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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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Polarity defects of suppressed sec3Δ strains. (A) Percentage of yeast mother cells with wild-type-like elongated morphology was determined as described in Materials and methods. For each dataset the yeast strains were grown in parallel and DIC images were taken to measure length to width ratios. The average and SD were derived from four independent datasets. At least 50 cells were measured per strain and dataset (total >200 cells per strain). (B) Comparison of morphology after α-factor treatment. The indicated yeast strains of mating type a were incubated in the presence of α-factor for 6 h in SC medium. DIC pictures of representative examples of shmooing yeast cells are shown. Arrowheads point to cells extending aberrant round shmoos typical for sec3Δ cells. (C) To quantitatively compare the shmoos of the different yeast mutants, the curvature of the shmoo tips was measured as indicated with the white circles in B. For each strain 100 shmoos were measured for the analysis. The mean radius of shmoo curvature with a 99% confidence interval is shown. (D) Sec4p localization in yeast buds (top panels) and the corresponding DIC pictures (bottom panels). (E) Sec4p-positive areas in the bud were measured. For each strain the mean values and the 99% confidence interval from 100 measured cells are shown.
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fig2: Polarity defects of suppressed sec3Δ strains. (A) Percentage of yeast mother cells with wild-type-like elongated morphology was determined as described in Materials and methods. For each dataset the yeast strains were grown in parallel and DIC images were taken to measure length to width ratios. The average and SD were derived from four independent datasets. At least 50 cells were measured per strain and dataset (total >200 cells per strain). (B) Comparison of morphology after α-factor treatment. The indicated yeast strains of mating type a were incubated in the presence of α-factor for 6 h in SC medium. DIC pictures of representative examples of shmooing yeast cells are shown. Arrowheads point to cells extending aberrant round shmoos typical for sec3Δ cells. (C) To quantitatively compare the shmoos of the different yeast mutants, the curvature of the shmoo tips was measured as indicated with the white circles in B. For each strain 100 shmoos were measured for the analysis. The mean radius of shmoo curvature with a 99% confidence interval is shown. (D) Sec4p localization in yeast buds (top panels) and the corresponding DIC pictures (bottom panels). (E) Sec4p-positive areas in the bud were measured. For each strain the mean values and the 99% confidence interval from 100 measured cells are shown.

Mentions: In an earlier study we found several phenotypes of sec3Δ cells suggesting a defect in polarized secretion. Although Sec4p is concentrated in a very small area at the bud tip of wild-type cells, it is broadly distributed in the buds of sec3Δ cells. Unlike the elongated wild-type cells, sec3Δ cells are also round and are unable to extend normal mating projections (Wiederkehr et al., 2003). Therefore, we tested whether overproduction of Sec1p or Sec4p, in addition to stimulating secretion, would also restore the polarity of sec3Δ cells. The sec3Δ cells overproducing Sec4p were round and showed defects in mating projection formation similar to sec3Δ cells (Fig. 2, A–C). As Sec4p was overexpressed the Sec4p staining was stronger, but was still distributed broadly in the bud as in sec3Δ cells (Fig. 2, D and E). In a wild-type background, Sec4p overexpression did not significantly affect the focal localization of Sec4p in the bud, although a fraction of the cells expressing very high levels of Sec4p showed additional cytoplasmic Sec4p staining. Surprisingly, overproduction of Sec1p led to a partial restoration of these sec3Δ defects. A much larger fraction of sec3Δ cells overproducing Sec1p were elongated, similar to the morphology of wild-type cells (Fig. 2 A). The sec3Δ cells carrying the SEC1 multi-copy plasmid were also better at forming mating projections than sec3Δ cells, although quite a few cells in the culture still showed aberrant, rounded projections (Fig. 2, B and C). Sec4p localization remained partially delocalized in sec3Δ cells overexpressing Sec1p, but was more restricted at sites of polarized secretion than in the corresponding sec3Δ strain (Fig. 2, D and E). It was surprising to find that overproduction of Sec1p restored secretion to a similar extent as Sec4p, yet unlike Sec4p also partially restored polarity. The final parameter we examined was the inheritance of cortical ER into the yeast bud. The sec3Δ cells extend ER tubules into the bud, but the cortical ER fails to be established in the daughter cells. Overproduction of either Sec1p or Sec4p in the sec3Δ cells failed to completely restore inheritance of the ER into the bud. Although most small buds still lacked cortical ER, in both cases overproduction did improve ER inheritance, as a significant fraction of the cells were able to establish cortical ER by the time the cells were large budded (Fig. 3, A and B). Tubule number, dynamics, and orientation appeared normal (Table II).


