Fus2p is a key regulator of cell fusion, which localizes to the shmoo tip in pheromone-arrested cells. A previous study demonstrated that the localization of Fus2p-GFP is surprisingly dynamic (Paterson et al., 2008
) and was altered in mutants affecting mating and cell fusion. Fus2p-GFP was found to be nuclear in fus3
mutants or when expressed under the GAL1
promoter in mitotic cells, i.e., when the pheromone pathway is compromised or not active. In this study, we dissect the conditions necessary for nuclear versus cytoplasmic localization and show that both the pheromone pathway and the cell cycle play a role in regulating Fus2p localization. Pheromone signaling dependent on Fus3p plays a positive regulatory role in Fus2p exit, whereas cell cycle signaling via Cdc28p plays a negative role.
When asynchronous cultures are exposed to pheromone, most cells will be post-START and must complete the current round of cell division before cell cycle arrest. Immediately after START, Clns associated with Cdc28p block pheromone signaling and downstream cellular responses (Strickfaden et al., 2007
). However, as the cells enter G2, the pheromone signaling pathway becomes active, and the transcription of pheromone-regulated genes is allowed to proceed. Along with other pheromone-induced genes, Fus2p-GFP is expressed in G2 but localized to the nucleus until after mitosis. Because Fus2p is required for cell fusion and localizes to the site of cell fusion during conjugation, it is likely that the nuclear form is inactive with Fus2p sequestered away from its site of action. Although the specific function of Fus2p is not yet known, its homology with Rho–guanine exchange factor proteins suggests that it regulates a Rho-type G protein at the cell cortex. Because all of the Rho-type G proteins are constitutively expressed and play important roles during mitosis, premature activation by a pheromone-dependent regulatory protein would likely interfere with normal cell cycle progression. Thus, cells are faced with the problem of how to stockpile adequate levels of proteins required for mating while limiting their activity until after they have completed mitosis and are ready to mate.
We envision that four simple ways allow for delayed export and activation of Fus2p. First, Fus2p expression might be a delayed response to pheromone, perhaps by making expression dependent on an intermediate transcription factor like Kar4p (Lahav et al., 2007
). Second, a protein required for Fus2p exit may be expressed in a delayed manner. Third, Fus2p exit might depend on its reaching a critical concentration that triggers export. Fourth, exit may be coupled to cell cycle–dependent regulation. Because the timing of Fus2p exit was correlated with the time of cell division in an asynchronous population and not with the length of time in pheromone, the first two scenarios are unlikely. Separating expression of Fus2p from pheromone signaling response using the GAL1
promoter also had no effect. Thus, export was not a consequence of increased Fus2p levels, making the third scenario unlikely. In contrast, the observed coupling of exit to posttranslational cell cycle regulation provides an elegant solution to the problem of transition between mitotic growth and cell cycle arrest. Proteins required for mating can be synthesized before they are needed and sequestered until their activation will not compromise cell cycle progression.
We do not know how general the mechanism of cell cycle–dependent nuclear localization is to regulate mating-specific proteins. Several proteins are known that show cell cycle–regulated (e.g., Swi5p and Cdc24p; Moll et al., 1991
; Nern and Arkowitz, 2000
) or signaling-regulated nuclear localization (e.g., Msn2p and Msn4p; Jacquet et al., 2003
), and several proteins required for pheromone signaling shuttle between the nucleus and cytoplasm (e.g., Fus3p and Ste5p; Choi et al., 1999
; Mahanty et al., 1999
; van Drogen et al., 2001
). To our knowledge, Fus2p is unique in being subject to two different pathways of regulation. Two other proteins with different roles in mitosis and mating, Kar3p and Far1p, also show mating-regulated localization patterns being largely nuclear in mitotic cells but cytoplasmic in mating cells (Meluh and Rose, 1990
; Nern and Arkowitz, 1999
). It is not yet known whether the transition between these patterns is restricted during the cell cycle.
Cell cycle requirement for exit
Prolonged arrest in the cell cycle by itself is not required for Fus2p exit. Instead, it appears that Fus2p exit is negatively correlated with Cln levels in G1. First, far1
mutants do not arrest in the presence of pheromone, yet many far1
cells showed transient Fus2p exit as they progressed from cytokinesis into G1. Fus2p usually returned to the nucleus as the cells progressed into the cell cycle when Cln levels would be rising. Second, we observed frequent mother–daughter asymmetry in far1
mutants, with daughters showing exit more often than mothers. This is most likely because mothers have higher Cln levels immediately after cell division and progress into the cell cycle before daughter cells (Laabs et al., 2003
). Third, introduction of the cln2
deletion, which removes a G1 Cln, allowed cell cycle arrest and Fus2p exit in all far1
mutant cells. Moreover, in the far1 cln2
mutant, we observed Fus2p at the bud tip only in small-budded cells when the G1 Cln would be expected to be active. Fourth, inhibition of Cdc28p allowed Fus2p export in all stages of the cell cycle but only in the presence of pheromone. Thus, high Cdc28p activity is correlated with Fus2p nuclear localization, and low Cdc28p activity is permissive for Fus2p nuclear exit.
