Several lines of evidence previously suggested an interaction between Cdc42p and Fus2p. First, they interact by two-hybrid assay (Nelson et al., 2004
). Second, BEM1
, encoding a scaffold for Cdc42p and its effectors, is a high-copy suppressor of fus2
(Fitch et al., 2004
). Third, high-copy FUS2
suppresses the mating defects of bem1
mutations (Leberer et al., 1996
). Finally, Fus2p contains a Dbl-homology domain (Paterson et al., 2008
), which is shared with activators of Rho-GTPases. However, Cdc42p has numerous functions and interaction partners and so could potentially have multiple direct and indirect roles during mating.
To implicate CDC42 specifically in cell fusion, we sought mutant alleles defective in cell fusion but not other steps of mating. One mutation, cdc42-138, caused a strong defect in cell fusion but not in mitotic growth, which was not due to impaired pheromone signaling or cell polarization. The mutant phenotype closely resembled fus2 mutations and analysis of double mutants showed that cdc42-138 and fus2 mutations conferred defects in the same step in cell fusion.
The Dbl-homology domain in Fus2p is required for as much as 90% of Fus2p's function in cell fusion. The presence of residual function suggests that Fus2p may have other roles in cell fusion or more than one interaction with Cdc42p. Indeed, we found that Cdc42p interacts with the Dbl-homology domain and additional sequences in Fus2p.
Although Fus2p contains a Dbl-homology domain found in other Cdc42p GEFs, we did not detect GEF activity (unpublished data). Instead, we found that Fus2p interacted more strongly with Cdc42-Q61L, the GTP-bound form of Cdc42p, in vitro, consistent with the two-hybrid data (Nelson et al., 2004
). Because GEFs are expected to bind more strongly to the GDP-bound G-protein, these findings suggest that Fus2p is not a GEF but may instead serve either as a mating-specific effector for Cdc42p or to localize the activated Cdc42p to the cell fusion zone. It is notable that another putative GEF, Lte1p, requires its GEF domain for cortical localization and not for activation of Tem1p (Geymonat et al., 2009
If Fus2p is an effector, what might be the role of the Fus2p–Cdc42p interaction in promoting cell fusion? Fus2p binds the BAR domain–containing protein Rvs161p, and the interaction is essential for cell fusion (Brizzio et al., 1998
). BAR-domain proteins preferentially bind to curved membranes (Peter et al., 2004
), and Rvs161p, together with Rvs167p, binds to membranes and promotes curvature in vitro (Friesen et al., 2006
; Youn et al., 2009
). Mutations in Rvs161p that inhibit membrane binding also inhibit mating (Youn et al., 2009
), suggesting that Fus2p/Rvs161p's role in mating requires membrane interaction.
Fus2p localizes near clustered vesicles, and both Fus2p and the vesicles are partly delocalized in fus1
mutants (Gammie et al., 1998
; Paterson et al., 2008
). Deletion of neither Fus2p's Dbl-homology domain nor cdc42-138
affected Fus2p localization. Moreover, vesicles clustered normally in cdc42-138
. These data suggest that the Cdc42p interaction might regulate an activity associated with Fus2p/Rvs161p rather than its localization. Recent work showed that membrane bending by the vesicle protein synaptotagmin is required for vesicle fusion (Hui et al., 2009
). Perhaps Cdc42p regulates the degree of curvature induced by Fus2p/Rvs161p, deforming vesicle and/or target membranes in aid of membrane fusion. Alternatively, Fus2p/Rvs161p might interact with the curved outer surface of the vesicle fusion pore, stabilizing it to promote fusion. In either case, Fus2p/Rvs161p may play a role in facilitating the exocytosis of secreted proteins required for cell wall dissolution.
A role for the Rho-insert domain
The Rho-insert domain is a ~20-residue loop containing a 13-residue α-helix unique to the Rho subfamily of small G proteins (Feltham et al., 1997
). The residues of Cdc42p required for cell fusion (D121 and D122) are within four charged amino acids at one end of the Rho-insert domain. Previous work in yeast was not informative as to possible function; individual mutations to alanine did not affect growth; mutation of all four was lethal (Kozminski et al., 2000
). A role in cell fusion represents the first specific function identified for this region of the protein in yeast.
