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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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Distribution of gold particles after immunoelectron microscopy. Gold particles that were found to be in the bud tips of cells were counted, and the distances between all such particles and the plasma membrane were calculated. (A) Histogram showing the distribution of gold particle labeling when Spa2p-13myc was used as a negative control for vesicle association. Approximately 74% of the particles were found within 50 nm of the plasma membrane. Average distance between the plasma membrane and particles was found to be 55.0 ± 5.7 nm. (B) Histogram of the distribution of gold particles in a strain harboring the Sec3p-13myc fusion. 62% of gold particles were found to be within 50 nm of the plasma membrane, indicating that the majority of Sec3p-13myc was associated with the plasma membrane. Average distance between the plasma membrane and gold particles was 58.7 ± 13.8 nm. (C) Histogram of plasma membrane-gold particle distances in the Sec15p-13myc strain after immunoelectron microscopy. Less than half of the signal, 40%, was within 50 nm of the plasma membrane. These presumably represent Sec15p-13myc fusion proteins that are actively engaged in tethering vesicles to the plasma membrane in preparation for membrane fusion. The average distance from the plasma membrane to gold particles in the tip was found to be 119 ± 17 nm.
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fig5: Distribution of gold particles after immunoelectron microscopy. Gold particles that were found to be in the bud tips of cells were counted, and the distances between all such particles and the plasma membrane were calculated. (A) Histogram showing the distribution of gold particle labeling when Spa2p-13myc was used as a negative control for vesicle association. Approximately 74% of the particles were found within 50 nm of the plasma membrane. Average distance between the plasma membrane and particles was found to be 55.0 ± 5.7 nm. (B) Histogram of the distribution of gold particles in a strain harboring the Sec3p-13myc fusion. 62% of gold particles were found to be within 50 nm of the plasma membrane, indicating that the majority of Sec3p-13myc was associated with the plasma membrane. Average distance between the plasma membrane and gold particles was 58.7 ± 13.8 nm. (C) Histogram of plasma membrane-gold particle distances in the Sec15p-13myc strain after immunoelectron microscopy. Less than half of the signal, 40%, was within 50 nm of the plasma membrane. These presumably represent Sec15p-13myc fusion proteins that are actively engaged in tethering vesicles to the plasma membrane in preparation for membrane fusion. The average distance from the plasma membrane to gold particles in the tip was found to be 119 ± 17 nm.

Mentions: We also calculated the average distance in the bud tips from the plasma membrane to Spa2p-13myc, Sec3p-13myc, and Sec15p-13myc (as an exemplar of the exocyst subunits other than Sec3p). The results are plotted in Fig. 5. The average distance of gold particles from the plasma membrane for Spa2p-13myc and Sec3p-13myc are quite similar, indicating that Sec3p-13myc is associated with the membrane. In contrast, Sec15p-13myc is on average twice as distant from the plasma membrane as Spa2p-13myc. Histograms showing the distribution of each set of gold particles are also shown in Fig. 5. These data show that the distribution of Sec3p-13myc more closely resembles that of Spa2p-13myc rather than Sec15p-13myc, again indicating that the localization of Sec3p-13myc is more similar to that of Spa2p than Sec15p. The broader distribution of gold particles associated with Sec15p-13myc presumably reflects that some particles are found in the middle regions of bud tips as they are transported on vesicles to sites of exocytosis.


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)

Distribution of gold particles after immunoelectron microscopy. Gold particles that were found to be in the bud tips of cells were counted, and the distances between all such particles and the plasma membrane were calculated. (A) Histogram showing the distribution of gold particle labeling when Spa2p-13myc was used as a negative control for vesicle association. Approximately 74% of the particles were found within 50 nm of the plasma membrane. Average distance between the plasma membrane and particles was found to be 55.0 ± 5.7 nm. (B) Histogram of the distribution of gold particles in a strain harboring the Sec3p-13myc fusion. 62% of gold particles were found to be within 50 nm of the plasma membrane, indicating that the majority of Sec3p-13myc was associated with the plasma membrane. Average distance between the plasma membrane and gold particles was 58.7 ± 13.8 nm. (C) Histogram of plasma membrane-gold particle distances in the Sec15p-13myc strain after immunoelectron microscopy. Less than half of the signal, 40%, was within 50 nm of the plasma membrane. These presumably represent Sec15p-13myc fusion proteins that are actively engaged in tethering vesicles to the plasma membrane in preparation for membrane fusion. The average distance from the plasma membrane to gold particles in the tip was found to be 119 ± 17 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172445&req=5

fig5: Distribution of gold particles after immunoelectron microscopy. Gold particles that were found to be in the bud tips of cells were counted, and the distances between all such particles and the plasma membrane were calculated. (A) Histogram showing the distribution of gold particle labeling when Spa2p-13myc was used as a negative control for vesicle association. Approximately 74% of the particles were found within 50 nm of the plasma membrane. Average distance between the plasma membrane and particles was found to be 55.0 ± 5.7 nm. (B) Histogram of the distribution of gold particles in a strain harboring the Sec3p-13myc fusion. 62% of gold particles were found to be within 50 nm of the plasma membrane, indicating that the majority of Sec3p-13myc was associated with the plasma membrane. Average distance between the plasma membrane and gold particles was 58.7 ± 13.8 nm. (C) Histogram of plasma membrane-gold particle distances in the Sec15p-13myc strain after immunoelectron microscopy. Less than half of the signal, 40%, was within 50 nm of the plasma membrane. These presumably represent Sec15p-13myc fusion proteins that are actively engaged in tethering vesicles to the plasma membrane in preparation for membrane fusion. The average distance from the plasma membrane to gold particles in the tip was found to be 119 ± 17 nm.
Mentions: We also calculated the average distance in the bud tips from the plasma membrane to Spa2p-13myc, Sec3p-13myc, and Sec15p-13myc (as an exemplar of the exocyst subunits other than Sec3p). The results are plotted in Fig. 5. The average distance of gold particles from the plasma membrane for Spa2p-13myc and Sec3p-13myc are quite similar, indicating that Sec3p-13myc is associated with the membrane. In contrast, Sec15p-13myc is on average twice as distant from the plasma membrane as Spa2p-13myc. Histograms showing the distribution of each set of gold particles are also shown in Fig. 5. These data show that the distribution of Sec3p-13myc more closely resembles that of Spa2p-13myc rather than Sec15p-13myc, again indicating that the localization of Sec3p-13myc is more similar to that of Spa2p than Sec15p. The broader distribution of gold particles associated with Sec15p-13myc presumably reflects that some particles are found in the middle regions of bud tips as they are transported on vesicles to sites of exocytosis.

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