We have previously characterized a mutant in the Cdc42 GTPase, which is specifically defective in post-Golgi vesicle docking and fusion with the plasma membrane but exhibits no detectable defect in the polarized localization of the exocytic machinery. Based on these results, we suggested that Cdc42 acts as a positive allosteric regulator of the late secretory apparatus at sites of polarized growth. Here, we show that another Rho GTPase in yeast, Rho3, acts in a similar manner to positively regulate exocytosis independent of any effect in the polarization of the exocytic machinery. Together, these findings suggest a novel mechanism for the action of Rho GTPases in polarized secretion. This new model, depicted in , is distinct from previous models in which Rho proteins were thought to act in polarization of exocytosis by sequestering components of the secretion machinery at a specific site on the plasma membrane (, top; Guo et al., 2001
; Symons and Rusk, 2003
). In this model, a polarized patch of activated Rho GTPase would act directly on the initially unpolarized late secretory machinery (, bottom). This localized signal would increase the activity of the machinery and hence the likelihood of a productive secretory vesicle fusion event at a precise site on the membrane. Because many components of the docking and fusion machinery—including the exocyst complex (Guo et al., 1999
; Folsch et al., 2003
; Vik-Mo et al., 2003
), Cdc42, and Rho1 (Abe et al., 2003
; Wedlich-Soldner et al., 2003
)—have been found to associate with secretory vesicles and other transport intermediates, ongoing allosteric regulation would be expected to lead to the polarization of the secretion apparatus and to a further increase in allosteric activation. According to this view, the polarization of the secretory machinery would then be a consequence, rather than a cause, of ongoing polarized delivery of vesicles to the plasma membrane. Based on this model, the activated Rho GTPases, rather than their effectors, would serve as the major spatial landmarks for polarized secretion.
Figure 10. Models for the spatial regulation of exocytosis by Rho GTPases. (A) Recruitment model for regulation of exocytosis. A polarized patch of GTP-bound Rho protein would recruit specific components of the exocytic machinery, including the exocyst complex, (more ...)
Previous work on the Sec3 component of the exocyst complex has led to the hypothesis that the localization of this protein might serve as a spatial landmark for polarization of vesicle docking and fusion events on the plasma membrane (Finger et al., 1998
). However, in the current study, we found that polarization of Sec3, through its NH2
-terminal RBD, is dispensable for its overall function in the cell, although a positive role for this domain was detected in combination with certain other secretory defective mutants. Indeed, although the NH2
-terminal RBD domain is essential for the polarization of Sec3, localization and function of the exocytic machinery is not dependent on Sec3 polarization. Our analyses of Sec3 localization using antibodies to examine the localization of native Sec3 suggest that its polarization is determined in the same way as the rest of the exocyst complex. This is in strong agreement with our model for Rho-dependent polarization of exocytosis. It is not presently clear why we see such distinct differences in the polarization of Sec3 to those seen in the previous studies done primarily with GFP-tagged forms of the protein. It is possible that either the presence of the GFP tag itself influences the localization of Sec3 or that our Sec3 antibodies recognize an epitope whose accessibility is sensitive to a conformational change in the protein.
Guo et al. (2001)
has suggested that, in the absence of polarized Sec3, the rest of the exocyst complex may be polarized through a parallel pathway involving Exo70. Because Exo70 binds to Rho3, this interaction could represent a redundant pathway for exocyst localization. We tested this hypothesis directly using an allele of Rho3 that has been shown to be specifically defective in interacting with Exo70. We find no evidence for redundancy between the Rho3–Exo70 and Rho1–Sec3 pathways. To exclude an indirect effect due to sec3-
overexpression, we performed similar experiments using a strain producing a wild-type level of Sec3-ΔN protein as the only source of Sec3. Again, no redundancy between the two pathways was observed (unpublished data). Thus, we find no evidence to suggest that either Sec3 alone or the exocyst as a whole act as spatial landmarks for exocytosis. Rather, we propose that the exocyst polarization is a consequence of ongoing polarized exocytosis (, bottom). Sec3 is the only component of the exocyst whose localization has been reported to be independent of the secretory pathway and the actin cytoskeleton (Finger et al., 1998
). In yeast, the Sec8 and Sec15 subunits are delivered to the plasma membrane through the secretory pathway, and the other exocyst subunits, with the exception of Sec3, act similarly (Finger et al., 1998
; Guo et al., 1999
, Boyd et al., 2004
). However, we report here that the mechanism by which Sec3 becomes polarized appears to be similar to that of the other exocyst subunits and involves polarized delivery of vesicles to the plasma membrane. Thus, it is unlikely that additional interactions between Rho GTPases and the exocyst would be involved in the initial polarization of this complex, but these signals would serve to regulate the assembled complex at sites of growth.
