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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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Immunoelectron microscopy of 13myc-tagged exocyst subunits. Haploid cells containing 13myc tags were grown in SC medium and cryosectioned as described in Materials and methods. (A) Immunoelectron micrograph of strain NY2508, which contains Spa2p fused to 13 consecutive 9E10 epitope tags in tandem. Fixed cryosections were labeled with anti-9E10 rabbit polyclonal antibody and 10-nm gold particles conjugated to protein A. (B) Immunoelectron micrograph of strain NY2504, which contains Sec10p fused to the 13myc epitope. The graph shows a tip with several vesicles visible at some distance from the plasma membrane. Gold particles associated with these vesicles are indicated by solid black arrows. Gold particles that are near both the plasma membrane and a vesicle are denoted by a white arrow, and gold particles that are not associated with any vesicle are denoted by the double arrows. (C) Histogram displaying the statistical significance of the association of gold particles with vesicles. Statistical significance was established by determining the surface areas of the cell and vesicles, counting gold particles, and then calculating the ratio of gold particle densities in each area; this factor is termed the labeling ratio (see Materials and methods). A labeling ratio of one indicates no association with vesicles, whereas a ratio >1 indicates colocalization of vesicles and gold particles, and indicates that exocyst subunits are associated with vesicles. Error bars represent 95% confidence intervals.
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fig4: Immunoelectron microscopy of 13myc-tagged exocyst subunits. Haploid cells containing 13myc tags were grown in SC medium and cryosectioned as described in Materials and methods. (A) Immunoelectron micrograph of strain NY2508, which contains Spa2p fused to 13 consecutive 9E10 epitope tags in tandem. Fixed cryosections were labeled with anti-9E10 rabbit polyclonal antibody and 10-nm gold particles conjugated to protein A. (B) Immunoelectron micrograph of strain NY2504, which contains Sec10p fused to the 13myc epitope. The graph shows a tip with several vesicles visible at some distance from the plasma membrane. Gold particles associated with these vesicles are indicated by solid black arrows. Gold particles that are near both the plasma membrane and a vesicle are denoted by a white arrow, and gold particles that are not associated with any vesicle are denoted by the double arrows. (C) Histogram displaying the statistical significance of the association of gold particles with vesicles. Statistical significance was established by determining the surface areas of the cell and vesicles, counting gold particles, and then calculating the ratio of gold particle densities in each area; this factor is termed the labeling ratio (see Materials and methods). A labeling ratio of one indicates no association with vesicles, whereas a ratio >1 indicates colocalization of vesicles and gold particles, and indicates that exocyst subunits are associated with vesicles. Error bars represent 95% confidence intervals.

Mentions: Fig. 4 shows that Spa2p-13myc is concentrated at the bud tip near the plasma membrane, but is not associated with vesicles. For the exocyst subunits and Sec2-13myc, we determined the density of gold particle labeling on vesicles versus the remainder of the cell, including the plasma membrane, to determine if there was a significant increase in labeling near vesicles. In contrast to Spa2p, an association of gold particles with vesicles was observed for Sec2p-13myc and all exocyst subunits with the exception of Sec3p-13myc. Fig. 4 also shows the differences in label density, expressed as the ratio of labeling in the cell as a whole to labeling near vesicles (see Materials and methods). For the untagged, wild-type control strain, this ratio should be unity because random background labeling should have no bias toward or against vesicles, and this is in fact what was observed. Other strains expressing various exocyst subunits fused to 13myc tags showed a range of labeling ratios, from approximately two- to fourfold above background levels. The conclusion we draw from this experiment is that all exocyst subunits, except for Sec3p, are associated with vesicles in transit 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)

Immunoelectron microscopy of 13myc-tagged exocyst subunits. Haploid cells containing 13myc tags were grown in SC medium and cryosectioned as described in Materials and methods. (A) Immunoelectron micrograph of strain NY2508, which contains Spa2p fused to 13 consecutive 9E10 epitope tags in tandem. Fixed cryosections were labeled with anti-9E10 rabbit polyclonal antibody and 10-nm gold particles conjugated to protein A. (B) Immunoelectron micrograph of strain NY2504, which contains Sec10p fused to the 13myc epitope. The graph shows a tip with several vesicles visible at some distance from the plasma membrane. Gold particles associated with these vesicles are indicated by solid black arrows. Gold particles that are near both the plasma membrane and a vesicle are denoted by a white arrow, and gold particles that are not associated with any vesicle are denoted by the double arrows. (C) Histogram displaying the statistical significance of the association of gold particles with vesicles. Statistical significance was established by determining the surface areas of the cell and vesicles, counting gold particles, and then calculating the ratio of gold particle densities in each area; this factor is termed the labeling ratio (see Materials and methods). A labeling ratio of one indicates no association with vesicles, whereas a ratio >1 indicates colocalization of vesicles and gold particles, and indicates that exocyst subunits are associated with vesicles. Error bars represent 95% confidence intervals.
© Copyright Policy
Related In: Results  -  Collection

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fig4: Immunoelectron microscopy of 13myc-tagged exocyst subunits. Haploid cells containing 13myc tags were grown in SC medium and cryosectioned as described in Materials and methods. (A) Immunoelectron micrograph of strain NY2508, which contains Spa2p fused to 13 consecutive 9E10 epitope tags in tandem. Fixed cryosections were labeled with anti-9E10 rabbit polyclonal antibody and 10-nm gold particles conjugated to protein A. (B) Immunoelectron micrograph of strain NY2504, which contains Sec10p fused to the 13myc epitope. The graph shows a tip with several vesicles visible at some distance from the plasma membrane. Gold particles associated with these vesicles are indicated by solid black arrows. Gold particles that are near both the plasma membrane and a vesicle are denoted by a white arrow, and gold particles that are not associated with any vesicle are denoted by the double arrows. (C) Histogram displaying the statistical significance of the association of gold particles with vesicles. Statistical significance was established by determining the surface areas of the cell and vesicles, counting gold particles, and then calculating the ratio of gold particle densities in each area; this factor is termed the labeling ratio (see Materials and methods). A labeling ratio of one indicates no association with vesicles, whereas a ratio >1 indicates colocalization of vesicles and gold particles, and indicates that exocyst subunits are associated with vesicles. Error bars represent 95% confidence intervals.
Mentions: Fig. 4 shows that Spa2p-13myc is concentrated at the bud tip near the plasma membrane, but is not associated with vesicles. For the exocyst subunits and Sec2-13myc, we determined the density of gold particle labeling on vesicles versus the remainder of the cell, including the plasma membrane, to determine if there was a significant increase in labeling near vesicles. In contrast to Spa2p, an association of gold particles with vesicles was observed for Sec2p-13myc and all exocyst subunits with the exception of Sec3p-13myc. Fig. 4 also shows the differences in label density, expressed as the ratio of labeling in the cell as a whole to labeling near vesicles (see Materials and methods). For the untagged, wild-type control strain, this ratio should be unity because random background labeling should have no bias toward or against vesicles, and this is in fact what was observed. Other strains expressing various exocyst subunits fused to 13myc tags showed a range of labeling ratios, from approximately two- to fourfold above background levels. The conclusion we draw from this experiment is that all exocyst subunits, except for Sec3p, are associated with vesicles in transit 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