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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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Videomicrography of exocyst subunits fused to triple GFP tags. Haploid cells containing fusions of exocyst subunits to triple GFP tags were grown in SC medium and prepared for viewing as described in Materials and methods. Movies of 50 to 100 frames captured at five frames per second were analyzed for moving puncta consistent with vesicles in transit along actin cables. The velocity of individual puncta was calculated by determining the number of pixels traveled in a given number of frames and converting to micrometers per second. (A) Average speed of movement of puncta was determined for exocyst subunits fused to a triple-GFP tag as well as for GFP-Sec4p. We did not count movement near the mother-bud neck because movement rates there tend to be reduced as vesicles transition from the mother to the daughter cell. We did not calculate movement rates for Sec3p-3xGFP, which had no visible puncta movement, or Sec15p-3xGFP, which had puncta that were too faint to reliably track. Error bars represent 95% confidence intervals. (B) Videomicrographs of strain NY2510 containing exocyst subunit Sec5p fused to a 3xGFP tag. Capture rate is five frames per second. Contrast has been enhanced to visualize puncta. Several frames are shown here, with arrows indicating a punctum that travels from a mid-mother cell location near the upper shoulder into the bud tip during the course of about 4 s of video capture. Several frames captured during the time the punctum paused near the mother-bud neck were excised from the series. Quicktime videos of this movie and movies of all other visible subunits fused to 3xGFP are available in the online supplemental material (available at http://www/jcb.org/cgi/content/full/jcb.200408124/DC1).
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fig6: Videomicrography of exocyst subunits fused to triple GFP tags. Haploid cells containing fusions of exocyst subunits to triple GFP tags were grown in SC medium and prepared for viewing as described in Materials and methods. Movies of 50 to 100 frames captured at five frames per second were analyzed for moving puncta consistent with vesicles in transit along actin cables. The velocity of individual puncta was calculated by determining the number of pixels traveled in a given number of frames and converting to micrometers per second. (A) Average speed of movement of puncta was determined for exocyst subunits fused to a triple-GFP tag as well as for GFP-Sec4p. We did not count movement near the mother-bud neck because movement rates there tend to be reduced as vesicles transition from the mother to the daughter cell. We did not calculate movement rates for Sec3p-3xGFP, which had no visible puncta movement, or Sec15p-3xGFP, which had puncta that were too faint to reliably track. Error bars represent 95% confidence intervals. (B) Videomicrographs of strain NY2510 containing exocyst subunit Sec5p fused to a 3xGFP tag. Capture rate is five frames per second. Contrast has been enhanced to visualize puncta. Several frames are shown here, with arrows indicating a punctum that travels from a mid-mother cell location near the upper shoulder into the bud tip during the course of about 4 s of video capture. Several frames captured during the time the punctum paused near the mother-bud neck were excised from the series. Quicktime videos of this movie and movies of all other visible subunits fused to 3xGFP are available in the online supplemental material (available at http://www/jcb.org/cgi/content/full/jcb.200408124/DC1).

Mentions: We reasoned that if exocyst subunits associate with vesicles before their arrival at the bud tip, it might be possible to capture video sequences of vesicles bearing GFP-tagged subunits in transit to sites of exocytosis. Previous reports have shown that this is possible with a GFP-Sec4p fusion protein, when it is overexpressed (Schott et al., 2002), so we reasoned that with improvements in camera technology it might be possible for exocyst subunits as well, even under conditions in which they are expressed at wild-type levels, which is thought to be from several hundred to a thousand copies per cell (Ghaemmaghami et al., 2003). However, we found that triple-GFP tags were necessary to boost the signal level from fluorescent fusions, other than GFP-Sec4p, to reliably capture movies of vesicles in motion. In strains harboring triple-GFP–tagged exocyst fusion proteins, we observed small puncta of fluorescence moving in a manner consistent with secretory vesicles. The average rates of puncta movement for each fusion protein are tabulated in Fig. 6 A; they are all nearly equal to the movement rate of an overexpressed GFP-Sec4p fusion protein as previously reported (Schott et al., 2002). Fig. 6 B shows several stills from a movie of a Sec5p-GFP fusion construct in an otherwise wild-type cell. A Quicktime movie of the complete sequence, as well as movies of the other exocyst-3xGFP fusion proteins in wild-type cells, is available in the online supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200408124/DC1).


