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Correspondence to Peter Novick: peter.novick{at}yale.edu
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Abstract |
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Introduction |
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The exocyst was first purified from yeast by coimmunoprecipitation using an epitope-tagged allele of Sec8p (TerBush et al., 1996). All members of the exocyst except Sec3p are essential for viability, whereas loss of Sec3p results in slow growth and decreased spatial control of exocytosis at 25°C and inhibition of both growth and secretion at 37°C (Bowser and Novick, 1991; TerBush and Novick, 1995; Guo et al., 1999a; Wiederkehr et al., 2003). The exocyst displays a characteristic, cell cycledependent localization pattern that begins with the appearance of a small cap at sites of bud formation. This cap persists at the apical tip of the growing bud until the time of nuclear division when it shifts to an isotropic distribution over the surface of the growing bud. Finally, there is an abrupt shift in localization to the mother-bud neck near the time of cytokinesis, where it remains until cell separation (TerBush and Novick, 1995, Finger et al., 1998). This pattern of localization mirrors the pattern of deposition of newly synthesized material at the cell surface (Field and Schekman, 1980).
Although all exocyst subunits share the same pattern of localization, they exhibit strikingly different requirements for this localization. The polarized localization of Sec3p is insensitive to blocks in membrane traffic as well as to disruption of the actin cytoskeleton (Finger et al., 1998). Rather, its localization involves a direct interaction with the Rho family GTPases, Rho1p and Cdc42p, in their GTP-bound form on the plasma membrane (Drgonova et al., 1999; Guo et al., 2001; Zhang et al., 2001). In contrast, Sec8p requires both ongoing membrane traffic as well as actin function for its localization (Ayscough et al., 1997; Finger et al., 1998). Sec15p, a direct effector of the Rab GTPase Sec4p (Guo et al., 1999b), was found, like Sec4p, to associate with secretory vesicles, at least upon overexpression. Based on these observations, we have proposed that Sec3p serves as a spatial landmark on the plasma membrane for incoming secretory vesicles (Finger et al., 1998). Other subunits, we speculated, either ride the vesicles to these sites or are recruited to the sites directly from the cytosol in response to the arrival of the secretory vesicles. Assembly of the complex would then occur as vesicles arrive at the sites marked by Sec3p and would serve to tether the vesicles to the plasma membrane at these sites. In support of the model, we have shown that in a sec4-8 mutant, in which vesicle delivery is blocked, a pool of Sec3p develops that sediments more slowly than the fully assembled exocyst complex (Guo et al., 2001).
Several lines of evidence from mammalian epithelial and neuronal cell lines also contribute to a model in which exocyst subunits arrive at sites of exocytosis on vesicles. In rat brain lysates (Brymora et al., 2001) and in PC12 neuronal cell lysates (Moskalenko et al., 2002, 2003), the Sec5 and Exo84 subunits of the mammalian exocyst have been found to associate with RalA, which in turn is found on synaptic and axonal vesicles (Bielinski et al., 1993). Elements of the exocyst have also been found associated with other compartments in the secretory pathway, including the endoplasmic reticulum in MDCK cells (Grindstaff et al., 1998), the trans-Golgi network of NRK cell lysates (Yeaman et al., 2001), vesicles in PC12 cell and rat brain lysates (Moskalenko et al., 2002; Sans et al., 2003), and complexed with GTP-bound ARF6 on endosomes found in MDCK and NRK cell lysates (Prigent et al., 2003).
The detailed mechanism by which the exocyst functions in tethering is under investigation. Central to this question is how the exocyst comes to be assembled at exocytic sites. Related to this issue is the dynamic behavior of the exocyst subunits, and how the behavior of individual exocyst subunits bears on the mechanism of tethering at a molecular level. Here, we systematically examine the dynamic behavior, in live yeast cells, of each of the exocyst subunits fused to GFP by photobleaching recovery experiments. We have also used immunogold electron microscopy and video fluorescence microscopy to show that a subset of exocyst subunits is transported on vesicles to sites of exocytosis at the plasma membrane where they assemble with the remaining subunits.
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Results |
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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 13 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
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/
. For each subunit, at least 12 lines were generated using IGOR and used to calculate an average
. 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
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.
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We found no evidence to support the existence of an immobile fraction of exocyst subunits at sites of exocytosis. Recovery efficiency of photobleached tips depends on the fraction of the total cellular signal bleached. In all cases, the recovery efficiency measured was approximately equal to the expected recovery efficiency as determined by measuring the fraction of total signal bleached.