A prediction of the allostery model is that the function of Rho GTPases in exocytosis would not require GTP hydrolysis, as allosteric regulation is expected to be maximal in the GTP-bound state (Buck et al., 2004
; Peterson et al., 2004
). In support of this model, we found that Rho3 efficiently fulfills its function in exocytosis when locked in a GTP-bound form, as measured by suppression of specific late secretory mutants. In fact, the GTPase-deficient mutant can fulfill all the functions of Rho3 in the cell because it completely rescues a deletion in the gene in a manner that is genetically and morphologically indistinguishable from the wild-type RHO3
gene. In contrast, Cdc42 is known to require GTP hydrolysis for many of its functions in the cell, including septin ring assembly and early polarization (Gladfelter et al., 2002
; Irazoqui et al., 2003
). Consistent with this, we and others have found that most cdc42
mutant alleles fail to be rescued by a mutant form of Cdc42 unable to hydrolyze GTP. In contrast, we found that the cdc42-6
allele, which has a very specific defect in exocytosis, is well complemented by a GTPase-deficient form of Cdc42. These results provide further support for the fact that Rho3 and Cdc42 function in a similar allosteric fashion in the spatial regulation of exocytosis.
Overall, our analysis of the rho3-V51
mutants show that these Rho GTPases regulate secretion independent of their ability to polarize the exocytic machinery and to hydrolyze GTP. These observations lead us to propose a model in which Rho proteins work as allosteric regulators of the unpolarized secretion machinery by activating the fusion of vesicles with the plasma membrane. As a consequence of this activation, components of the secretion machinery, carried on post-Golgi vesicles, would themselves become polarized (Boyd et al., 2004
). This would be expected to result in the reinforcement of the polarization of this process by a positive feedback mechanism. Importantly, this model is applicable to polarization events observed in mammalian cells. In particular, the basolateral membrane recruitment of the Sec6/8 complex in epithelial cells is known to be a consequence of cell–cell adhesion. The polarized localization of the Sec6/8 complex correlates with, but does not precede, the development of polarized transport to the basolateral membrane (Grindstaff et al., 1998
). In the developing neuron, the polarized patches of Sec6/8 observed in the synaptic region disappear upon maturation of the synapse (Hazuka et al., 1999
). This phenomenon may simply reflect a decrease in the delivery of the exocyst components to the membrane, as trafficking strongly decreases after maturation of the synapse.
Determining the precise nature of allosteric regulation by Rho3 and Cdc42 on the exocytic machinery and the exocyst will be critical to understanding the molecular mechanism by which Rho proteins regulate polarized secretion. A likely mechanism for allosteric regulation would involve relief of an autoinhibitory interaction similar to that found for the effect of Rho GTPases in regulating the activity of members of the formin family (Zigmond, 2004
). By analogy, binding of the Rho3 GTPase to the RBD of Exo70 (for example) would relieve an inhibitory interaction within Exo70 or perhaps between Exo70 and another exocyst subunit. The conformational change created by the binding of Rho3 would then promote the ability of this complex to drive downstream events through direct effects on SNAREs (Sivaram et al., 2005
) or through an intermediary (Lehman et al., 1999
). In either case, the result would be a direct increase in the rate of vesicle docking and fusion at sites populated by the activated Rho GTPase.