Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit*

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G1 phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G2/M. Accordingly, phosphorylation of Gpa1 in G2/M phase leads to polyubiquitination in G1 phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.

Plasmid Construction-Yeast shuttle plasmids pRS315-ELM1 and pRS316-ELM1 were constructed by PCR amplification of ELM1 Ϯ 500 bp flanking the open reading frame (primers SalI-ELM1-F and SacI-ELM1-R) and directional cloning into the SalI and SacI sites of pRS315 or pRS316. Single point mutations (Gpa1 S200A , Gpa1 S200E , and Elm1 K117R ) were constructed by QuikChange (Stratagene) mutagenesis using the indicated primers (supplemental Table 2). Plasmids containing gpa1 15S/T-A were constructed by chemical synthesis of a 413-bp fragment of GPA1 (base pairs 392-786 of the open reading frame starting from the naturally occurring HindIII restriction site of GPA1) within which all serine and threonine codons were mutated to alanine, and a silent BglII site was introduced by mutation of base pairs 781 and 783 (GenScript). The synthesized fragment was cloned into GPA1 plasmids in which the same silent BglII mutation was introduced by QuikChange (using primer GPA1-BglII-F/R), followed by restriction digestion with HindIII and BglII. pYEX 4T-1-GST-ELM1 was purified from the yeast GST-fusion library host strain EJ 758 (28) using a yeast plasmid miniprep kit (Zymo Research) and then retransformed into BY4741 elm1⌬ cells for expression of GST-Elm1. All plasmids were verified by DNA sequencing.
Growth Arrest and Release-Log-phase cell cultures were arrested in G 1 phase with a synthetic ␣-factor peptide (30 M final concentration, CHI Scientific 53424), in S phase by addition of hydroxyurea (added in powder form to 10 mg/ml final concentration, Sigma Aldrich H8627), and in G 2 /M phase by addition of nocodazole (15 g/ml final concentration, Sigma Aldrich M1404). Note that for G 2 /M phase arrest, cells were first treated with 1% dimethyl sulfoxide for 30 min at 30°C, followed by addition of nocodazole (100ϫ stock at 1.5 mg/ml in dimethyl sulfoxide), resulting in a 2% final concentration of dimethyl sulfoxide in the cell culture. Each arrest was allowed to proceed for 2.5 h at 30°C unless otherwise noted. Cells were released from arrest by centrifugation and washing with 3 ϫ 100 ml of sterile water followed by resuspension in fresh medium to an A 600 nm of 0.7 and growth at 30°C.
In Vivo Ubiquitination Assays-Gpa1 polyubiquitination was detected by constitutive (ADH1 promoter) expression of Gpa1 in yeast harboring a temperature-sensitive proteasome mutation (cim3-1) or by coexpression of Gpa1 and Myc-ubiquitin as described previously (22). For cim3-1 and isogenic wild type cells, log-phase cultures were grown at the permissive temperature (25°C) to an A 600 nm of 0.5-0.6, followed by transition to the restrictive temperature (37°C) for 3 h. Inducible Mycubiquitin strains were grown at 30°C to an A 600 nm of 0.5-0.6, followed by addition of CuSO 4 to 100 M for 3 h at 30°C as described previously (29). Detection of Gpa1 ubiquitination at different cell cycle stages was accomplished by arresting logphase cells followed by induction of Myc-ubiquitin expression for 3 h.
Phosphatase Assays-Cells grown to an A 600 nm of 1.0 were harvested by centrifugation at 2000 ϫ g and stored at Ϫ80°C. Cell pellets were resuspended in 1ϫ phosphatase buffer (New England Biolabs) containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, 1 mM MnCl 2 , and 1ϫ EDTAfree protease inhibitors (Roche). Each resuspended pellet was split in half and subjected to glass bead lysis in the presence or absence of phosphatase inhibitors (50 mM NaF and 1.3 mM sodium orthovanadate) (S6508, Sigma Aldrich). Lysates were centrifuged at 21,000 ϫ g for 15 min, and the supernatant fraction was then collected into a fresh tube with or without 60 units (2.25 units/l final concentration) of protein phosphatase (New England Biolabs) for 30 min at 30°C. The reaction was stopped by addition of 6ϫ SDS-PAGE loading buffer, and the samples were immediately subjected to SDS-PAGE and immunoblot analysis. Alternatively, phosphatase assays were conducted on purified protein. Briefly, 1 l of yeast-purified Gpa1-FLAG was diluted in 1ϫ -phosphatase buffer with or without 20 units (2 units/l final concentration) protein phosphatase.