FRAP in the presence of Latrunculin A indicates that recovery of one class depends on the actin cytoskeleton
Because the recovery times for those subunits that appeared to use the fast mode of recovery were more similar to the recovery time for GFP-Sec4p than the recovery times for Sec3p-GFP or the slow mode for Exo70p-GFP, we decided to investigate whether or not all of the fast-recovering group used the same mechanism of recovery as GFP-Sec4p. Because GFP-Sec4p is present on vesicles that are transported to the bud tip along actin cables, we decided to test if recovery from photobleaching for all exocyst subunits depends on the presence of an intact actin cytoskeleton. As one test of this hypothesis, we treated cells with the actin-depolymerizing agent Latrunculin A. At a concentration of 200 µM, Latrunculin A treatment abolishes actin-based structures within 5 min in yeast (Ayscough et al., 1997). Vesicle transport to the bud tip halts, as shown by the effect on GFP-Sec4p localization, which showed a time-dependent loss of GFP-Sec4p from the bud tip (unpublished data). We examined by FRAP the population of cells that had not yet lost a focus of localized GFP in the time frame from 5 to 30 min after addition of Latrunculin A. We found that recovery of exocyst-GFP fusions was substantially inhibited (Fig. 3). In most cases there was only a small amount of recovery observed, <5%, but based on this low efficiency and the speed of this fraction's recovery (complete within 10 s), it may represent a freely diffusible population of each subunit, though we have no information on the size of this population in Latrunculin Atreated cells. Because we observed little recovery from photobleaching in the presence of Latrunculin A during the 5 to 30 min window after Latrunculin A addition, our conclusion is that delivery of these exocyst subunits to the bud tip depends on actin.
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Two results from the FRAP analysis stand out in significance: first, in the case of all fusions except Sec3p-GFP and Exo70p-GFP, an assembled actin network is necessary to recover from photobleaching; second, those subunits that are dependent on actin normally have a recovery time that is more similar to that of the GFP-Sec4 fusion protein than to the actin-independent subunit Sec3p-GFP or the slow mode of Exo70p-GFP recovery. Both of these results are consistent with the hypothesis that six of the eight exocyst subunits (and approximately half of Exo70p-GFP) are delivered to sites of exocytosis on secretory vesicles. We further tested this hypothesis by two methods: first, we ascertained the distribution of the exocyst subunits using immunogold electron microscopy and correlated that distribution with the presence of vesicular structures; second, we imaged the movement of exocyst-GFP fusion proteins in live cells.
Electron microscopy reveals association of vesicles with exocyst proteins
To use immunoelectron microscopy confidently to assess the association of exocyst proteins with secretory vesicles, we needed both a negative and positive control. As a negative control, we looked for a protein that is concentrated at bud tips, but is not associated with vesicles. Spa2p helps to maintain the polarization of actin cables by facilitating the localization of the formin Bni1p (Fujiwara et al., 1998). Because the localization of Spa2p to the bud tip is known to be independent of actin (Ayscough et al., 1997), we would not expect it to be delivered on secretory vesicles. As a positive control we chose Sec2p, the exchange factor for Sec4p, because it has been shown to be associated with secretory vesicles (Walch-Solimena et al., 1997). Sec4p itself was not used as a positive control for two reasons. First, the 13myc-Sec4p fusion protein was not fully functional, and when present as the sole copy of Sec4p in the cell caused buildup of unfused vesicles (unpublished data). Second, although Sec4p maintains its membrane association with a carboxy terminal lipid anchor, Sec2p, as a peripheral membrane protein, is more similar to the exocyst proteins. Spa2p, Sec2p, and all eight exocyst subunits were tagged at their carboxy termini with 13 myc epitopes. Table I shows the numbers of gold particles, cells, and vesicles counted as part of the experiment for each fusion protein examined.
