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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 of GFP-tagged exocyst subunits in the presence of Latrunculin A. Cells were grown in SC medium and prepared as described in Materials and methods. (A) Micrographs of strains containing Sec8-GFP and Sec3-GFP (NY2442 and NY2439, respectively) in cells with or without 200 mM Latrunculin A added to the medium. (B) Bud tips in Latrunculin A–treated cells were bleached and monitored for recovery. Sample recovery graphs with DMSO and DMSO + 200 μM Latrunculin A are shown for each exocyst subunit fused to GFP. (C) 20 bleach/recovery procedures for each of the GFP fusion strains were performed, and the frequencies of good (>20%), medium (5 to 20%), or poor (<5%) recovery with and without Latrunculin A present were plotted. (D) In the presence of Latrunculin A, both Sec3p-GFP and Exo70p-GFP displayed recovery kinetics characterized by a single mode of recovery. Sec3p-GFP had a recovery τ of 59 ± 11 s, whereas Exo70p-GFP had a recovery τ of 57 ± 13 s.
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fig3: Photobleaching recovery of GFP-tagged exocyst subunits in the presence of Latrunculin A. Cells were grown in SC medium and prepared as described in Materials and methods. (A) Micrographs of strains containing Sec8-GFP and Sec3-GFP (NY2442 and NY2439, respectively) in cells with or without 200 mM Latrunculin A added to the medium. (B) Bud tips in Latrunculin A–treated cells were bleached and monitored for recovery. Sample recovery graphs with DMSO and DMSO + 200 μM Latrunculin A are shown for each exocyst subunit fused to GFP. (C) 20 bleach/recovery procedures for each of the GFP fusion strains were performed, and the frequencies of good (>20%), medium (5 to 20%), or poor (<5%) recovery with and without Latrunculin A present were plotted. (D) In the presence of Latrunculin A, both Sec3p-GFP and Exo70p-GFP displayed recovery kinetics characterized by a single mode of recovery. Sec3p-GFP had a recovery τ of 59 ± 11 s, whereas Exo70p-GFP had a recovery τ of 57 ± 13 s.

Mentions: Because the recovery times for those subunits that appeared to use the fast mode of recovery were more similar to the recovery time for GFP-Sec4p than the recovery times for Sec3p-GFP or the slow mode for Exo70p-GFP, we decided to investigate whether or not all of the fast-recovering group used the same mechanism of recovery as GFP-Sec4p. Because GFP-Sec4p is present on vesicles that are transported to the bud tip along actin cables, we decided to test if recovery from photobleaching for all exocyst subunits depends on the presence of an intact actin cytoskeleton. As one test of this hypothesis, we treated cells with the actin-depolymerizing agent Latrunculin A. At a concentration of 200 μM, Latrunculin A treatment abolishes actin-based structures within 5 min in yeast (Ayscough et al., 1997). Vesicle transport to the bud tip halts, as shown by the effect on GFP-Sec4p localization, which showed a time-dependent loss of GFP-Sec4p from the bud tip (unpublished data). We examined by FRAP the population of cells that had not yet lost a focus of localized GFP in the time frame from 5 to 30 min after addition of Latrunculin A. We found that recovery of exocyst-GFP fusions was substantially inhibited (Fig. 3). In most cases there was only a small amount of recovery observed, <5%, but based on this low efficiency and the speed of this fraction's recovery (complete within 10 s), it may represent a freely diffusible population of each subunit, though we have no information on the size of this population in Latrunculin A–treated cells. Because we observed little recovery from photobleaching in the presence of Latrunculin A during the 5 to 30 min window after Latrunculin A addition, our conclusion is that delivery of these exocyst subunits to the bud tip depends on actin.


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 of GFP-tagged exocyst subunits in the presence of Latrunculin A. Cells were grown in SC medium and prepared as described in Materials and methods. (A) Micrographs of strains containing Sec8-GFP and Sec3-GFP (NY2442 and NY2439, respectively) in cells with or without 200 mM Latrunculin A added to the medium. (B) Bud tips in Latrunculin A–treated cells were bleached and monitored for recovery. Sample recovery graphs with DMSO and DMSO + 200 μM Latrunculin A are shown for each exocyst subunit fused to GFP. (C) 20 bleach/recovery procedures for each of the GFP fusion strains were performed, and the frequencies of good (>20%), medium (5 to 20%), or poor (<5%) recovery with and without Latrunculin A present were plotted. (D) In the presence of Latrunculin A, both Sec3p-GFP and Exo70p-GFP displayed recovery kinetics characterized by a single mode of recovery. Sec3p-GFP had a recovery τ of 59 ± 11 s, whereas Exo70p-GFP had a recovery τ of 57 ± 13 s.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172445&req=5

fig3: Photobleaching recovery of GFP-tagged exocyst subunits in the presence of Latrunculin A. Cells were grown in SC medium and prepared as described in Materials and methods. (A) Micrographs of strains containing Sec8-GFP and Sec3-GFP (NY2442 and NY2439, respectively) in cells with or without 200 mM Latrunculin A added to the medium. (B) Bud tips in Latrunculin A–treated cells were bleached and monitored for recovery. Sample recovery graphs with DMSO and DMSO + 200 μM Latrunculin A are shown for each exocyst subunit fused to GFP. (C) 20 bleach/recovery procedures for each of the GFP fusion strains were performed, and the frequencies of good (>20%), medium (5 to 20%), or poor (<5%) recovery with and without Latrunculin A present were plotted. (D) In the presence of Latrunculin A, both Sec3p-GFP and Exo70p-GFP displayed recovery kinetics characterized by a single mode of recovery. Sec3p-GFP had a recovery τ of 59 ± 11 s, whereas Exo70p-GFP had a recovery τ of 57 ± 13 s.
Mentions: Because the recovery times for those subunits that appeared to use the fast mode of recovery were more similar to the recovery time for GFP-Sec4p than the recovery times for Sec3p-GFP or the slow mode for Exo70p-GFP, we decided to investigate whether or not all of the fast-recovering group used the same mechanism of recovery as GFP-Sec4p. Because GFP-Sec4p is present on vesicles that are transported to the bud tip along actin cables, we decided to test if recovery from photobleaching for all exocyst subunits depends on the presence of an intact actin cytoskeleton. As one test of this hypothesis, we treated cells with the actin-depolymerizing agent Latrunculin A. At a concentration of 200 μM, Latrunculin A treatment abolishes actin-based structures within 5 min in yeast (Ayscough et al., 1997). Vesicle transport to the bud tip halts, as shown by the effect on GFP-Sec4p localization, which showed a time-dependent loss of GFP-Sec4p from the bud tip (unpublished data). We examined by FRAP the population of cells that had not yet lost a focus of localized GFP in the time frame from 5 to 30 min after addition of Latrunculin A. We found that recovery of exocyst-GFP fusions was substantially inhibited (Fig. 3). In most cases there was only a small amount of recovery observed, <5%, but based on this low efficiency and the speed of this fraction's recovery (complete within 10 s), it may represent a freely diffusible population of each subunit, though we have no information on the size of this population in Latrunculin A–treated cells. Because we observed little recovery from photobleaching in the presence of Latrunculin A during the 5 to 30 min window after Latrunculin A addition, our conclusion is that delivery of these exocyst subunits to the bud tip depends on actin.

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