Affinity Purification of Gpa1-FLAG-Yeast harboring pRS316-ADH-GPA1-FLAG was grown to early log-phase and then harvested by centrifugation. The resulting cell pellet was lysed, and Gpa1-FLAG was purified as described previously (4).
Gene Transcription Assay-Pheromone-dependent transcription reporter assays were conducted as described previously (34). Briefly, cell cultures at an A 600 nm of 0.8 were dispensed (90 l into each of 48 wells of a 96-well plate) and mixed with 10 l of ␣-factor peptide at the indicated concentration for 90 min at 30°C. Next, each well was mixed with 20 l of FDG solution (130 mM PIPES (pH 7.2), 0.25% Triton-X100, 0.5 mM fluorescein di-␤-galactopyranoside (Marker Gene Technologies, M0250)) for 1 h at 37°C. The reaction was stopped by the addition of 20 l of 1 M sodium bicarbonate followed by fluorescence quantification using a fluorescence plate reader (Spec-traMax M5, Molecular Devices).

RESULTS
Gpa1 Is a Phosphoprotein-Gpa1 undergoes a variety of posttranslational modifications, including myristoylation, palmitoylation, and ubiquitination (21,36). The myristoylation state of Gpa1 can be distinguished by an electrophoretic mobility shift following SDS-PAGE and immunoblotting. Because only a fraction of Gpa1 is mobility-shifted under these conditions, the prevailing view has been that Gpa1 exists in both myristoylated and non-myristoylated states (37). However, upon close inspection of overexpressed protein, we found that even non-myristoylated Gpa1 G2A exhibits differential mobility by immunoblotting, indicating the presence of another modification (Fig.  1A). Given that phosphorylation can likewise alter the electrophoretic mobility of proteins, we asked whether phosphorylation rather than myristoylation might account for the second form of Gpa1. Consistent with this hypothesis, phosphatase treatment of whole cell extracts (Fig. 1B) or of purified Gpa1 (C) resulted in complete loss of the slower migrating form of Gpa1. We also found that mutation of serine 200, which was identified as a phosphorylation site by mass spectrometry (38), altered the mobility of Gpa1. Substitution of Ser-200 with alanine (S200A) FIGURE 1. Phosphorylation induces an electrophoretic mobility shift in Gpa1. Immunoblot analysis of Gpa1 using Gpa1-specific antibodies. A, cells over-expressing wild-type GPA1 (pAD4M-GPA1) (WT) or the myristoylationsite mutant GPA1 G2A (G2A) analyzed by immunoblotting. The arrow marks the position of the presumptive phosphorylated form of Gpa1. B, soluble protein extracts from cells overexpressing wild-type Gpa1 were split into two separate aliquots, and one half was treated (ϩ) with -protein phosphatase. p-Gpa1, phosphorylated Gpa1. C, Gpa1-FLAG purified from nocodazole-arrested cells and treated (ϩ) with -protein phosphatase (ppase). D, wild type cells overexpressing wild-type Gpa1 (WT) or phosphorylation site mutants (S200A, ALA or S200E, GLU). *, nonspecific immunoblot band.
or glutamate (S200E) replicated the mobility of dephosphorylated and phosphorylated Gpa1, respectively (Fig. 1D). We conclude that Gpa1 is a phosphorylated protein.
A Yeast Kinome Screen Reveals Elm1 as a G Protein Kinase-To identify the Gpa1 kinase, we monitored the phosphorylation-dependent mobility shift of endogenously expressed Gpa1 in gene deletion strains representing the majority of the yeast kinome. Of the 109 strains tested, deletion of ELM1 alone resulted in a significant observable loss of phosphorylated Gpa1. Phosphorylation of Gpa1 was restored by plasmid-borne expression of ELM1 ( Fig. 2A). We observed no such differences in the absence of kinases that act downstream of Elm1 or that are functionally similar to Elm1 (see discussion, supplemental Fig. 1A) (33,39). In addition to testing kinase deletions, we also monitored Gpa1 phosphorylation in each of 31 phosphatasedeletion strains, including all those involved in the pheromone response, but did not observe any difference in the abundance of phosphorylated Gpa1 (supplemental Fig. 1C). We conclude that Elm1 phosphorylates Gpa1 in vivo.
