Localization of a protein to the bud tip represents a balance between targeting of the protein to this region, and diffusion or recycling of the protein from this site. The exocyst protein Sec15p is a direct downstream effector of Sec4p (Guo et al., 1999b
). We show here that the localization of Sec15p to the bud tip requires active Sec4p, suggesting that Sec4p controls the delivery and/or assembly of the exocyst complex at the bud. But in contrast to Sec4p, treatment of cells with latrunculin does not affect Sec15p localization. The initial targeting of Sec15p needs actin cables as Sec15p could not be found in the emerging buds in the tpm
mutant cells released from G0
. However, once Sec15p reaches its destination, its association with the plasma membrane seems to be quite stable. This hypothesis is consistent with the previous biochemical fractionation data that the majority of the endogenous Sec15p is associated with the plasma membrane (Bowser and Novick, 1991
) even though Sec15p clearly has the ability to associate with the vesicles (Salminen and Novick, 1989
; Guo et al., 1999b
). The rab protein Sec4p is also targeted to the bud along actin cables (Walch-Solimena et al., 1997
; Pruyne et al., 1998
). However, unlike Sec15p, Sec4p is rapidly depolarized in the presence of latrunculin (; also see Ayscough et al., 1997
) or in the tpm
mutant shifted to the restrictive temperature (Pruyne et al., 1998
). GTP-loaded Sec4p is transported via post-Golgi vesicles to the plasma membrane. Once reaching the bud tip membrane, its GAPs, Msb3p and Msb4p, promote GTP hydrolysis and Sec4p becomes GDP-bound (Gao et al., 2003
) at the bud. The GDP-bound form of Sec4p is then extracted from the lipids by Sec19p, the GDI for the rab GTPases, for subsequent recycling (Garrett et al., 1994
). Sec19p not only regulates Sec4p, its GDI function is toward all the rab proteins at different stages of membrane traffic. Therefore, in sec19
mutants, early stage trafficking defects were also detected (Novick et al., 1980
). It has been shown that polarized Sec4p localization needs functional upstream traffic (Walch-Solimena et al., 1997
). However, in the sec19
mutant, Sec4p accumulates in the bud (). This result suggests that the recycling of Sec4-GDP was blocked in the GDI mutant; therefore the remaining Sec4p was accumulated in the bud in a seemingly “polarized” manner. We have also treated the sec19-1
mutant cells by latrunculin and examined the localization of Sec4p in these cells. We expect that Sec4p will not be accumulated in the buds in sec19-1
cells as both the targeting and recycling of Sec4p are blocked. We indeed observed the loss of Sec4p in the bud (unpublished data). However, we also found that DMSO (used to dissolve latrunculin, as a control) treatment alone led to Sec4p delocalization in ~40% of the sec19-1
cells. One possibility is that DMSO, as an organic solvent, may modify the lipids in the plasma membrane, therefore complicating our interpretation of the GDI function at the plasma membrane.
The recycling of Sec4p in the presence of functional GDI is a fast process compared with that of Sec15p, as disruption of the actin cytoskeleton by latrunculin leads to rapid depolarization of Sec4p but not Sec15p. Sec15p, though controlled by Sec4p, is mislocalized in the GDI mutant. This mislocalization probably resulted from disruption of upstream trafficking in the sec19 mutant. The mechanisms for the recycling or diffusion of Sec15p and other exocyst proteins are unknown. Future experiments are needed to address this question.
We have also compared the localization of Sec15p with another exocyst protein, Sec3p, in the cells. The relationship between Sec3p and actin was rigorously tested previously (Finger et al., 1998
). Like Sec15p, the maintenance of Sec3p at the bud tip is not sensitive to latrunculin treatment. However, the targeting of Sec3p was also shown to be independent of actin as Sec3-GFP can be targeted as a patch-like structure even in G0
released cells in the presence of latrunculin (Finger et al., 1998
). Also in the tpm
mutant cells, Sec3p is much more stable than Sec15p at the bud tip, remaining mostly polarized after a 90-min shift to the restrictive temperature (). The time course of Sec15p localization closely resembles that of Sec8p in the tpm
mutant previously reported (Pruyne et al., 1998
; Guo laboratory, unpublished results). These results revealed that different mechanisms direct the targeting of Sec3p versus other members of the exocyst complex. We previously found that Cdc42p and Rho1p control the localization of Sec3p through their interaction with the N-terminus of Sec3p (Guo et al., 2001
; Zhang et al., 2001
). Rho1p and Cdc42p compete with each other in their binding to this region of Sec3p (Zhang et al., 2001
). This interaction is necessary for the polarized localization of Sec3p, as deletion of the N-terminus of Sec3p (Sec3ΔN) leads to mislocalization of Sec3p without affecting the assembly of the exocyst complex (Guo et al., 2001
). Interestingly, in the sec3
mutant strain, Sec15p is still polarized (Guo laboratory, unpublished results). Therefore, there must be parallel pathways that target different exocyst components to sites of active secretion. Here we demonstrate that Cdc42p controls Sec15p localization in the cells () similar to Sec3p. Although Cdc42p controls Sec3p localization through its direct binding with Sec3p N-terminus, its role for Sec15p and other exocyst components must be mediated through a Sec3p-independent pathway. Future works are needed to further compare these proteins in the same cells using time-lapse microscopy so that their kinetics differences can be better studied. These results revealed the diversity of exocyst targeting in yeast cells. As the exocyst functions in a step proceeding SNARE-mediated membrane fusion, the eight proteins are targets for cellular regulators (Lipschutz and Mostov, 2002
; Novick and Guo, 2002
; Hsu et al., 2004
). The assembly of the exocyst may integrate various sources of cellular information to ensure that exocytosis occurs at the right time and place.
