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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 and Sec4p overexpression stimulate growth and secretion of a sec3Δ mutant strain. (A) Wild-type (left) and sec3Δ mutant strains (right) carrying the indicated multi-copy (2μ) plasmids were grown on SC plates for 3 d at 25°C. After the loss of the 2μSEC1 plasmid on the 5FOA plate (bottom left), the previously suppressed sec3Δ strain is slow growing comparable to the strain with the control empty plasmid (top left). (B) Invertase secretion was measured in wild-type, sec3Δ, and sec3Δ strains suppressed by either 2μSEC1 or 2μSEC4. To derepress invertase, the cells were shifted to low glucose (0.1%) containing SC medium at 25°C. After 90 min incubation in this medium, invertase activity was measured. The graph shows the amount of intracellular invertase as a percentage of the total newly synthesized invertase. For each strain the average and SD from four independent experiments are shown.
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fig1: Sec1p and Sec4p overexpression stimulate growth and secretion of a sec3Δ mutant strain. (A) Wild-type (left) and sec3Δ mutant strains (right) carrying the indicated multi-copy (2μ) plasmids were grown on SC plates for 3 d at 25°C. After the loss of the 2μSEC1 plasmid on the 5FOA plate (bottom left), the previously suppressed sec3Δ strain is slow growing comparable to the strain with the control empty plasmid (top left). (B) Invertase secretion was measured in wild-type, sec3Δ, and sec3Δ strains suppressed by either 2μSEC1 or 2μSEC4. To derepress invertase, the cells were shifted to low glucose (0.1%) containing SC medium at 25°C. After 90 min incubation in this medium, invertase activity was measured. The graph shows the amount of intracellular invertase as a percentage of the total newly synthesized invertase. For each strain the average and SD from four independent experiments are shown.

Mentions: Multi-copy plasmids used to overexpress the genes of interest were introduced into a sec3Δ/SEC3 heterozygous diploid strain. The transformants were then sporulated and dissected. After dissection and marker analysis, wild-type and sec3Δ mutant haploids that retained the URA3 based multi-copy plasmid were struck out for single colonies on synthetic complete (SC)-Ura plates at 25°C. Overproduction of Sec1p or Sec4p clearly suppressed the growth defect of sec3Δ cells (Fig. 1 A and Table I). However, the suppressed sec3Δ strains remain temperature-sensitive at 37°C (unpublished data and Table I). As expected, a control strain overproducing Sec3p restored growth at all temperatures. An empty plasmid control (Fig. 1 A) or multi-copy plasmids carrying SEC5 or SEC6, encoding two other subunits of the exocyst, had no effect on sec3Δ growth. Interestingly, the multi-copy SSO2 or SEC9 plasmids also improved sec3Δ growth but less strikingly than either SEC1 or SEC4 (Fig. 1 A). These genetic results show that Sec1p, Sec4p and, to a lesser extent SNAREs, can compensate for the absence of Sec3p from the exocyst complex suggesting a functional connection between the exocyst, Sec1p, Sec4p, and SNARE proteins.


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 and Sec4p overexpression stimulate growth and secretion of a sec3Δ mutant strain. (A) Wild-type (left) and sec3Δ mutant strains (right) carrying the indicated multi-copy (2μ) plasmids were grown on SC plates for 3 d at 25°C. After the loss of the 2μSEC1 plasmid on the 5FOA plate (bottom left), the previously suppressed sec3Δ strain is slow growing comparable to the strain with the control empty plasmid (top left). (B) Invertase secretion was measured in wild-type, sec3Δ, and sec3Δ strains suppressed by either 2μSEC1 or 2μSEC4. To derepress invertase, the cells were shifted to low glucose (0.1%) containing SC medium at 25°C. After 90 min incubation in this medium, invertase activity was measured. The graph shows the amount of intracellular invertase as a percentage of the total newly synthesized invertase. For each strain the average and SD from four independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Sec1p and Sec4p overexpression stimulate growth and secretion of a sec3Δ mutant strain. (A) Wild-type (left) and sec3Δ mutant strains (right) carrying the indicated multi-copy (2μ) plasmids were grown on SC plates for 3 d at 25°C. After the loss of the 2μSEC1 plasmid on the 5FOA plate (bottom left), the previously suppressed sec3Δ strain is slow growing comparable to the strain with the control empty plasmid (top left). (B) Invertase secretion was measured in wild-type, sec3Δ, and sec3Δ strains suppressed by either 2μSEC1 or 2μSEC4. To derepress invertase, the cells were shifted to low glucose (0.1%) containing SC medium at 25°C. After 90 min incubation in this medium, invertase activity was measured. The graph shows the amount of intracellular invertase as a percentage of the total newly synthesized invertase. For each strain the average and SD from four independent experiments are shown.
Mentions: Multi-copy plasmids used to overexpress the genes of interest were introduced into a sec3Δ/SEC3 heterozygous diploid strain. The transformants were then sporulated and dissected. After dissection and marker analysis, wild-type and sec3Δ mutant haploids that retained the URA3 based multi-copy plasmid were struck out for single colonies on synthetic complete (SC)-Ura plates at 25°C. Overproduction of Sec1p or Sec4p clearly suppressed the growth defect of sec3Δ cells (Fig. 1 A and Table I). However, the suppressed sec3Δ strains remain temperature-sensitive at 37°C (unpublished data and Table I). As expected, a control strain overproducing Sec3p restored growth at all temperatures. An empty plasmid control (Fig. 1 A) or multi-copy plasmids carrying SEC5 or SEC6, encoding two other subunits of the exocyst, had no effect on sec3Δ growth. Interestingly, the multi-copy SSO2 or SEC9 plasmids also improved sec3Δ growth but less strikingly than either SEC1 or SEC4 (Fig. 1 A). These genetic results show that Sec1p, Sec4p and, to a lesser extent SNAREs, can compensate for the absence of Sec3p from the exocyst complex suggesting a functional connection between the exocyst, Sec1p, Sec4p, and SNARE proteins.

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