To identify the protein(s) responsible for Fus2p transport, yeast strains harboring mutations in the various myosins were examined. Among the yeast myosin genes, only mutations in the class V myosin, Myo2p, compromised Fus2p localization and transport. The strongest evidence in support of a role for Myo2p in Fus2p transport was the observation that the average maximum speed of Fus2p puncta was significantly reduced in mutants in which the length of the Myo2p neck domain was decreased. We conclude that Myo2p transports Fus2p along actin cables to the shmoo tip during yeast mating.
During polarization in response to pheromone, the actin cytoskeleton becomes oriented toward the shmoo tip. Consequently, new cell surface growth and secretion occurs primarily at the shmoo tip, and proteins associated with the secretory pathway and/or actin transport will necessarily become initially concentrated in the mating projection. Retention of cell fusion proteins at the shmoo tip is required to prevent mislocalized and/or ectopic activation of the fusion machinery. Retention at the shmoo tip may arise from multiple mechanisms, including slow diffusion coupled with endocytic recycling (e.g., Snc1p; Valdez-Taubas and Pelham, 2003
), stable association with distinct lipid microdomains (e.g., Fus1p; Bagnat and Simons, 2002
; Jin et al., 2008
), and protein–protein interactions. In the case of Fus2p, retention is dependent on both Fus1p and one or more components of the actin cytoskeleton. We cannot yet determine whether Myo2p plays a direct role in Fus2p retention, because all mutations that cause loss of Fus2p-GFP localization also cause loss of actin polarization.
An unexpected result of the work described in this study is the differential stability of actin polarization in mitotic cells and in shmoos. We found that mitotic myo2-16
cells retained normal cytoskeletal polarization after short incubations at the restrictive temperature, whereas actin polarity was very rapidly lost in pheromone-treated myo2-16
cells under identical conditions. Furthermore, actin polarization in shmoos was specifically impaired in the myo2
mutants with defects in vesicle binding and not mutants with defects in vacuole and/or mitochondria binding. These findings, along with other recent results, suggest a positive feedback loop between actin-dependent transport and polarization of the cytoskeleton (Pruyne et al., 1998
; Wedlich-Soldner et al., 2003
; Aronov and Gerst, 2004
; Pruyne et al., 2004
). According to one model, cell polarity in yeast is established by the Rho-GTPase Cdc42p, which activates actin assembly via the formin family of proteins (Park and Bi, 2007
). Localized deposition of Cdc42p at intrinsic spatial markers (i.e., the bud site in mitotic yeast) is supplemented by the actin-dependent delivery of Cdc42p, creating a feedback loop for robust cell polarization. Cdc42p is a putative vesicle-associated cargo of Myo2p (Wedlich-Soldner et al., 2003
), and mutations that break this feedback loop (i.e., by blocking steps in the late secretory pathway) cause aberrant cytoskeletal polarity (Aronov and Gerst, 2004
). Thus, the loss of polarization that we observed in myosin and tropomyosin mutants may be due to the mislocalization of Cdc42p.
The different kinetics for the loss of actin polarity between mitotic cells and shmoos suggest that mitotic cells have a more robust system to maintain cell polarization. One major difference between the two cell types is the structure of the bud neck. Mitotic yeast cells have a ring of septins, a specialized structure formed by the copolymerization of four GTPases, at the cortex at the mother-bud neck (Longtine et al., 1996
). The septin ring is required for the maintenance of components of the yeast polarisome in the bud, and cells with temperature-sensitive mutations in the septins lose actin polarity at the restrictive temperature (Barral et al., 2000
). It has been proposed that the septin ring forms a passive diffusion barrier which prevents the flow of polarizing factors out of the daughter cell. Thus, when the cytoskeleton is perturbed by the inactivation of Tpm1p or Myo2p in mitotic cells, polarity may be partially maintained by the septin ring. In mating cells, septins form a more diffuse band of fibers around the neck of the shmoo oriented toward the shmoo tip (Longtine et al., 1998
; L. Silverstein and M. Rose, unpublished observations). Although the role of the septins during conjugation is not clear, they do not appear to function as a diffusion barrier for cortical membrane proteins between the tip of the shmoo and the cell body (Proszynski et al., 2006
). Therefore, in shmoos, once the actomyosin transport system is disrupted, there would be no barrier to prevent the rapid diffusion of polarity proteins (presumably including Cdc42p) throughout the cell.
