed by streptavidin coupled to HRP. The lysates were prepared from wt (A), sec3Δ (B), sec5Δ (C), or exo70Δ (D) strains carrying either a multi-copy 2μSEC1 or 2μSEC4 plasmid. In each panel a wild-type control without plasmid is included. The SNARE complex isolations grouped in each panel were performed in parallel. 30% of the immunoprecipitations (IP) and 1% lysate controls were run on 12% SDS-PAGE gels.
Mentions: Members of the SM family bind to SNARE proteins and may regulate SNARE complex assembly, stability, or function (Kosodo et al., 2002; Peng and Gallwitz, 2002; Toonen and Verhage, 2003; Scott et al., 2004). Ongoing membrane traffic is essential for SNARE complex formation, and temperature-sensitive sec4 and exocyst mutants lead to the rapid loss of exocytic SNARE complexes after a shift to the restrictive temperature (Carr et al., 1999; Grote et al., 2000). Therefore, we tested how SNARE complex levels are affected by the absence of Sec3p, Sec5p, or Exo70p, as well as by Sec1p or Sec4p overproduction. For this analysis, steady-state levels of SNARE complexes were measured in the different mutants (Fig. 9). The SNARE complexes were isolated using an antibody against the v-SNARE Snc, and the relative amount of Sso in the immunoprecipitates was determined. Consistent with our earlier results, ∼1% of Sso was co-isolated with Snc from a wild-type strain (Grote et al., 2000). Upstream inhibition of membrane traffic and the concomitant slowed formation of SNARE complexes leads to a decrease in steady-state levels as Sec18p-mediated disassembly of SNARE complexes continues. As secretory function is only partially affected in the sec3Δ mutant, SNARE complex levels were only reduced in this strain (Fig. 9 B, lane 6). In a sec3Δ strain about half as much Sso was isolated in SNARE complexes together with Snc (40% ± 10; n = 5), compared with a wild-type strain. Consistent with its ability to restore secretion, Sec4p overproduction also restored the amount of SNARE complexes that can be isolated from a sec3Δ mutant background (Fig. 9 B, lane 8).

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