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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 or Sec4p overproduction can bypass a deletion of the essential exocyst genes SEC5 and EXO70. (A) Wild-type, sec5Δ, and exo70Δ strains were struck for single colonies on either SC or SC plates containing 5FOA (to select against the plasmid) as indicated. The growth of sec5Δ mutants (left plates) and exo70Δ (right plates) in the presence (SC plates) or absence of the plasmid (5FOA plates) was directly compared with the growth of a corresponding wild-type strain. On each plate the mutant strains are shown on the right. (B) Invertase secretion defects of suppressed sec5Δ and exo70Δ strains were measured as described in Fig. 1 B.
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fig4: Sec1p or Sec4p overproduction can bypass a deletion of the essential exocyst genes SEC5 and EXO70. (A) Wild-type, sec5Δ, and exo70Δ strains were struck for single colonies on either SC or SC plates containing 5FOA (to select against the plasmid) as indicated. The growth of sec5Δ mutants (left plates) and exo70Δ (right plates) in the presence (SC plates) or absence of the plasmid (5FOA plates) was directly compared with the growth of a corresponding wild-type strain. On each plate the mutant strains are shown on the right. (B) Invertase secretion defects of suppressed sec5Δ and exo70Δ strains were measured as described in Fig. 1 B.

Mentions: Given the efficient suppression of the secretion defect of a sec3Δ mutant, we determined if overproduction of Sec1p or Sec4p could bypass the requirement for any other exocyst subunits. Dissection of sec6Δ/SEC6, sec8Δ/SEC8, sec10Δ/SEC10, sec15Δ/SEC15, or exo84Δ/EXO84 heterozygous diploids strains overproducing either Sec1p or Sec4p did not result in any viable haploid strains disrupted for these exocyst genes. However, dissection of sec5Δ/SEC5 and exo70Δ/EXO70 strains gave rise to viable haploid sec5Δ and exo70Δ strains in the presence of either the SEC1 or the SEC4 multi-copy plasmid (Fig. 4 A). The sec5Δ and exo70Δ strains were strictly dependent on Sec1p or Sec4p overproduction for viability. Tetrads in which the multi-copy plasmids were lost during sporulation only gave rise to two wild-type haploid strains. Furthermore, no sec5Δ and exo70Δ colonies were observed after selection against the URA3 plasmid marker on 5-fluoroorotic acid (5FOA) plates (Fig. 4 A). Only wild-type cells, which do not require the URA3-based plasmids, grew on SC plates containing 5FOA.


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 or Sec4p overproduction can bypass a deletion of the essential exocyst genes SEC5 and EXO70. (A) Wild-type, sec5Δ, and exo70Δ strains were struck for single colonies on either SC or SC plates containing 5FOA (to select against the plasmid) as indicated. The growth of sec5Δ mutants (left plates) and exo70Δ (right plates) in the presence (SC plates) or absence of the plasmid (5FOA plates) was directly compared with the growth of a corresponding wild-type strain. On each plate the mutant strains are shown on the right. (B) Invertase secretion defects of suppressed sec5Δ and exo70Δ strains were measured as described in Fig. 1 B.
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Related In: Results  -  Collection

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fig4: Sec1p or Sec4p overproduction can bypass a deletion of the essential exocyst genes SEC5 and EXO70. (A) Wild-type, sec5Δ, and exo70Δ strains were struck for single colonies on either SC or SC plates containing 5FOA (to select against the plasmid) as indicated. The growth of sec5Δ mutants (left plates) and exo70Δ (right plates) in the presence (SC plates) or absence of the plasmid (5FOA plates) was directly compared with the growth of a corresponding wild-type strain. On each plate the mutant strains are shown on the right. (B) Invertase secretion defects of suppressed sec5Δ and exo70Δ strains were measured as described in Fig. 1 B.
Mentions: Given the efficient suppression of the secretion defect of a sec3Δ mutant, we determined if overproduction of Sec1p or Sec4p could bypass the requirement for any other exocyst subunits. Dissection of sec6Δ/SEC6, sec8Δ/SEC8, sec10Δ/SEC10, sec15Δ/SEC15, or exo84Δ/EXO84 heterozygous diploids strains overproducing either Sec1p or Sec4p did not result in any viable haploid strains disrupted for these exocyst genes. However, dissection of sec5Δ/SEC5 and exo70Δ/EXO70 strains gave rise to viable haploid sec5Δ and exo70Δ strains in the presence of either the SEC1 or the SEC4 multi-copy plasmid (Fig. 4 A). The sec5Δ and exo70Δ strains were strictly dependent on Sec1p or Sec4p overproduction for viability. Tetrads in which the multi-copy plasmids were lost during sporulation only gave rise to two wild-type haploid strains. Furthermore, no sec5Δ and exo70Δ colonies were observed after selection against the URA3 plasmid marker on 5-fluoroorotic acid (5FOA) plates (Fig. 4 A). Only wild-type cells, which do not require the URA3-based plasmids, grew on SC plates containing 5FOA.

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