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)

Polarity defects of suppressed sec3Δ strains. (A) Percentage of yeast mother cells with wild-type-like elongated morphology was determined as described in Materials and methods. For each dataset the yeast strains were grown in parallel and DIC images were taken to measure length to width ratios. The average and SD were derived from four independent datasets. At least 50 cells were measured per strain and dataset (total >200 cells per strain). (B) Comparison of morphology after α-factor treatment. The indicated yeast strains of mating type a were incubated in the presence of α-factor for 6 h in SC medium. DIC pictures of representative examples of shmooing yeast cells are shown. Arrowheads point to cells extending aberrant round shmoos typical for sec3Δ cells. (C) To quantitatively compare the shmoos of the different yeast mutants, the curvature of the shmoo tips was measured as indicated with the white circles in B. For each strain 100 shmoos were measured for the analysis. The mean radius of shmoo curvature with a 99% confidence interval is shown. (D) Sec4p localization in yeast buds (top panels) and the corresponding DIC pictures (bottom panels). (E) Sec4p-positive areas in the bud were measured. For each strain the mean values and the 99% confidence interval from 100 measured cells are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172455&req=5

fig2: Polarity defects of suppressed sec3Δ strains. (A) Percentage of yeast mother cells with wild-type-like elongated morphology was determined as described in Materials and methods. For each dataset the yeast strains were grown in parallel and DIC images were taken to measure length to width ratios. The average and SD were derived from four independent datasets. At least 50 cells were measured per strain and dataset (total >200 cells per strain). (B) Comparison of morphology after α-factor treatment. The indicated yeast strains of mating type a were incubated in the presence of α-factor for 6 h in SC medium. DIC pictures of representative examples of shmooing yeast cells are shown. Arrowheads point to cells extending aberrant round shmoos typical for sec3Δ cells. (C) To quantitatively compare the shmoos of the different yeast mutants, the curvature of the shmoo tips was measured as indicated with the white circles in B. For each strain 100 shmoos were measured for the analysis. The mean radius of shmoo curvature with a 99% confidence interval is shown. (D) Sec4p localization in yeast buds (top panels) and the corresponding DIC pictures (bottom panels). (E) Sec4p-positive areas in the bud were measured. For each strain the mean values and the 99% confidence interval from 100 measured cells are shown.
Mentions: In an earlier study we found several phenotypes of sec3Δ cells suggesting a defect in polarized secretion. Although Sec4p is concentrated in a very small area at the bud tip of wild-type cells, it is broadly distributed in the buds of sec3Δ cells. Unlike the elongated wild-type cells, sec3Δ cells are also round and are unable to extend normal mating projections (Wiederkehr et al., 2003). Therefore, we tested whether overproduction of Sec1p or Sec4p, in addition to stimulating secretion, would also restore the polarity of sec3Δ cells. The sec3Δ cells overproducing Sec4p were round and showed defects in mating projection formation similar to sec3Δ cells (Fig. 2, A–C). As Sec4p was overexpressed the Sec4p staining was stronger, but was still distributed broadly in the bud as in sec3Δ cells (Fig. 2, D and E). In a wild-type background, Sec4p overexpression did not significantly affect the focal localization of Sec4p in the bud, although a fraction of the cells expressing very high levels of Sec4p showed additional cytoplasmic Sec4p staining. Surprisingly, overproduction of Sec1p led to a partial restoration of these sec3Δ defects. A much larger fraction of sec3Δ cells overproducing Sec1p were elongated, similar to the morphology of wild-type cells (Fig. 2 A). The sec3Δ cells carrying the SEC1 multi-copy plasmid were also better at forming mating projections than sec3Δ cells, although quite a few cells in the culture still showed aberrant, rounded projections (Fig. 2, B and C). Sec4p localization remained partially delocalized in sec3Δ cells overexpressing Sec1p, but was more restricted at sites of polarized secretion than in the corresponding sec3Δ strain (Fig. 2, D and E). It was surprising to find that overproduction of Sec1p restored secretion to a similar extent as Sec4p, yet unlike Sec4p also partially restored polarity. The final parameter we examined was the inheritance of cortical ER into the yeast bud. The sec3Δ cells extend ER tubules into the bud, but the cortical ER fails to be established in the daughter cells. Overproduction of either Sec1p or Sec4p in the sec3Δ cells failed to completely restore inheritance of the ER into the bud. Although most small buds still lacked cortical ER, in both cases overproduction did improve ER inheritance, as a significant fraction of the cells were able to establish cortical ER by the time the cells were large budded (Fig. 3, A and B). Tubule number, dynamics, and orientation appeared normal (Table II).

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