Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p

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.

Figure 1.

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.

Figure 2.

Determination of kinetic components of recovery. Recovery from photobleaching is described by Eq. 1 (see Materials and methods): I_∞ − I_t = (I_∞ − I₀)e^−kt, where I_∞ is the signal intensity at full recovery, I_t 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.

Figure 3.

Photobleaching recovery of GFP-tagged exocyst subunits in the presence of Latrunculin A. Cells were grown in SC medium and prepared as described in Materials and methods. (A) Micrographs of strains containing Sec8-GFP and Sec3-GFP (NY2442 and NY2439, respectively) in cells with or without 200 mM Latrunculin A added to the medium. (B) Bud tips in Latrunculin A–treated cells were bleached and monitored for recovery. Sample recovery graphs with DMSO and DMSO + 200 μM Latrunculin A are shown for each exocyst subunit fused to GFP. (C) 20 bleach/recovery procedures for each of the GFP fusion strains were performed, and the frequencies of good (>20%), medium (5 to 20%), or poor (<5%) recovery with and without Latrunculin A present were plotted. (D) In the presence of Latrunculin A, both Sec3p-GFP and Exo70p-GFP displayed recovery kinetics characterized by a single mode of recovery. Sec3p-GFP had a recovery τ of 59 ± 11 s, whereas Exo70p-GFP had a recovery τ of 57 ± 13 s.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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).

Figure 7.

Photobleaching recovery experiments on strains containing a Sec8p-GFP fusion protein with various alleles of sec4. (A) Photobleaching recovery rates. Recovery from photobleaching in strain NY2443, which harbors Sec8p-GFP and the wild-type SEC4 allele, had a τ of 23 ± 4.5 s. Compared with the wild-type background, recovery of the Sec8-GFP fusion was much slower in a strain harboring the sec4-8 allele (NY2518), which had a recovery τ of 56 ± 14 s. Photobleaching recovery was measured in NY2518 at RT, substantially below the restrictive temperature of 30°C. Strain NY2519, which expresses approximately fivefold more Sec4p than NY2443, was also examined and found to have a τ of 22.5 ± 11.5 s. Error bars represent 95% confidence intervals. (B) Western blot of whole-cell extracts from strains NY1210 (wild type) and NY2520 (otherwise wild-type that contains the integrating Sec4p overexpression vector pNB170), showing that Sec4p is overproduced approximately fivefold in NY2520.

Figure 8.

Model of targeting and tethering in S. cerevisiae. The exocyst subunits (except for Sec3p) associate with vesicles before movement to the bud tip. Once the vesicle has arrived at the bud tip, Sec3p and Exo70p bind to the rest of the exocyst to complete the formation of the tethering complex. Exo70p is also found to ride vesicles to sites of exocytosis in addition to localizing there by interacting with Rho3p.