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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 Sec8p. The exocyst was isolated from lysates of wild-type (B and controls in A, C, and D), sec3Δ (A), sec5Δ (C), and exo70Δ (D) mutant strains as described in Materials and methods. Except for the control strains (no tag), all strains express Sec8p with a carboxy-terminal 13myc epitope. For the exocyst isolation, myc epitope–tagged Sec8p was immunoprecipitated with a monoclonal myc antibody. Co-immunoprecipitation of Sec1p, Sec6p, Sec10p, Sec15p, and Ssop was detected by Western blotting. 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. 30% of the immunoprecipitates (IP, right panels) is compared with 1% of the lysates (left panels). Major nonspecific bands in the lysates are marked with an asterisk. Two exposures of the Sec1p Western blot are shown in A.
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fig7: Co-isolation of Sec1p and exocyst subunits with myc epitope tagged Sec8p. The exocyst was isolated from lysates of wild-type (B and controls in A, C, and D), sec3Δ (A), sec5Δ (C), and exo70Δ (D) mutant strains as described in Materials and methods. Except for the control strains (no tag), all strains express Sec8p with a carboxy-terminal 13myc epitope. For the exocyst isolation, myc epitope–tagged Sec8p was immunoprecipitated with a monoclonal myc antibody. Co-immunoprecipitation of Sec1p, Sec6p, Sec10p, Sec15p, and Ssop was detected by Western blotting. 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. 30% of the immunoprecipitates (IP, right panels) is compared with 1% of the lysates (left panels). Major nonspecific bands in the lysates are marked with an asterisk. Two exposures of the Sec1p Western blot are shown in A.

Mentions: Our working model of exocyst function is that the complex assembles to mediate vesicle tethering at the plasma membrane. By this model, the stably assembled exocyst assures that vesicles are tethered to the correct subdomain of the plasma membrane to allow the vesicles to undergo membrane fusion. Several of the temperature-sensitive exocyst mutants form a less stable complex or are missing specific subunits from the complex (TerBush and Novick, 1995). We analyzed the assembly state of the exocyst in the deletion mutants. For this purpose endogenous Sec8p was myc epitope tagged and isolated from different mutant backgrounds. In the absence of Sec3p, Sec5p, or Exo70p there was a clear reduction in the yield of exocyst subunits that were coprecipitated with Sec8myc (Fig. 7). A large fraction of the exocyst complex (50–80%) was isolated by immunoprecipitation of Sec8myc from a wild-type strain (Fig. 7 A, lane 4). Although similar amounts of Sec8myc were isolated from a sec3Δ strain, only 2–6% of Sec6p, Sec10p, or Sec15p was co-isolated (Fig. 7 A, lane 5). No background of exocyst subunits was observed when the isolation was conducted in parallel from an untagged control strain (Fig. 7 A, lane 3). These results demonstrate that in a sec3Δ strain only a small fraction of the exocyst is assembled and sufficiently stable to be isolated by immunoprecipitation. Therefore, Sec3p is important for exocyst assembly or stability. Overproduction of Sec1p or Sec4p improves secretion in a sec3Δ strain, but has no effect on the coprecipitation of the other exocyst subunits with Sec8myc (Fig. 7 A, lanes 6 and 7). Similar effects on exocyst assembly state were observed with the sec5Δ and exo70Δ mutants (Fig. 7, C and D). Only 2–6% of Sec6p, Sec10p, and Sec15p was co-isolated with Sec8myc from these strains, regardless of the suppressing plasmid (Fig. 7 C, lanes 6 and 12; Fig. 7 D, lane 6). Overproduction of Sec1p or Sec4p in a wild-type background had no effect on exocyst isolation (Fig. 7 C, lanes 5 and 11).


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 Sec8p. The exocyst was isolated from lysates of wild-type (B and controls in A, C, and D), sec3Δ (A), sec5Δ (C), and exo70Δ (D) mutant strains as described in Materials and methods. Except for the control strains (no tag), all strains express Sec8p with a carboxy-terminal 13myc epitope. For the exocyst isolation, myc epitope–tagged Sec8p was immunoprecipitated with a monoclonal myc antibody. Co-immunoprecipitation of Sec1p, Sec6p, Sec10p, Sec15p, and Ssop was detected by Western blotting. 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. 30% of the immunoprecipitates (IP, right panels) is compared with 1% of the lysates (left panels). Major nonspecific bands in the lysates are marked with an asterisk. Two exposures of the Sec1p Western blot are shown in A.
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fig7: Co-isolation of Sec1p and exocyst subunits with myc epitope tagged Sec8p. The exocyst was isolated from lysates of wild-type (B and controls in A, C, and D), sec3Δ (A), sec5Δ (C), and exo70Δ (D) mutant strains as described in Materials and methods. Except for the control strains (no tag), all strains express Sec8p with a carboxy-terminal 13myc epitope. For the exocyst isolation, myc epitope–tagged Sec8p was immunoprecipitated with a monoclonal myc antibody. Co-immunoprecipitation of Sec1p, Sec6p, Sec10p, Sec15p, and Ssop was detected by Western blotting. 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. 30% of the immunoprecipitates (IP, right panels) is compared with 1% of the lysates (left panels). Major nonspecific bands in the lysates are marked with an asterisk. Two exposures of the Sec1p Western blot are shown in A.
Mentions: Our working model of exocyst function is that the complex assembles to mediate vesicle tethering at the plasma membrane. By this model, the stably assembled exocyst assures that vesicles are tethered to the correct subdomain of the plasma membrane to allow the vesicles to undergo membrane fusion. Several of the temperature-sensitive exocyst mutants form a less stable complex or are missing specific subunits from the complex (TerBush and Novick, 1995). We analyzed the assembly state of the exocyst in the deletion mutants. For this purpose endogenous Sec8p was myc epitope tagged and isolated from different mutant backgrounds. In the absence of Sec3p, Sec5p, or Exo70p there was a clear reduction in the yield of exocyst subunits that were coprecipitated with Sec8myc (Fig. 7). A large fraction of the exocyst complex (50–80%) was isolated by immunoprecipitation of Sec8myc from a wild-type strain (Fig. 7 A, lane 4). Although similar amounts of Sec8myc were isolated from a sec3Δ strain, only 2–6% of Sec6p, Sec10p, or Sec15p was co-isolated (Fig. 7 A, lane 5). No background of exocyst subunits was observed when the isolation was conducted in parallel from an untagged control strain (Fig. 7 A, lane 3). These results demonstrate that in a sec3Δ strain only a small fraction of the exocyst is assembled and sufficiently stable to be isolated by immunoprecipitation. Therefore, Sec3p is important for exocyst assembly or stability. Overproduction of Sec1p or Sec4p improves secretion in a sec3Δ strain, but has no effect on the coprecipitation of the other exocyst subunits with Sec8myc (Fig. 7 A, lanes 6 and 7). Similar effects on exocyst assembly state were observed with the sec5Δ and exo70Δ mutants (Fig. 7, C and D). Only 2–6% of Sec6p, Sec10p, and Sec15p was co-isolated with Sec8myc from these strains, regardless of the suppressing plasmid (Fig. 7 C, lanes 6 and 12; Fig. 7 D, lane 6). Overproduction of Sec1p or Sec4p in a wild-type background had no effect on exocyst isolation (Fig. 7 C, lanes 5 and 11).

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