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Discussion |
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Exo70p can apparently use two distinct pathways to arrive at the bud tip, as seen by the biphasic nature of photobleaching recovery graphs. Supporting this possibility, Exo70p-GFP was found to maintain its localization in the presence of 200 µM Latrunculin A, which blocks vesicle delivery. Additionally, the frequency of full recovery from photobleaching for Exo70p-GFP was reduced in the presence of Latrunculin A, but not abolished (Fig. 3 C). In fact, the frequency of recovery in the presence of Latrunculin was similar to that for Sec3p-GFP in Latrunculin A, whereas when Latrunculin A was absent it was greater than that of Sec3p-GFP. The rate of recovery from photobleaching for Exo70p in the presence of Latrunculin ( = 57 ± 17 s) was almost identical to the rate determined for Sec3p-GFP in both Latrunculin-treated and untreated cells (
= 59 ± 11 s) and equivalent to the slow rate of Exo70p recovery as determined by analysis of photobleaching recovery curves generated in the absence of Latrunculin A. Finally, the association of Exo70p-13myc with vesicles in transit was shown by both immunoelectron microscopy and by video microscopy of cells harboring a triple-GFP tag fused to the COOH terminus of Exo70p. Our conclusion is that a portion of Exo70p is transported to sites of exocytosis on vesicles, possibly as part of a partially assembled exocyst complex, but approximately half also localizes independently of vesicle traffic through direct association with Rho proteins (Adamo et al., 1999; Robinson et al., 1999). In this case, it may be responsible for the Sec3p-independent route of exocyst localization that has previously been reported (Guo et al., 2001).
In total, our data support a model in which a subset of subunits is delivered on vesicles and exocyst assembly is completed only as the vesicles arrive at sites of exocytosis marked by the remaining subunits, Sec3p and Exo70p (Fig. 8). An important implication of this model is that there must be a cycle of assembly for the exocyst. If we consider the cycle to begin with exocyst assembly and vesicle tethering, then a mechanism must exist for disassembly and recycling of the exocyst subunits (apart from Sec3p). We presently have no information on the mechanisms of disassembly and recycling. There must also be a mechanism for recruiting certain members of the exocyst onto newly formed, or forming, secretory vesicles. It has been speculated that exocyst components are loaded onto vesicles at the trans-Golgi (Munro, 2004), but this has yet to be shown experimentally.
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We have shown that the rate of recovery from photobleaching of a Sec8p-GFP fusion is dramatically reduced in a strain harboring the sec4-8 allele. We feel that this result validates the use of FRAP as a diagnostic tool for investigating the affects of mutations on subunit dynamics, and provides evidence that the affect on recovery rates caused by Latrunculin A treatment are accurately representing changes in vesicle movement rates and are not due to secondary effects of depolymerizing actin. Although it is not surprising that loss of Sec4p function would alter the rate of recovery, it will be interesting to see how that effect is mediated. Does the sec4-8 mutant fail to load exocyst subunits on secretory vesicles? Or, does Myo2p bind less effectively to vesicles in the sec4-8 mutant, leading to a delay in delivery? A more thorough analysis of how Sec4p affects photobleaching recovery rates may help answer questions concerning the role of Rab GTPases in vesicle generation, association with motor proteins, and tethering.
Although Sec3p is the most stable member of the exocyst, the rate of recovery from photobleaching for the Sec3p-GFP fusion ( = 59 ± 11 s) indicates that a rapid remodeling capability is maintained throughout the cell cycle. This finding is consistent with the requirement for dramatic remodeling of the exocytic machinery seen at two points in the cell cycle. The first transition is from a small region in the bud tip to an isotropic distribution in the large bud, and the second occurs when secretion moves from the bud surface to a ring around the mother-bud neck near the time of cytokinesis. Each of these transitions occurs in only a few minutes, so if Sec3p were substantially less dynamic, remodeling would be affected.
Secretory vesicles are targeted to sites of exocytosis in several distinct steps. Vesicles are transported along actin cables by the type V myosin, Myo2p (Govindan et al., 1995; Pruyne et al., 1998; Karpova et al., 2000). We have shown that most of the exocyst subunits are associated with the vesicles as they are transported. Once vesicles reach the end of the actin cable, they are tethered at sites of exocytosis through the interaction of the vesicle-associated exocyst subunits with Sec3p and Exo70p on the plasma membrane. Rho proteins coordinate vesicle delivery with vesicle tethering by interacting with both the formins, which assemble actin cables (Dong et al., 2003), as well as with Sec3p (Guo et al., 2001) and Exo70p (Adamo et al., 1999; Robinson et al., 1999), which mediate tethering. Assembly of the exocyst may serve to tether vesicles to tightly focused sites in preparation for membrane fusion, a step catalyzed by SNARE complexes. A key event of molecular recognition between target membrane and vesicles is initiated by the exocyst before SNARE complex assembly.