Based on its localization to cellular regions containing high concentrations of vesicles and the membrane affinity of its binding partner, Rvs161p, it has been proposed that Fus2p is associated with vesicles that carry hydrolytic enzymes required for cell wall degradation (Paterson et al., 2008
). At the zone of cell fusion, Fus2p may trigger the release of the vesicular cargo via interaction between its Rho-GEF domain and a Rho-GTPase. The finding that Myo2p, a transporter of membrane-bound organelles, also transports Fus2p puncta supports the hypothesis that Fus2p is associated with membrane-bound organelles.
In S. cerevisiae
, vesicle trafficking is controlled by a family of 11 Rab GTPases that function in vesicle budding, vesicle tethering, and the fusion of vesicles with their target membranes (Lazar et al., 1997
; Grosshans et al., 2006
). Each Rab GTPase is believed to act at a defined step in the trafficking pathway, and Sec4p has been identified as the Rab GTPase specifically required for the fusion of exocytotic secretory vesicles with the plasma membrane (Walch-Solimena et al., 1997
; Novick and Guo, 2002
). We found that Fus2p puncta and Sec4p puncta were transported at significantly different speeds, and Fus2p-mCherryFP did not appear to colocalize with Sec4p-GFP in cytoplasmic puncta. We have also found that Fus2p is able to stay associated with the shmoo tip under conditions in which Sec4p does not, including in myo2-16
shmoos. At this time, we cannot rule out the possibility that an interaction occurs between Sec4p and Fus2p-marked cargo specifically at the shmoo tip or that a minor fraction of Sec4p associates with Fus2p-marked cargo during transport. However, taken together, our observations suggest that Fus2p and Sec4p localize predominantly to different populations of vesicles or organelles that are transported by Myo2p.
The existence of multiple classes of vesicles has been demonstrated in mitotic yeast cells (Harsay and Bretscher, 1995
); however, trafficking in shmoos remains relatively unexplored. Interestingly, two groups have reported that the average size of vesicles found at the shmoo tip is significantly smaller than the average size of vesicles found in budding cells (Baba et al., 1989
; Breton et al., 2001
). Thus it is possible that vesicle formation and trafficking occur via somewhat different mechanisms in pheromone-treated cells and in mitotic cells. In support of this hypothesis, Fus1p, which is not expressed in mitotic cells, appears to play a role in the clustering or anchoring of vesicles to the shmoo tip during mating (Gammie et al., 1998
Our findings therefore suggest that during the pheromone response, Myo2p transports at least two distinct classes of membrane-bound cargo. The first class constitutes the canonical, Sec4p-associated vesicles bound for the plasma membrane. These vesicles presumably carry cell wall components that are required for the formation of a mating projection and are tethered to the plasma membrane by the exocyst complex (Whyte and Munro, 2002
). The second class of cargo is marked by Fus2p and is specifically required for cell fusion. This cargo may include hydrolytic enzymes necessary for cell wall degradation and would be anchored to the cortex by a complex including Fus1p, rather than the exocyst. Fusion of this cargo to the plasma membrane would be triggered by a signal generated by prezygote formation and may require the activity of the Fus2p GEF domain. The existence of distinct classes of vesicles or organelles would allow cells to fulfill the two opposing requirements for efficient mating: first, cells must grow and add cell wall in the direction of the pheromone gradient and second, cells must be able to specifically degrade their cell wall during zygote formation.