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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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Determination of kinetic components of recovery. Recovery from photobleaching is described by Eq. 1 (see Materials and methods): I∞ − It = (I∞ − I0)e−kt, where I∞ is the signal intensity at full recovery, It is the signal intensity at time t, I0 is the signal intensity immediately after photobleaching, k is the rate constant (equivalent to −1/τ), and t is time. (A) Plot of kt versus time will be characterized by a single line if recovery has one component or segments with two different slopes if recovery has two components. All exocyst components except Exo70p-GFP show a fit to a single line. Exo70p-GFP has two segments with differing slopes, indicating that there are two modes of photobleaching recovery for this subunit. (B) Histogram comparing FRAP recovery times for exocyst subunits and GFP-Sec4p. For each subunit, a best-fit curve determination was made for each photobleaching experiment, and the average τ of 12 recovery curves was calculated. In the case of Exo70p-GFP, both the fast and slow components are shown. Error bars represent 95% confidence intervals.
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fig2: Determination of kinetic components of recovery. Recovery from photobleaching is described by Eq. 1 (see Materials and methods): I∞ − It = (I∞ − I0)e−kt, where I∞ is the signal intensity at full recovery, It is the signal intensity at time t, I0 is the signal intensity immediately after photobleaching, k is the rate constant (equivalent to −1/τ), and t is time. (A) Plot of kt versus time will be characterized by a single line if recovery has one component or segments with two different slopes if recovery has two components. All exocyst components except Exo70p-GFP show a fit to a single line. Exo70p-GFP has two segments with differing slopes, indicating that there are two modes of photobleaching recovery for this subunit. (B) Histogram comparing FRAP recovery times for exocyst subunits and GFP-Sec4p. For each subunit, a best-fit curve determination was made for each photobleaching experiment, and the average τ of 12 recovery curves was calculated. In the case of Exo70p-GFP, both the fast and slow components are shown. Error bars represent 95% confidence intervals.

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)

Determination of kinetic components of recovery. Recovery from photobleaching is described by Eq. 1 (see Materials and methods): I∞ − It = (I∞ − I0)e−kt, where I∞ is the signal intensity at full recovery, It is the signal intensity at time t, I0 is the signal intensity immediately after photobleaching, k is the rate constant (equivalent to −1/τ), and t is time. (A) Plot of kt versus time will be characterized by a single line if recovery has one component or segments with two different slopes if recovery has two components. All exocyst components except Exo70p-GFP show a fit to a single line. Exo70p-GFP has two segments with differing slopes, indicating that there are two modes of photobleaching recovery for this subunit. (B) Histogram comparing FRAP recovery times for exocyst subunits and GFP-Sec4p. For each subunit, a best-fit curve determination was made for each photobleaching experiment, and the average τ of 12 recovery curves was calculated. In the case of Exo70p-GFP, both the fast and slow components are shown. Error bars represent 95% confidence intervals.
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Related In: Results  -  Collection

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fig2: Determination of kinetic components of recovery. Recovery from photobleaching is described by Eq. 1 (see Materials and methods): I∞ − It = (I∞ − I0)e−kt, where I∞ is the signal intensity at full recovery, It is the signal intensity at time t, I0 is the signal intensity immediately after photobleaching, k is the rate constant (equivalent to −1/τ), and t is time. (A) Plot of kt versus time will be characterized by a single line if recovery has one component or segments with two different slopes if recovery has two components. All exocyst components except Exo70p-GFP show a fit to a single line. Exo70p-GFP has two segments with differing slopes, indicating that there are two modes of photobleaching recovery for this subunit. (B) Histogram comparing FRAP recovery times for exocyst subunits and GFP-Sec4p. For each subunit, a best-fit curve determination was made for each photobleaching experiment, and the average τ of 12 recovery curves was calculated. In the case of Exo70p-GFP, both the fast and slow components are shown. Error bars represent 95% confidence intervals.
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