Elm1 Is Required for Maximal Pheromone-induced Gene Transcription-Elm1 is best known as a regulator of cell morphology during G 2 /M phase of the cell cycle. During G 2 /M, Elm1 phosphorylates proteins required for the morphogenesis checkpoint that coordinates bud emergence and mitosis (40,41) as well as for organization of septins during cytokinesis (33,39,42). Yeast harboring elm1 mutations exhibit a morphologi-cally distinct growth pattern in which cells delay cytokinesis and undergo elongated bud growth, a process that also occurs under conditions of nitrogen starvation and filamentous growth (43). Elm1 has been proposed to inhibit the filamentous growth response that includes multiple signaling pathway branches, including (but not limited to) the MAP kinases Ste20, Ste11, Ste7, and Kss1 (43,44), all of which participate as well in the pheromone response pathway. Accordingly, we found that Kss1 was more highly expressed and activated in elm1⌬ compared with wild-type cells (Fig. 2B). In addition, deletion of KSS1 in elm1⌬ cells reduced the filamentous-like phenotype (elongated buds and flocculation) typical of elm1⌬ cells (Fig.  2C). In contrast, the other pheromone-responsive MAP kinase, Fus3, was largely unaffected by the absence of Elm1 (Fig. 2B).
We next determined whether Elm1 regulates pheromonedependent gene transcription using a ␤-galactosidase reporter fused with the promoter of the mating-specific gene FUS1. We found that elm1⌬ cells exhibit a significantly reduced maximum level of pheromone-induced gene transcription. Signaling was restored upon plasmid-borne expression of wild-type Elm1 but not kinase-inactive Elm1 K117R (Fig. 2D). Thus, Elm1 represses activation of the MAP kinase branch of the filamentous growth response, including Kss1, and is required for maximum response to the pheromone.
Gpa1 Is Phosphorylated Directly by Elm1-Elm1 clearly plays a role in multiple signaling pathways, including the filamentous growth and pheromone response pathways, any of which could be indirectly responsible for the diminished Gpa1 phosphorylation in elm1⌬ cells. Therefore, we asked whether Elm1 acts directly on Gpa1. We established that purified Elm1 can bind to and phosphorylate recombinant Gpa1 in vitro (Fig. 3). Elm1 is capable of autophosphorylation (43), and we show that this activity is exhibited as well by GST-Elm1 (Fig. 3, A-C). Both Elm1 autophosphorylation and Gpa1 transphosphorylation were blocked when catalytically inactive GST-Elm1 K117R was substituted for the wild-type kinase (Fig. 3A). Consistent with our findings in vivo, Ser-200 is required for maximum phosphorylation of Gpa1 (Fig. 3B). Similar results were observed upon mutation of all 15 Ser and Thr residues in the ubiquitination domain (data not shown). Finally, Elm1 was able to bind to, but not effectively phosphorylate, Gpa1 ⌬UD , a mutant that lacks the ubiquitination domain of the protein (residues 129 -236) (Fig.  3, C and D). We conclude that Elm1 phosphorylates Gpa1 directly at Ser-200 and multiple other sites throughout the protein.