Pheromone requirement for exit
Previous results showed that Fus2p does not exit the nucleus in fus3
mutants (Paterson et al., 2008
). This is not simply a result of the inability of fus3
mutants to cell cycle arrest because the cln3
mutation suppressed arrest but not Fus2p exit. Moreover, it is not likely that Fus3p is only required for the transcriptional induction of a protein required for Fus2p exit. First, the fus3
mutant was not suppressed by high copy STE12
. Second, Fus2p that had already localized to the shmoo tip returned very rapidly to the nucleus when Fus3p kinase activity was chemically inhibited. It is much more likely that Fus2p is regulated directly by Fus3p. First, the Fus2NTD
fragment is phosphorylated at S84 in vivo in response to pheromone. Second, the phosphorylation is dependent on Fus3p, and Fus3p can phosphorylate Fus2p in vitro.
Third, mutation of Ser84 to alanine prevented export of both Fus2NTD
and full-length Fus2p. Conversely, mutation of Ser84 to glutamate or aspartate (negatively charged residues that mimic a phosphoryl group) allowed constitutive export of Fus2p-GFP in mitotic cells. Collectively, these results are most consistent with a direct requirement for Fus3p activity for Fus2p exit.
Because general inhibition of the mating signaling pathway is known to occur in late G1 through S phase, down-regulation of Fus3p would account for inhibition of Fus2p exit during this part of the cell cycle. Consistent with this, Fus2p was frequently found at the bud tip of small-budded STE5-8A cells, which are defective in the G1/S inhibition of Fus3p. Inhibition of Fus3p most likely accounts for the return of Fus2p to the nucleus observed in far1 mutant cells; as Cln levels accumulate at the end of G1, the mating pathway would be shut off and Fus3p made inactive.
Interestingly, pheromone regulation plays a separate role in the recruitment of cytoplasmic Fus2p to the plasma membrane because Fus2pS84E is diffusely cytoplasmic in mitotic cells, whereas it is tip localized in shmoos. Moreover, cytoplasmic Fus2pS84E also fails to localize to the cortex in the fus3 cln3 mutant treated with pheromone, suggesting that the defect is not simply a result of the absence of pheromone-induced proteins.
Mechanism of cell cycle regulation in late G2
Inhibition of Fus3p activation via Ste5p is limited to G1/S. Even upon extended pheromone treatment, active Cdc28p has, at best, a very minor effect on pheromone-dependent transcription during G2, arguing that Fus3p is fully active during G2. Accordingly, we favor a model in which the pheromone pathway and Cdc28p converge to regulate Fus2p localization.
How could such regulation work mechanistically? We envisage two general classes of models that differ in whether Cdc28p acts directly or indirectly. As an example of direct regulation, Cdc28p might phosphorylate Fus2p, causing nuclear import (or blocking export) independent of Fus3p phosphorylation. Alternatively, Cdc28p phosphorylation of Fus2p might block phosphorylation by Fus3p. If Fus2p is phosphorylated by Cdc28p, it is unlikely to occur within the NTD based on the mobility of the mitotically expressed protein in Phos-tag polyacrylamide gels. Arguing against the first model is the fact that the S84E mutant has constitutively greater cytoplasmic distribution than the wild-type protein, making it unlikely that Cdc28p phosphorylation can override S84 phosphorylation.
Examples of indirect regulation include the possibility that Cdc28p-dependent regulation controls the specificity of nuclear import or export receptors, thereby controlling Fus2p localization independent of Fus3p regulation. Alternatively, Cdc28p might regulate Fus3p substrate specificity in such a way that targets required for the transcriptional response to pheromone (Dig1p and Dig2p) can be phosphorylated, but Fus2p and possibly other targets required for later events in mating are not.
Ultimately, phosphorylation at S84 impacts the nuclear/cytoplasmic distribution of the protein. Phosphorylation is known to regulate the nuclear import of many proteins. For example, in Msn2p, phosphorylation by cAMP-dependent protein kinase interferes with NLS function (Gorner et al., 2002
). The S84 residue in Fus2p does not lie in a predicted NLS; indeed, there is no predicted NLS within the Fus2NTD
fragment that is both necessary and sufficient for nuclear localization. Elucidation of the mechanism of Fus2p localization will require identification of the true NLS and nuclear export signal and determining the impact of S84 phosphorylation and possibly phosphorylation at other sites.
We have described a novel mechanism regulating mating-dependent processes in yeast. Before pheromone-treated cells complete mitosis, FUS2 becomes active at the transcriptional level, but Fus2p is negatively regulated at the level of localization. As cells enter G1, two antagonistic signaling pathways converge to regulate the export Fus2p to the cytoplasm: positive regulation by the pheromone response pathway and relief of negative regulation by the cell cycle. We suggest that similar patterns of regulation impact other genes involved in the mating pathway as well as other differentiated states of the cell. Elucidation of the mechanism of Fus2p control will help us further understand the intricacies of this highly regulated biological process.