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Materials and methods |
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To establish how many modes of action were contributing to recovery for each subunit, we manipulated the basic equation that describes recovery (Salmon et al., 1984):
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Fluorescence and video microscopy
Epifluorescence microscopy and video microscopy of strains harboring exocyst proteins fused to a triple-GFP tag were conducted on cells grown overnight in SC medium at 25°C, and then diluted to OD = 0.2 and allowed to recover in fresh SC medium for 2 h at 25°C. Samples were collected by centrifugation, resuspended in fresh SC medium, and 7 µl of the suspension placed on a slide with coverslip. The cells were allowed to settle on the bottom surface of the slide for 5 to 10 min and examined on an Axioplan2 upright microscope (Carl Zeiss MicroImaging, Inc.) with 63x Plan Neofluor apochromatic oil-immersion objective lens with NA 1.3 and 100 W xenon excitation lamp. Images were captured with a cooled-CCD camera (model ORCA ER; Hamamatsu) and analyzed, and, if appropriate, enhanced with Openlab software (Improvision) running on an Apple G4 Macintosh computer. Still images were exposed for 0.25 to 1 s depending on sample brightness. Video images were collected with an exposure time of 160 ms per frame, resulting in a frame rate of five frames per second with no pixel binning. Individual movies varied in length from 50 to 100 frames (10 to 20 s).
EM
Strains analyzed by EM harbored a fusion of the relevant exocyst genes with 13 consecutive myc 9E10 epitopes, again as the sole copy and under the control of the wild-type promoter specific for the gene. Cells were grown overnight in SC medium, diluted in the morning to OD = 0.2 and grown to OD = 1.0, all at 25°C. Cells were fixed by addition of 37% formaldehyde to a concentration of 3.7% for 10 min, pelleted, resuspended in fixation medium (PBS + 2% glucose, 20 mM EGTA, and 3.7% formaldehyde), and incubated with shaking for 1 h at 25°C. Cells were washed twice in fixation medium without formaldehyde, resuspended in 250 mM Hepes, pH 7.4, with 8% PFA, and incubated overnight at 4°C without shaking. The fixed cells were pelleted and resuspended in 20% sucrose with 2% gelatin. After the gelatin had hardened, the gelatin cones were flash frozen in liquid nitrogen and thin cryo-sections were cut with a cooled ultramicrotome. Sections were incubated with commercial antibody against the 9E10 epitope (Invitrogen), labeled with 5- or 10-nm gold particles conjugated to protein A, and observed with an electron microscope (model 410; Philips). Images were captured at 16,500x on film (Kodak) and developed, and the negatives were examined to determine the surface area of cells and vesicles, as well as the number and distribution of gold particles compared with vesicles. Gold particles were considered to be associated with vesicles if they were within 50 nm of a subcellular structure unambiguously identifiable as a vesicle, as measured by examining negatives with a 7x loupe with submillimeter scale. However, if a gold particle was found to be within 50 nm of the plasma membrane, it was not counted as being associated with any vesicle. In this way, we eliminated association with the plasma membrane as a complicating factor when calculating vesicle-labeling ratio. The data yielded the cell area, the vesicle area (assuming an average vesicle radius of 40 nm), and the number of gold particles in each, and from these the density of gold particles in the cell as a whole (largely a function of washing efficiency) and the density of gold particles within 50 nm of a vesicle. We chose 50 nm as a conservative cutoff based on the size of the assembled mammalian exocyst (30 to 35 nm; Hsu et al., 1998) and the lengths of the antibody (10 to 15 nm; Amit et al., 1985) and gold-conjugated protein A (5 to 10 nm).
Online supplemental material
Videos 17 show time-lapse movies, acquired at five frames per second and displayed at 25 frames per second, of otherwise wild-type cells (strain background NY1210) harboring GFP-Sec4p or exocyst subunits fused to a 3xGFP tag at their COOH termini. All videos were captured and processed as detailed in the Materials and methods section Fluorescence and video microscopy. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408124/DC1.
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Acknowledgments |
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C. Boyd was supported by National Institutes of Health (NIH) training grant DK0717-28. This work was supported by NIH grants GM35370 and CA 46128 awarded to P. Novick.
Submitted: 20 August 2004
Accepted: 19 October 2004
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