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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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Cortical ER inheritance in sec5Δ and exo70Δ mutant strains. The appearance of the ER marker Hmg1-GFP in the yeast bud during ER inheritance. (A) Hmg1-GFP fluorescence pictures (left) were taken at different stages of yeast bud growth. The three categories were as described in Fig. 3. DIC pictures of the small budded (top), large budded (middle), and large budded cells with nuclear ER (bottom) are shown on the right. (B) Quantification of the ER inheritance phenotype of suppressed sec5Δ and exo70Δ strains as described in Fig. 3 B.
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fig6: Cortical ER inheritance in sec5Δ and exo70Δ mutant strains. The appearance of the ER marker Hmg1-GFP in the yeast bud during ER inheritance. (A) Hmg1-GFP fluorescence pictures (left) were taken at different stages of yeast bud growth. The three categories were as described in Fig. 3. DIC pictures of the small budded (top), large budded (middle), and large budded cells with nuclear ER (bottom) are shown on the right. (B) Quantification of the ER inheritance phenotype of suppressed sec5Δ and exo70Δ strains as described in Fig. 3 B.

Mentions: Although the exocyst works as a complex in secretion, specific subunits might confer different aspects of exocyst function. Therefore, we tested whether Sec5p and Exo70p, like Sec3p, are required for polarized secretion and ER inheritance. Cells lacking SEC5 have the broad Sec4p distribution and morphology defects observed for the sec3Δ cells (Fig. 5). The sec5Δ mutants also have a severe ER inheritance defect, similar to the sec3Δ strain. At each stage during bud growth, a large fraction of the sec5Δ cells have little or no cortical ER (Fig. 6, A and B), although the number of tubules is equal or higher than in the wild-type cells and tubule dynamics and orientation appear normal (Table II). In summary, Sec5p appears to be as important for polarized secretion and ER inheritance as Sec3p. With regard to its Sec4p localization and morphology phenotypes, the exo70Δ strain overproducing Sec4p is similar to the sec3Δ and the suppressed sec5Δ mutant strains (Fig. 5). However, ER inheritance is only delayed in this mutant strain, as the defect is restricted to small budded cells (Fig. 6, A and B). By the time larger buds have formed, most exo70Δ cells have inherited cortical ER. Tubule number, dynamics, and orientation appear normal (Table II). This distinction from the sec3Δ and sec5Δ mutants is even more striking in an exo70Δ mutant overproducing Sec1p, where ER inheritance is close to normal even in small budded cells (Fig. 6, A and B). In contrast, the sec3Δ and sec5Δ mutant overproducing Sec1p have very dramatic defects in ER inheritance, suggesting that the function of the Exo70p is less directly linked to ER inheritance than Sec3p and Sec5p. In addition, exo70Δ cells overproducing Sec1p are mostly elongated, similar to wild-type yeast cells (Fig. 5 A). Furthermore, the mating projections of the exo70Δ strain overproducing Sec1p are even more pronounced than those of the wild-type cells or wild-type cells overproducing Sec1p. Of all the mutants analyzed here, Sec4p localization was most highly polarized in the exo70Δ 2μSEC1 cells, although compared with wild-type cells, Sec4p was still partially delocalized (Fig. 5, D and E). Sec1p overproduction appears to improve polarized secretion, as in both the sec3Δ and exo70Δ mutant backgrounds Sec1p, but not Sec4p, clearly improves the morphology of the cells. The differences observed for the various strains, especially when overproducing Sec1p, show that Exo70p contributes differently to polarized secretion and ER inheritance than do Sec3p or Sec5p (Table III). In summary, Sec5p and Exo70p carry out essential functions in the exocyst, but their function can be bypassed when secretion is stimulated by the overproduction of either Sec1p or Sec4p.


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)

Cortical ER inheritance in sec5Δ and exo70Δ mutant strains. The appearance of the ER marker Hmg1-GFP in the yeast bud during ER inheritance. (A) Hmg1-GFP fluorescence pictures (left) were taken at different stages of yeast bud growth. The three categories were as described in Fig. 3. DIC pictures of the small budded (top), large budded (middle), and large budded cells with nuclear ER (bottom) are shown on the right. (B) Quantification of the ER inheritance phenotype of suppressed sec5Δ and exo70Δ strains as described in Fig. 3 B.
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fig6: Cortical ER inheritance in sec5Δ and exo70Δ mutant strains. The appearance of the ER marker Hmg1-GFP in the yeast bud during ER inheritance. (A) Hmg1-GFP fluorescence pictures (left) were taken at different stages of yeast bud growth. The three categories were as described in Fig. 3. DIC pictures of the small budded (top), large budded (middle), and large budded cells with nuclear ER (bottom) are shown on the right. (B) Quantification of the ER inheritance phenotype of suppressed sec5Δ and exo70Δ strains as described in Fig. 3 B.
Mentions: Although the exocyst works as a complex in secretion, specific subunits might confer different aspects of exocyst function. Therefore, we tested whether Sec5p and Exo70p, like Sec3p, are required for polarized secretion and ER inheritance. Cells lacking SEC5 have the broad Sec4p distribution and morphology defects observed for the sec3Δ cells (Fig. 5). The sec5Δ mutants also have a severe ER inheritance defect, similar to the sec3Δ strain. At each stage during bud growth, a large fraction of the sec5Δ cells have little or no cortical ER (Fig. 6, A and B), although the number of tubules is equal or higher than in the wild-type cells and tubule dynamics and orientation appear normal (Table II). In summary, Sec5p appears to be as important for polarized secretion and ER inheritance as Sec3p. With regard to its Sec4p localization and morphology phenotypes, the exo70Δ strain overproducing Sec4p is similar to the sec3Δ and the suppressed sec5Δ mutant strains (Fig. 5). However, ER inheritance is only delayed in this mutant strain, as the defect is restricted to small budded cells (Fig. 6, A and B). By the time larger buds have formed, most exo70Δ cells have inherited cortical ER. Tubule number, dynamics, and orientation appear normal (Table II). This distinction from the sec3Δ and sec5Δ mutants is even more striking in an exo70Δ mutant overproducing Sec1p, where ER inheritance is close to normal even in small budded cells (Fig. 6, A and B). In contrast, the sec3Δ and sec5Δ mutant overproducing Sec1p have very dramatic defects in ER inheritance, suggesting that the function of the Exo70p is less directly linked to ER inheritance than Sec3p and Sec5p. In addition, exo70Δ cells overproducing Sec1p are mostly elongated, similar to wild-type yeast cells (Fig. 5 A). Furthermore, the mating projections of the exo70Δ strain overproducing Sec1p are even more pronounced than those of the wild-type cells or wild-type cells overproducing Sec1p. Of all the mutants analyzed here, Sec4p localization was most highly polarized in the exo70Δ 2μSEC1 cells, although compared with wild-type cells, Sec4p was still partially delocalized (Fig. 5, D and E). Sec1p overproduction appears to improve polarized secretion, as in both the sec3Δ and exo70Δ mutant backgrounds Sec1p, but not Sec4p, clearly improves the morphology of the cells. The differences observed for the various strains, especially when overproducing Sec1p, show that Exo70p contributes differently to polarized secretion and ER inheritance than do Sec3p or Sec5p (Table III). In summary, Sec5p and Exo70p carry out essential functions in the exocyst, but their function can be bypassed when secretion is stimulated by the overproduction of either Sec1p or Sec4p.

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