In this report, we have presented evidence that Exo70 and Myo2 are downstream effectors of the Rho3 GTPase, which plays a critical role in the regulation of bud growth. Together with the effector Bni1, it appears that this GTPase has at least three different effector molecules which have all been implicated in aspects of actin cytoskeletal organization and exocytosis, as shown in Fig. .
Schematic showing the various effectors of Rho3 and the cellular processes that they regulate. See Discussion for an explanation of each interaction.
Rho3 directly interacts with Exo70, a component of the exocyst (a complex of Exo70, Sec3, Sec5, Sec6, Sec8, Sec10, and Sec15), which appears to reside in the acceptor membrane and is thought to facilitate docking of secretory vesicles, since a high concentration of Sec6, Sec8, and Sec15 was found at the tips of small buds, which represent active sites of exocytosis (53
). We have shown that the interaction of Rho3 with Exo70 requires the intact effector domain of Rho3 and is dependent on the GTP-bound form of Rho3 and that the patterns of localization of the two proteins overlap. It is possible that the interaction between Rho3 and Exo70 serves to properly localize Exo70. This hypothesis is supported by our demonstration that the localization patterns of these proteins overlap and that the localization of Exo70 is altered by the expression of an activated allele of RHO3
. This activated allele of RHO3
causes cells to become cold sensitive and elongated as well as bent at the sites where actin patches are localized (23
). Exo70 appears to accumulate at various regions, including the bud tips in Rho3E129,A131
-expressing cells. This localization pattern is also observed for Rho3E129,A131
. Thus, activated Rho3E129,A131
appears to affect the localization of Exo70, which may result in misdirection of the exocytosis machinery. Rho3 may exert additional effects on exocyst function by influencing the process of vesicle fusion; however, additional work is needed to clarify these points.
Rho3 also interacts with Myo2, an essential myosin in yeast that appears to play critical roles in the organization of the actin cytoskeleton. During polarized growth of yeast cells, actin patches, which represent the cytoskeleton-plasma membrane interface (42
), accumulate in the bud where cell growth is taking place; in addition, actin cables orient toward the bud (28
). These specialized structures do not form in the absence of Myo2 function, as the temperature-sensitive myo2-66
mutant displays delocalized actin patches at the restrictive temperature (26
). In addition, the mutants arrest as large unbudded cells. These phenotypes are similar to the rho3-1
mutant in which the cells become enlarged and rounded at the restrictive temperature, and actin patches lie scattered throughout the cell surface (23
). Furthermore, both the myo2-66
and rho3 rho4
mutants display delocalized deposition of chitin as well as a multinucleate phenotype (37
). This overlap of myo2-66
phenotypes is consistent with our assignment of Myo2 as an effector of Rho3 and raises the possibility that Rho3 affects the activity of Myo2. Finally, Rho3 may also influence changes in the actin cytoskeleton through its interaction with the Bni1 protein. The Bni1 protein contains two formin homology domains which are responsible for the binding of profilin, an actin binding protein (24
In addition to its role in actin cytoskeletal organization, Myo2 appears to be critical for the transport of a class of secretory vesicles to the bud, as secretory vesicles accumulate predominantly in the mother cell in the myo2-66
). Myo2 belongs to the class V unconventional myosin family which includes mouse dilute (39
) and chicken brain myosin V (14
), proteins also implicated in the transport of membrane-bound organelles (55
). Genetic interactions between MYO2
and seven late-acting sec
genes including four components of the exocyst have been detected (18
). Furthermore, both rho3-1
cells display synthetic lethality with sec4
). While the precise mechanism remains unclear, these observations raise the possibility that Myo2 and the exocyst work in concert to ensure polarized cell growth by properly depositing newly synthesized proteins at the bud site. Rho3 may be the G protein which regulates this process. It is interesting that the region of Myo2 where Rho3 interacts is located in the coiled-coil and non-α-helical C-terminal domain, a region that is thought to specify cargo loading in the class V myosins (40
How might Myo2, Rho3, and Exo70 work in concert to bring about proper bud growth? As discussed above, Myo2 may be responsible for transporting vesicles to the bud. Once in the bud, the vesicles then interact with proteins necessary to promote vesicle fusion, a process requiring both Sec4 and likely the exocyst (15
). Rho3 could function in this process early on by assisting Myo2 in transport of vesicles to the bud. This hypothesis is supported by the fact that Rho3-depleted cells undergo lysis with small buds, which could be indicative of the cargo never reaching its destination. One possibility is that Rho3 is actually inserted into the vesicular membrane and functions as the anchor to which both the vesicle and Myo2 bind. Upon arriving at the bud tip, Rho3 could then interact with Exo70 to facilitate the fusion event. Another possibility is that Rho3 located at the bud tip membrane can interact with Exo70. Upon arrival of the vesicle, Rho3, through its interaction with Myo2, could then function to facilitate vesicle fusion. The validity of these ideas awaits the results of genetic analyses which will give further insight into the exact role that these proteins play in the process of polarized cell growth.