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)

Videomicrography of exocyst subunits fused to triple GFP tags. Haploid cells containing fusions of exocyst subunits to triple GFP tags were grown in SC medium and prepared for viewing as described in Materials and methods. Movies of 50 to 100 frames captured at five frames per second were analyzed for moving puncta consistent with vesicles in transit along actin cables. The velocity of individual puncta was calculated by determining the number of pixels traveled in a given number of frames and converting to micrometers per second. (A) Average speed of movement of puncta was determined for exocyst subunits fused to a triple-GFP tag as well as for GFP-Sec4p. We did not count movement near the mother-bud neck because movement rates there tend to be reduced as vesicles transition from the mother to the daughter cell. We did not calculate movement rates for Sec3p-3xGFP, which had no visible puncta movement, or Sec15p-3xGFP, which had puncta that were too faint to reliably track. Error bars represent 95% confidence intervals. (B) Videomicrographs of strain NY2510 containing exocyst subunit Sec5p fused to a 3xGFP tag. Capture rate is five frames per second. Contrast has been enhanced to visualize puncta. Several frames are shown here, with arrows indicating a punctum that travels from a mid-mother cell location near the upper shoulder into the bud tip during the course of about 4 s of video capture. Several frames captured during the time the punctum paused near the mother-bud neck were excised from the series. Quicktime videos of this movie and movies of all other visible subunits fused to 3xGFP are available in the online supplemental material (available at http://www/jcb.org/cgi/content/full/jcb.200408124/DC1).
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Related In: Results  -  Collection

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fig6: Videomicrography of exocyst subunits fused to triple GFP tags. Haploid cells containing fusions of exocyst subunits to triple GFP tags were grown in SC medium and prepared for viewing as described in Materials and methods. Movies of 50 to 100 frames captured at five frames per second were analyzed for moving puncta consistent with vesicles in transit along actin cables. The velocity of individual puncta was calculated by determining the number of pixels traveled in a given number of frames and converting to micrometers per second. (A) Average speed of movement of puncta was determined for exocyst subunits fused to a triple-GFP tag as well as for GFP-Sec4p. We did not count movement near the mother-bud neck because movement rates there tend to be reduced as vesicles transition from the mother to the daughter cell. We did not calculate movement rates for Sec3p-3xGFP, which had no visible puncta movement, or Sec15p-3xGFP, which had puncta that were too faint to reliably track. Error bars represent 95% confidence intervals. (B) Videomicrographs of strain NY2510 containing exocyst subunit Sec5p fused to a 3xGFP tag. Capture rate is five frames per second. Contrast has been enhanced to visualize puncta. Several frames are shown here, with arrows indicating a punctum that travels from a mid-mother cell location near the upper shoulder into the bud tip during the course of about 4 s of video capture. Several frames captured during the time the punctum paused near the mother-bud neck were excised from the series. Quicktime videos of this movie and movies of all other visible subunits fused to 3xGFP are available in the online supplemental material (available at http://www/jcb.org/cgi/content/full/jcb.200408124/DC1).
Mentions: We reasoned that if exocyst subunits associate with vesicles before their arrival at the bud tip, it might be possible to capture video sequences of vesicles bearing GFP-tagged subunits in transit to sites of exocytosis. Previous reports have shown that this is possible with a GFP-Sec4p fusion protein, when it is overexpressed (Schott et al., 2002), so we reasoned that with improvements in camera technology it might be possible for exocyst subunits as well, even under conditions in which they are expressed at wild-type levels, which is thought to be from several hundred to a thousand copies per cell (Ghaemmaghami et al., 2003). However, we found that triple-GFP tags were necessary to boost the signal level from fluorescent fusions, other than GFP-Sec4p, to reliably capture movies of vesicles in motion. In strains harboring triple-GFP–tagged exocyst fusion proteins, we observed small puncta of fluorescence moving in a manner consistent with secretory vesicles. The average rates of puncta movement for each fusion protein are tabulated in Fig. 6 A; they are all nearly equal to the movement rate of an overexpressed GFP-Sec4p fusion protein as previously reported (Schott et al., 2002). Fig. 6 B shows several stills from a movie of a Sec5p-GFP fusion construct in an otherwise wild-type cell. A Quicktime movie of the complete sequence, as well as movies of the other exocyst-3xGFP fusion proteins in wild-type cells, is available in the online supplemental material (available at http://www.jcb.org/cgi/content/full/jcb.200408124/DC1).

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