We have monitored the dynamic behavior of exocyst subunits fused to GFP by measuring fluorescence recovery from photobleaching at bud tips. We were able to divide exocyst subunits into two groups based on several criteria. The first group consists of Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p, which all recovered with τ near to that of the vesicle-associated Rab GTPase, Sec4p. Their τ ranged from 11 s for Exo84p to 26 s for Sec6p, with an average of 18 s, compared with 12 s for Sec4p. Sec3p and Exo70p define the second group, which recovered severalfold more slowly. A key distinction between the two groups is their recovery behavior in the presence of Latrunculin A. Both Sec3p and Exo70p recovered in the presence of Latrunculin A, whereas the members of the first group all failed to do so. Therefore, the first group is distinguished from Sec3p and Exo70p not only by the speed with which they recover, but also by their requirement for an intact actin cytoskeleton for their recovery from photobleaching. This distinction from Sec3p is also evident in the results from immunoelectron microscopy and in vivo video microscopy. These experiments were designed to detect association of exocyst components with vesicles, and, in both cases, all subunits except for Sec3p were found to be in association with vesicles and moving in a manner consistent with vesicular traffic. The conclusion that we draw from these data is that all exocyst subunits, except for Sec3p, are transported to sites of exocytosis on vesicles, where they interact with Sec3p to tether those vesicles to the plasma membrane.
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 ( 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 (τ = 57 ± 17 s) was almost identical to the rate determined for Sec3p-GFP in both Latrunculin-treated and untreated cells (τ = 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 (). 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.
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 (more ...)
Sec6p and Sec8p occupy intermediate positions with respect to some of the criteria that we have used to characterize exocyst components in this work. They have photobleaching recovery times that are somewhat longer than those for other actin-dependent exocyst subunits, and their association with vesicles as measured by immunoelectron microscopy is less robust than that for any other subunit except for Sec3p. But they both require actin for photobleaching recovery, and both are seen by videomicrography to move in a manner consistent with vesicular transport to sites of exocytosis. Why they should have photobleaching recovery times that are significantly greater than that for other exocyst subunits is unclear, but it may reflect a tendency to associate more strongly than other subunits with sites of exocytosis after tethering. With respect to the immunoelectron microscopy results, it may simply be the case that the tags are not readily accessible to the antibodies used to probe their locations in the cell. If the carboxy termini (and thus the 13myc tags) of Sec6p and Sec8p are less accessible than the carboxy termini of other subunits, then the detecting antibody would be less likely to bind the tag and this may well manifest itself as a reduced ratio of vesicle-specific labeling.
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 (τ = 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.