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Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.

Boyd C, Hughes T, Pypaert M, Novick P - J. Cell Biol. (2004)

Bottom Line: We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst.One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability.Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

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Photobleaching recovery experiments on strains containing a Sec8p-GFP fusion protein with various alleles of sec4. (A) Photobleaching recovery rates. Recovery from photobleaching in strain NY2443, which harbors Sec8p-GFP and the wild-type SEC4 allele, had a τ of 23 ± 4.5 s. Compared with the wild-type background, recovery of the Sec8-GFP fusion was much slower in a strain harboring the sec4-8 allele (NY2518), which had a recovery τ of 56 ± 14 s. Photobleaching recovery was measured in NY2518 at RT, substantially below the restrictive temperature of 30°C. Strain NY2519, which expresses approximately fivefold more Sec4p than NY2443, was also examined and found to have a τ of 22.5 ± 11.5 s. Error bars represent 95% confidence intervals. (B) Western blot of whole-cell extracts from strains NY1210 (wild type) and NY2520 (otherwise wild-type that contains the integrating Sec4p overexpression vector pNB170), showing that Sec4p is overproduced approximately fivefold in NY2520.
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fig7: Photobleaching recovery experiments on strains containing a Sec8p-GFP fusion protein with various alleles of sec4. (A) Photobleaching recovery rates. Recovery from photobleaching in strain NY2443, which harbors Sec8p-GFP and the wild-type SEC4 allele, had a τ of 23 ± 4.5 s. Compared with the wild-type background, recovery of the Sec8-GFP fusion was much slower in a strain harboring the sec4-8 allele (NY2518), which had a recovery τ of 56 ± 14 s. Photobleaching recovery was measured in NY2518 at RT, substantially below the restrictive temperature of 30°C. Strain NY2519, which expresses approximately fivefold more Sec4p than NY2443, was also examined and found to have a τ of 22.5 ± 11.5 s. Error bars represent 95% confidence intervals. (B) Western blot of whole-cell extracts from strains NY1210 (wild type) and NY2520 (otherwise wild-type that contains the integrating Sec4p overexpression vector pNB170), showing that Sec4p is overproduced approximately fivefold in NY2520.

Mentions: To verify that FRAP analysis can measure nonactin-mediated perturbations of the secretory system, we tested the effect of a temperature-sensitive sec4 mutation on the photobleach recovery rate of a Sec8p-GFP fusion. In a wild-type background, Sec8p-GFP recovers with a τ of ∼20 s, but when the Sec8p-GFP recovery rate was measured in a strain harboring the sec4-8 allele the result was quite different. This sec4 allele (G147D) has a reduced affinity for GTP, grows more slowly than wild-type at permissive temperatures, and is lethal at the restrictive temperature (Walworth et al., 1989). Fig. 7 A shows that at the permissive temperature we recorded a bleaching recovery rate for Sec8p-GFP of τ = 56 ± 14 s. No measurement at the restrictive temperature of the recovery rate of the Sec8p-GFP fusion was possible due to complications from heat shock and because bud tip localization of the Sec8p-GFP fusion was too infrequent at the restrictive temperature to bleach a representative sample. To test whether or not Sec4p function might be the rate-limiting step in recovery from photobleaching, we overexpressed Sec4p by a factor of 5–10-fold (Fig. 7 B). Under these conditions, there was no change from wild type in the rate of Sec8p-GFP recovery, indicating that under these conditions Sec4p is not the rate-limiting factor in the photobleaching recovery rate.


Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.

Boyd C, Hughes T, Pypaert M, Novick P - J. Cell Biol. (2004)

Photobleaching recovery experiments on strains containing a Sec8p-GFP fusion protein with various alleles of sec4. (A) Photobleaching recovery rates. Recovery from photobleaching in strain NY2443, which harbors Sec8p-GFP and the wild-type SEC4 allele, had a τ of 23 ± 4.5 s. Compared with the wild-type background, recovery of the Sec8-GFP fusion was much slower in a strain harboring the sec4-8 allele (NY2518), which had a recovery τ of 56 ± 14 s. Photobleaching recovery was measured in NY2518 at RT, substantially below the restrictive temperature of 30°C. Strain NY2519, which expresses approximately fivefold more Sec4p than NY2443, was also examined and found to have a τ of 22.5 ± 11.5 s. Error bars represent 95% confidence intervals. (B) Western blot of whole-cell extracts from strains NY1210 (wild type) and NY2520 (otherwise wild-type that contains the integrating Sec4p overexpression vector pNB170), showing that Sec4p is overproduced approximately fivefold in NY2520.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172445&req=5

fig7: Photobleaching recovery experiments on strains containing a Sec8p-GFP fusion protein with various alleles of sec4. (A) Photobleaching recovery rates. Recovery from photobleaching in strain NY2443, which harbors Sec8p-GFP and the wild-type SEC4 allele, had a τ of 23 ± 4.5 s. Compared with the wild-type background, recovery of the Sec8-GFP fusion was much slower in a strain harboring the sec4-8 allele (NY2518), which had a recovery τ of 56 ± 14 s. Photobleaching recovery was measured in NY2518 at RT, substantially below the restrictive temperature of 30°C. Strain NY2519, which expresses approximately fivefold more Sec4p than NY2443, was also examined and found to have a τ of 22.5 ± 11.5 s. Error bars represent 95% confidence intervals. (B) Western blot of whole-cell extracts from strains NY1210 (wild type) and NY2520 (otherwise wild-type that contains the integrating Sec4p overexpression vector pNB170), showing that Sec4p is overproduced approximately fivefold in NY2520.
Mentions: To verify that FRAP analysis can measure nonactin-mediated perturbations of the secretory system, we tested the effect of a temperature-sensitive sec4 mutation on the photobleach recovery rate of a Sec8p-GFP fusion. In a wild-type background, Sec8p-GFP recovers with a τ of ∼20 s, but when the Sec8p-GFP recovery rate was measured in a strain harboring the sec4-8 allele the result was quite different. This sec4 allele (G147D) has a reduced affinity for GTP, grows more slowly than wild-type at permissive temperatures, and is lethal at the restrictive temperature (Walworth et al., 1989). Fig. 7 A shows that at the permissive temperature we recorded a bleaching recovery rate for Sec8p-GFP of τ = 56 ± 14 s. No measurement at the restrictive temperature of the recovery rate of the Sec8p-GFP fusion was possible due to complications from heat shock and because bud tip localization of the Sec8p-GFP fusion was too infrequent at the restrictive temperature to bleach a representative sample. To test whether or not Sec4p function might be the rate-limiting step in recovery from photobleaching, we overexpressed Sec4p by a factor of 5–10-fold (Fig. 7 B). Under these conditions, there was no change from wild type in the rate of Sec8p-GFP recovery, indicating that under these conditions Sec4p is not the rate-limiting factor in the photobleaching recovery rate.

Bottom Line: We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst.One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability.Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

Show MeSH
Related in: MedlinePlus