Although our studies demonstrate that the post-Golgi secretory machinery is under the control of the actin cytoskeleton and polarity regulators, we also examined the localization of the polarity determinants in cells defective in exocytosis. We hypothesize that membrane traffic may be important for the delivery of polarity regulators to specific domains of the plasma membrane. We first examined Cdc42p, the “master” regulator for the establishment of polarity in yeast. Using affinity-purified antibodies, we found that the localization of endogenous Cdc42p at sites of polarized cell growth relies on a functional secretory pathway, as the sec
mutant cells lose Cdc42p staining at the bud tip over time. Cdc42p localization also needs actin cables, which are essential for polarized transport of secretory vesicles. Disruption of actin cables in the tpm
mutant results in loss of polarized localization of Cdc42p, consistent with the observation by Pruyne et al.
). Recently, Wedlich-Soldner et al.
) demonstrated that polarized localization of GFP-tagged Cdc42p required targeted secretion directed by the actin cytoskeleton. It was proposed that the F-actin–dependent transport of Cdc42p to the plasma membrane, which in turn is controlled by Cdc42p, provides a positive feedback loop that amplifies initial small signals for symmetry breaking in the early stages of yeast budding. Our study demonstrates that Cdc42p loses its localization in the established buds in exocytosis mutants. Our results suggest that a functional secretory pathway and actin cables are not only needed for bud emergence, but are also important for maintaining the localization of Cdc42p in the growing bud. Biochemical experiments (Wedlich-Soldner et al., 2003
) suggest that Cdc42p associates with secretory vesicles. It is possible that continued replenishment of Cdc42p to the bud to counter the endocytosis of bud membrane is important for the maintenance of Cdc42p at sites of cell polarization.
The function of Cdc42p is carried out in conjunction with Bem1p, which interacts with Cdc42p, its GEF Cdc24p, and other signaling molecules involved in cell polarity establishment. Here we found that Bem1p also loses its polarized localization in the sec
mutants, suggesting that the maintenance of Bem1p requires polarized transport. However, the time course of Bem1p depolarization is slower than that of Cdc42p. Especially, the slow depolarization of Bem1p in the tpm
mutant, which is known to rapidly lose cables after 1 min (Pruyne et al., 1998
), suggests that loss of actin cables affects Bem1p polarization indirectly over time. It is possible that the diffusion or recycling of Bem1p from the bud takes a time course different from Cdc42p. A recent finding suggests that the self-assembly of Bem1p leads to scaffold-mediated symmetry breaking in yeast (Irazoqui et al., 2003
). It is possible that the mislocalization of Bem1p observed in our study resulted indirectly from Cdc42p mislocalization in the mutants. Besides Bem1p, it was reported that proteins regulating actin polymerization, the motor protein Myo2p, and actin itself are disrupted in secretory mutants (Jin and Amberg, 2000
; Gao et al., 2003
; Aronov and Gerst, 2004
; Pruyne et al., 2004
). Overall these studies present a cyclical regulatory mechanism that may be important for the establishment and maintenance of polarized cell growth. In this regard, it is interesting to note that several yeast two-hybrid screenings from different sources revealed potential interactions between Sec15p and Bem1p (Drees et al., 2001
; also see SGD for more references). Here, we demonstrated that Sec15p indeed interacts with Bem1p in yeast cells. We have previously shown that Cdc42p directly interacts with Sec3p (Zhang et al., 2001
). Stringent cytological experiments suggest that Sec3p, unlike the other exocyst components, is not dependent on actin for its polarized localization (Finger et al., 1998
). Sec3p may associate with the plasma membrane and then assemble with other exocyst proteins, including Sec15p, that arrive at the bud tip via secretory vesicles along the actin cables (Finger and Novick, 1998
). The interaction of Sec15p with Bem1p and its assembly into the exocyst complex bring Cdc42p, its exchange factor Cdc24p, and the Cdc42p effector Sec3p into close proximity at the bud tip. These molecular interactions may represent a positive feedback loop coupling vesicle delivery and cell polarization. However, to test this hypothesis, the molecular details of the interactions clearly need to be elucidated first.
If secretion is important for the polarized localization of cell polarity regulators, why were cell morphology defects not observed in sec
mutants? Most of the sec
mutants previously isolated were “tight” alleles. On shifting to the restrictive temperature, bud emergence stopped quickly, as indicated by the nearly constant number of cells and buds (Novick et al., 1980
). In a heterogeneous population, cells were found to arrest at all stages of cell cycle, and no significant increase in cell size was noted (Novick et al., 1980
; Guo laboratory, unpublished observation). These characteristics of these sec
alleles are results of the screening strategy: only “dense” cells in which net cell surface growth stopped while cell mass increased, were selected (Novick et al., 1980
). Identification of new alleles of exocyst mutants may help reveal defects in both secretion and morphogenesis.
This cyclical regulatory mechanism between secretion and cell polarity has been observed in mammals. One recent example was presented by O'Brien et al.
). The authors demonstrated an autocrine loop, in which epithelial cells create their own extracellular polarity cue through exocytosis during cystogenesis. The secretion and assembly of extracellular laminin acts back on the cells to direct membrane traffic. Blocking the activity of the small GTPase Rac1 disrupts the loop, which leads to an inversion of the apical pole. Future studies using a variety of model systems will help us better understand the molecular basis and biological consequences underlying this cyclical regulatory mechanism.