Limits...
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.

Show MeSH

Related in: MedlinePlus

Fluorescence recovery of GFP-tagged exocyst subunits in S. cerevisiae. Haploid cells with integrated GFP fusion genes in an otherwise wild-type background were grown in SC medium at 25°C and mounted as described in Materials and methods. Bud tips were bleached with a fixed dye-tunable laser emitting at a wavelength of 440 nm, and images were recorded for analysis at various time points. (A) Two time series of recovery from photobleaching. GFP-Sec4p recovers completely in 45 s with a τ of 12 ± 4 s, whereas Sec3p-GFP requires about 3 min to recover completely, with a τ of 59 ± 17 s. Recovery is limited to ∼35% because approximately two-thirds of each exocyst subunit is located in the bud tip at any given time. (B) Best-fit recovery curve determination for the recoveries of GFP-Sec4p and Sec3p-GFP in part A. IGOR was used to generate a value for τ and to plot a recovery curve for each recovery experiment.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172445&req=5

fig1: Fluorescence recovery of GFP-tagged exocyst subunits in S. cerevisiae. Haploid cells with integrated GFP fusion genes in an otherwise wild-type background were grown in SC medium at 25°C and mounted as described in Materials and methods. Bud tips were bleached with a fixed dye-tunable laser emitting at a wavelength of 440 nm, and images were recorded for analysis at various time points. (A) Two time series of recovery from photobleaching. GFP-Sec4p recovers completely in 45 s with a τ of 12 ± 4 s, whereas Sec3p-GFP requires about 3 min to recover completely, with a τ of 59 ± 17 s. Recovery is limited to ∼35% because approximately two-thirds of each exocyst subunit is located in the bud tip at any given time. (B) Best-fit recovery curve determination for the recoveries of GFP-Sec4p and Sec3p-GFP in part A. IGOR was used to generate a value for τ and to plot a recovery curve for each recovery experiment.

Mentions: We photobleached bud tips with a fixed dye-tunable laser emitting light at a wavelength of 440 nm, focused to a small spot ∼1 μm in diameter. The absorption peak of GFP is 489 nm, but absorption extends far enough into the blue to allow the 440-nm laser to bleach GFP. Each bud tip was exposed to ∼10 pulses of 50 μs duration, for a total bleaching time of 0.5 ms. We observed recovery by collecting standard epifluorescence images at ∼10-s intervals for 1–3 min, depending on which exocyst subunit was being observed (Fig. 1). For each time point, we measured fluorescence intensity in the bud tip and calculated the time constant τ for each subunit by plotting the natural logarithm of the left side of Eq. 2 (see Materials and methods) against time. This plot should have a single straight line if there is only one component to the recovery, but a discontinuous line with two distinct slopes if there are two components that contribute to recovery. In these plots the slope(s) of each line is equal to −1/τ. For each subunit, at least 12 lines were generated using IGOR and used to calculate an average τ. All subunits except Exo70-GFP exhibited single-time constant kinetics (Fig. 2 A), whereas Exo70-GFP exhibited a recovery characterized by two modes of recovery, with τ of 22 ± 9 s for the fast mode and 57 ± 17 s for the slow mode. The overall rate of Exo70p-GFP recovery was 41 ± 13 s, implying that ∼46% of Exo70p-GFP recovers via the fast mode of recovery, whereas the remaining 54% uses the slow mode.


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)

Fluorescence recovery of GFP-tagged exocyst subunits in S. cerevisiae. Haploid cells with integrated GFP fusion genes in an otherwise wild-type background were grown in SC medium at 25°C and mounted as described in Materials and methods. Bud tips were bleached with a fixed dye-tunable laser emitting at a wavelength of 440 nm, and images were recorded for analysis at various time points. (A) Two time series of recovery from photobleaching. GFP-Sec4p recovers completely in 45 s with a τ of 12 ± 4 s, whereas Sec3p-GFP requires about 3 min to recover completely, with a τ of 59 ± 17 s. Recovery is limited to ∼35% because approximately two-thirds of each exocyst subunit is located in the bud tip at any given time. (B) Best-fit recovery curve determination for the recoveries of GFP-Sec4p and Sec3p-GFP in part A. IGOR was used to generate a value for τ and to plot a recovery curve for each recovery experiment.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Fluorescence recovery of GFP-tagged exocyst subunits in S. cerevisiae. Haploid cells with integrated GFP fusion genes in an otherwise wild-type background were grown in SC medium at 25°C and mounted as described in Materials and methods. Bud tips were bleached with a fixed dye-tunable laser emitting at a wavelength of 440 nm, and images were recorded for analysis at various time points. (A) Two time series of recovery from photobleaching. GFP-Sec4p recovers completely in 45 s with a τ of 12 ± 4 s, whereas Sec3p-GFP requires about 3 min to recover completely, with a τ of 59 ± 17 s. Recovery is limited to ∼35% because approximately two-thirds of each exocyst subunit is located in the bud tip at any given time. (B) Best-fit recovery curve determination for the recoveries of GFP-Sec4p and Sec3p-GFP in part A. IGOR was used to generate a value for τ and to plot a recovery curve for each recovery experiment.
Mentions: We photobleached bud tips with a fixed dye-tunable laser emitting light at a wavelength of 440 nm, focused to a small spot ∼1 μm in diameter. The absorption peak of GFP is 489 nm, but absorption extends far enough into the blue to allow the 440-nm laser to bleach GFP. Each bud tip was exposed to ∼10 pulses of 50 μs duration, for a total bleaching time of 0.5 ms. We observed recovery by collecting standard epifluorescence images at ∼10-s intervals for 1–3 min, depending on which exocyst subunit was being observed (Fig. 1). For each time point, we measured fluorescence intensity in the bud tip and calculated the time constant τ for each subunit by plotting the natural logarithm of the left side of Eq. 2 (see Materials and methods) against time. This plot should have a single straight line if there is only one component to the recovery, but a discontinuous line with two distinct slopes if there are two components that contribute to recovery. In these plots the slope(s) of each line is equal to −1/τ. For each subunit, at least 12 lines were generated using IGOR and used to calculate an average τ. All subunits except Exo70-GFP exhibited single-time constant kinetics (Fig. 2 A), whereas Exo70-GFP exhibited a recovery characterized by two modes of recovery, with τ of 22 ± 9 s for the fast mode and 57 ± 17 s for the slow mode. The overall rate of Exo70p-GFP recovery was 41 ± 13 s, implying that ∼46% of Exo70p-GFP recovers via the fast mode of recovery, whereas the remaining 54% uses the slow mode.

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