Gpa1 Phosphorylation Is Cell Cycle-dependent-We next determined how phosphorylation of Gpa1 is regulated. Elm1 is expressed primarily during S and G 2 /M phases of the cell cycle (42,43,45), a phenotype that we corroborated (supplemental Fig. 2). Further, longstanding evidence indicates that the mobility-shifted (phosphorylated) form of Gpa1 is significantly reduced upon pheromone stimulation (31), which induces cell cycle arrest in G 1 phase. Thus, we considered whether Gpa1 phosphorylation is regulated during the cell cycle. We directly compared Gpa1 from cells arrested in G 1 phase with ␣-factor (␣-F), in S phase with hydroxyurea, or in G 2 /M phase with nocodazole. Hydroxyurea inhibits deoxyribonucleotide synthesis, inducing a DNA replication checkpoint arrest in S phase (46). Nocodazole inhibits microtubule polymerization and induces a checkpoint arrest at the metaphase/anaphase transition of mitosis (47). We found that phosphorylated Gpa1 is most abundant in cells arrested in S and G 2 /M phase but lowest in cells arrested in G 1 phase (Fig. 4A). To determine the dynamics of phosphorylation during the cell cycle, we monitored Gpa1 during arrest and release in cells treated with nocodazole or hydroxyurea. To validate the arrest and release protocol, we monitored changes in the mitotic cyclin Clb2 (48). We found that phosphorylated Gpa1 accumulates in a coordinated fashion with Clb2 as cells arrest in either G 2 /M or S phase (Fig. 4, B  and D). Upon release from nocodazole, phosphorylated Gpa1 and Clb2 rapidly decrease in abundance and then increase concomitantly as cells reenter the cell cycle ( Fig. 4C and supplemental Fig. 3). Similar coordination between phosphorylated Gpa1 and Clb2 was evident in cells released from hydroxyureainduced cell cycle arrest (Fig. 4E). Finally, we observed temperature-dependent accumulation of phosphorylated Gpa1 in cdc6 -1 ts cells, which are incapable of DNA replication licensing, resulting in a post-START arrest in late G 1 /early S phase (Fig. 4A) (49,50). We conclude that Gpa1 phosphorylation is Elm1-dependent and cell cycle-regulated. Although phosphorylated Gpa1 accumulates throughout the S and G 2 /M phases, it is rapidly eliminated from cells during G 1 phase.
Elm1 Is Required for Gpa1 Polyubiquitination-We have shown previously that Gpa1 is polyubiquitinated by the SCF Cdc4 ubiquitin ligase (4) and that ubiquitination occurs primarily at lysine 165 within the ubiquitinated subdomain of Gpa1 (21). Typically, SCF recruits phosphorylated proteins as substrates for ubiquitination (24,25). Therefore, we asked whether Elm1 phosphorylation promotes Gpa1 polyubiquitination. We compared Gpa1 polyubiquitination in the presence and absence of ELM1 using a proteasome-deficient yeast strain, cim3-1 (22,23). For these experiments, Gpa1 was overexpressed to allow detection of the minor ubiquitinated species. Growth of cim3-1 cells at the restrictive temperature inacti-vates Cim3, an essential protein component of the 26 S proteasome (51), thereby stabilizing polyubiquitinated proteins and further enabling their detection by immunoblotting. Consistent with the hypothesis, Gpa1 polyubiquitination is considerably diminished in the absence of Elm1 or in the presence of plasmid-borne Elm1 K117R (Fig. 5). To validate the observations made in cim3-1 cells, we also monitored Gpa1 polyubiquitination in wild-type or elm1⌬ cells expressing myc-ubiquitin (proteins conjugated to myc-ubiquitin are degraded slowly) (supplemental Fig. 4) (52,53). Once again Gpa1 polyubiquitination was diminished in the absence of Elm1. We conclude that Elm1 is required for ubiquitination, as well as for phosphorylation, of Gpa1.
Gpa1 Ubiquitination Is Regulated during the Cell Cycle-Phosphorylation is a well established precursor to ubiquitination by the SCF (25) and serves as a signal for recruitment of target substrates by F-box proteins (54). Our previous findings indicate that Gpa1 is ubiquitinated by SCF Cdc4 . The data presented above indicate that Gpa1 is phosphorylated by Elm1 and that Elm1 is required for ubiquitination. Therefore, we postulated that phosphorylation by Elm1 precedes ubiquitination by SCF. To test this hypothesis, we compared the relative proportion of phosphorylated Gpa1 in cells lacking functional Cdc4 (F-box protein) or Cdc34 (ubiquitin-conjugating enzyme), which we previously identified as responsible for polyubiquitination of Gpa1 (4). Accordingly, we monitored Gpa1 in temperature-sensitive cdc4-1 and cdc34-2 mutants (27). Consistent with the hypothesis, we found the proportion of phosphorylated Gpa1 to be higher in asynchronous cells lacking active forms of either Cdc4 or Cdc34 but comparatively lower in identically treated wild-type cells (Fig. 6A, left panel). Similar results were obtained using Tet-repressible versions of CDC4 and CDC34 (data not shown). Thus, phosphorylated Gpa1 accumulates in the absence of functional SCF Cdc4 ubiquitin ligase.