Although little is known about Cdc42p's Rho-insert domain in yeast, hints may be gleaned from human Cdc42 and Rac1, where it is involved in the interactions with a variety of proteins, including Rho GDP dissociation inhibitors (Wu et al., 1997
; Richman et al., 2004
), formins (Richman et al., 2004
; Lammers et al., 2008
), and IQGAPs (McCallum et al., 1996
; Li et al., 1999
; Owen et al., 2008
). It is possible that in yeast other protein interactions may be affected in addition to Fus2p. Although the yeast formin Bni1p is required for cell fusion, it also is required for cell polarization (Evangelista et al., 1997
; Matheos et al., 2004
; Paterson et al., 2008
), likely acting prior to the function identified by cdc42-138
. Of interest, the yeast IQGAP, Iqg1p, regulates exocytosis by localizing Sec3p to polarized growth sites (Osman et al., 2002
), and Sec3p interacts with activated Cdc42p (Zhang et al., 2001
). Continuous secretion has been shown to be required for the late stages of cell fusion (Grote, 2010
). However, a specific requirement for either Iqpg1p or Sec3p in cell fusion has not been tested.
Cdc42 plays roles at multiple stages of mating and cell fusion
Analyzing the many roles of GTPases has required extensive use of point mutations to specifically interfere with individual functions (Mosch et al., 1999
; Kozminski et al., 2000
; Adamo et al., 2001
; Saka et al., 2001
; Barale et al., 2006
; Heinrich et al., 2007
). For example, this approach demonstrated that cdc42-V36M
affects cell fusion but not mitotic growth (Barale et al., 2006
). We found that cdc42-V36M
did not polarize in response to pheromone and showed unstable localization of Fus2p-GFP (), closely resembling spa2
. Fus1p-GFP also failed to localize in cdc42-V36M
(Barale et al., 2006
). We conclude that cdc42-V36M
is primarily defective for polarization and may affect cell fusion indirectly, possibly through Bni1p. The cdc42-V36M
mutant may also have a specific defect in cell fusion, which is masked by the earlier polarization defect.
In contrast, cdc42-138
mutant cells polarized normally and were specifically defective in cell fusion. Epistasis analysis showed that Spa2p acts upstream of Fus1p, which in turn acts upstream of Fus2p (Gammie et al., 1998
). The morphology of cdc42-138
most closely resembled fus2
, arguing that this allele disrupts a function downstream of Fus1p and Spa2p. In confirmation, double-mutant analysis showed that fus2
act at the same step.
We conclude that Cdc42p plays multiple roles during cell fusion. Roles in pheromone signaling, via Ste20p (Simon et al., 1995
; Zhao et al., 1995
), and polarization, via Bni1p (Evangelista et al., 1997
), are well established. We propose a separate function in cell fusion mediated by the Rho-insert domain in association with Fus2p/Rvs161p.
A conserved role of Cdc42 homologues in eukaryotic cell fusion?
Among metazoan cell fusion events, Drosophila
myoblast fusion may be the best understood (Chen and Olson, 2004
). Ultrastructural studies revealed clustered, electron-dense vesicles across the cell fusion zone (Doberstein et al., 1997
), similar to yeast cell fusion and vertebrate myoblast fusion (Lipton and Konigsberg, 1972
). Recent work showed that, as in yeast, fusion occurs at a single site, coincident with accumulation of actin (Sens et al., 2010
). Cdc42p homologues Drac1 and Drac2 are also required for myoblast fusion (Hakeda-Suzuki et al., 2002
) and most likely activated by an unconventional GEF (Erickson et al., 1997
; Brugnera et al., 2002
). Moreover, Cdc42p is required for mouse myoblast fusion (Vasyutina et al., 2009
). The conserved requirement for Cdc42p-related proteins in cell fusion suggests that yeast cell fusion will yield important insights into the mechanism in higher cells.