Finally, we considered whether ubiquitination, in addition to phosphorylation, might be regulated by the cell cycle. In support of this model, accumulation of phosphorylated Gpa1 in SCF-deficient cells is comparable with that observed in G 2 /Marrested cells (Fig. 6A, right panel). Moreover, we found that Gpa1 polyubiquitination is significantly higher in cells arrested in G 1 phase with ␣-factor mating pheromone or by temperature inactivation of cdc28-1 when compared with cells arrested in S phase or in G 2 /M phase (Fig. 6, B and C, and supplemental Fig.  5). Although treatment with ␣-factor results in Far1-mediated G 1 arrest, cells expressing cdc28-1 undergo G 1 arrest independent of pheromone pathway activation at the restrictive temperature (55). In either case, Gpa1 polyubiquitination is highest in cells arrested in G 1 phase. Taken together, the data presented here reveal that Gpa1 is dynamically regulated. Phosphorylation and ubiquitination are independent of the pheromone stimulus and therefore not the result of feedback regulation. Rather, these modifications occur in conjunction with the cell cycle. Thus, the G protein is simultaneously a regulator of, and regulated by, cell cycle progression.

DISCUSSION
It is now well established that the G protein ␣ subunit Gpa1 is ubiquitinated. Here we have begun to discern how this ubiquitination event is regulated. Our investigation began by showing that Gpa1 is phosphorylated and that phosphorylation results in an electrophoretic mobility shift of the protein. This property allowed us to determine that a single kinase (Elm1) is necessary for proper phosphorylation in vivo. Using purified components, we showed that Elm1 is also sufficient for Gpa1 phosphorylation in vitro. Elm1 is expressed primarily in S and G 2 /M phases of the cell cycle. Correspondingly, we found that the G protein is phosphorylated in a cell cycle-dependent manner. Gpa1 is polyubiquitinated by SCF, and phosphorylation is typically required for SCF-mediated ubiquitination. Thus, we investigated whether phosphorylation leads to ubiquitination of Gpa1 and whether ubiquitination also occurs in a cell cycle-dependent manner. Indeed, although Gpa1 phosphorylation peaks in G 2 /M phase, ubiquitination occurs in the subsequent G 1 phase. These findings establish that G proteins can be regulated by progression through the cell cycle. More broadly, they raise the possibility that other pathway components may also be subject to cell cycle regulation.
The participation of Elm1 in G protein signaling was unexpected. Elm1 is best known as a protein kinase that coordinates events leading to cell division, including bud emergence, mitosis, and cytokinesis (33, 39 -43). In this capacity, Elm1 phos-  phorylates protein kinases involved in septin organization and cytokinesis (Gin4 and Cla4) (33,39,42), another kinase that phosphorylates and deactivates the morphogenesis checkpoint protein Swe1 (Hsl1) (40,41), and a fourth kinase that inhibits the mitotic exit network when the spindle position checkpoint is activated (Kin4) (56). It is unlikely that Gpa1 is regulated by any of these Elm1 substrates, given that deletion of those genes has no effect on Gpa1 phosphorylation or pheromone responsiveness. Thus, Gpa1 represents a new target of Elm1.
Whereas elm1⌬ was the only mutant that exhibited a loss of Gpa1 phosphorylation, some residual phosphorylation could still be detected in vivo ( Fig. 2A). Therefore, we speculate that Gpa1 is phosphorylated by another kinase. Possibilities include kinases that are absent from the gene deletion array, kinases essential for cell viability, and kinases that have functions related to Elm1. Elm1 is one of three closely related kinases, including Tos3 and Sak1. All three proteins are known to phosphorylate and activate Snf1 (the yeast homolog of human AMPK (5Ј AMP-activated protein kinase), primarily under conditions of glucose starvation (57). Under the normal growth conditions used to assess mating pheromone responses however, deletion of TOS3, SAK1, or SNF1 does not appear to affect Gpa1 phosphorylation or pheromone signaling (supplemental Fig. 1A). Taken together, our findings indicate that Elm1 is largely responsible for Gpa1 phosphorylation, is uniquely able to regulate the pheromone response pathway, and does so in a cell cycle-dependent manner.
Other lines of evidence support the model that Elm1 phosphorylates Gpa1. First, Gpa1 is neither phosphorylated nor ubiquitinated when Elm1 is absent. In contrast, we did not observe any such differences in the absence of 108 other kinases. Second, we did not observe any differences in the absence of 31 different protein phosphatases. Thus it is unlikely that Elm1 acts indirectly by inhibiting the function of a Gpa1phosphatase. Third, we detected a substantial accumulation of phosphorylated Gpa1 in the absence of the SCF function. Finally, we detected a substantial enrichment of Gpa1 polyubiquitination in G 1 -arrested cells as compared with S or G 2 /M-arrested cell cultures. These data confirm that phosphor-ylation and ubiquitination occur in a cell cycle-dependent manner and that phosphorylation precedes ubiquitination.
As the primary negative regulator of the mating pathway, Gpa1 is a logical target for regulation by posttranslational modifications. We have shown that pheromone-dependent gene transcription is diminished in cells that lack Elm1 function. Similarly, pheromone-dependent gene transcription is diminished in cells expressing Gpa1 ⌬UD . Gpa1 ⌬UD lacks a domain (UD, residues 129 -236) that is required for ubiquitination by SCF as well as phosphorylation by Elm1 (4,21,22). The Gpa1 ubiquitination domain cannot be the only target of phosphorylation, however, because mutation of all 15 serine and threonine residues within this region failed to diminish phosphorylation in vitro (data not shown) or ubiquitination in vivo (supplemental Fig. 4). Thus, alternate phosphorylation sites are likely to exist, and these may likewise target the protein for polyubiquitination, at least under some circumstances. Taken together, these data indicate that phosphorylation can occur at multiple sites throughout the protein, whereas ubiquitination is restricted to a specific subdomain of the protein.
We have now identified the primary components necessary for Gpa1 phosphorylation (Elm1) and polyubiquitination (SCF). While much has been learned, substantive questions remain. For instance, we have yet to establish how Elm1 and SCF work together to modulate G protein function. All existing data indicate redundancy within this process because perturbations to phosphorylation or ubiquitination have modest effects on G protein stability. Selective pressure in yeast may have instilled this property because cells lacking Gpa1 cannot grow as a result of G 1 cell cycle arrest. Alternatively phosphorylation and ubiquitination may affect G protein signaling in other ways. For example, ubiquitination could affect G protein catalytic activity. Ubiquitination could also serve to restrict Gpa1 localization to specific signal transduction complexes. Considering that Elm1 is localized predominately to the bud neck between dividing cells, Gpa1 phosphorylation and subsequent polyubiquitination may occur only during cell division or within a specialized subdomain of the plasma membrane (Fig. 7). Indeed, FIGURE 7. Model of cell cycle G protein regulation. Elm1 phosphorylates a subpopulation of Gpa1 during S and G 2 /M phase. Phosphorylated Gpa1 is stable until entrance into the following G 1 phase, when it is targeted for polyubiquitination by the SCF Cdc4 ubiquitin ligase. The selective mechanism responsible for initiating phosphorylation of a fraction of the total G protein population is unknown (?) but may be due to cellular localization of signal transduction complexes near the bud neck of a dividing cell where Elm1 is localized during the S and G 2 /M phases. Localization-specific cell cycle-dependent ubiquitination of Gpa1 may provide a mechanism to optimize the local stoichiometry of G␣ relative to G␤␥ subunits and restrict signaling competency to G 1 phase of the cell cycle.
Gpa1 was recently shown to concentrate to the bud neck during G 2 /M phase, where Elm1 is located (58).
The yeast mating response is perhaps the best-characterized of any signal transduction system, yet it continues to reveal new mechanisms of signal regulation. It has long been known that pheromone stimulation activates the G protein and promotes cell cycle arrest in G 1 . We now find that the G protein ␣ subunit is phosphorylated and ubiquitinated in a manner that is contingent on cell cycle progression. The abundance of phosphorylated Gpa1 increases as cells progress through the S and G 2 /M phases and decreases rapidly after cells divide and enter G 1 phase. Taken together, these data show that the G protein can be dynamically regulated. More broadly, these findings reveal a previously unsuspected degree of coordination between G protein signaling and cell division.