Separating the Polarity Role of Gβγ from Its Signaling Role
The Gβγ dimer normally performs two roles in the mating pathway: it activates MAP kinase cascade signaling and it regulates proteins that control cell polarity. Our goal in this work was to study the polarity role of the receptor–Gαβγ module in isolation from its requirement in activating the MAP kinase cascade. Therefore, we used a variety of methods to activate signaling independent of pheromone and Gβγ, and then studied how perturbing Gαβγ function affects chemotropism and cell polarization. This strategy is an extension of one used previously in which overexpression of the transcription factor Ste12 was used to study the mating role of various MAP kinase pathway components while bypassing their role in transcriptional induction (Schrick et al., 1997
). Here, we used several newer reagents such as membrane-targeted versions of Ste5 (Ste5ΔN-CTM and Ste5ΔN-Sec22), which can promote robust MAP kinase cascade signaling, wild-type levels of mating, and normal polarized morphogenesis (Pryciak and Huntress, 1998
; Harris et al., 2001
). To ensure that communication between Gβγ and Ste5 was severed, these reagents used a truncated form of Ste5 that lacks the Gβγ-binding site (Ste5ΔN), and all assays were performed in strains where the genomic STE5
locus was deleted (ste5
Δ). For comparison, we also used a constitutively active form of Ste11 (Ste11ΔN) (Cairns et al., 1992
; Neiman and Herskowitz, 1994
) and overexpressed Ste12 (Dolan and Fields, 1990
; Schrick et al., 1997
). When signaling output was measured by induction of a transcriptional reporter construct (FUS1-lacZ
), we found that some bypass methods caused stronger activation than others, but all were independent of Ste4 (Gβ) and pheromone (A, right).
Figure 1. Chemotropism role of Gαβγ and receptor is separate from signaling. (A) Chemotropism requires Ste4 even when its signaling role is bypassed. Strains PPY858 (ste5Δ) and PPY886 (ste5Δ ste4Δ) harbored galactose-inducible (more ...)
We then used these signaling bypass methods to address the role of Gβγ in chemotropic mating. Chemotropism was monitored using a “pheromone confusion” assay (Dorer et al., 1995
; Nern and Arkowitz, 1998
), in which mating success is compared in the absence versus presence of excess exogenously added pheromone (α factor), which obscures natural pheromone gradients emanating from partner cells. Chemotropically proficient cells can use pheromone gradients to locate mating partners, and thus they mate with higher efficiency when pheromone gradients are left intact (−α factor) than when gradients are obscured (+α factor); cells defective at chemotropism cannot follow pheromone gradients, and thus they mate at the lower efficiency under either condition. Thus, this assay does not directly monitor directional growth, but rather it infers the ability of cells to detect and use pheromone gradients. Unlike earlier measures of “mating partner discrimination” (Jackson and Hartwell, 1990
; Jackson et al., 1991
), chemotropic proficiency in the pheromone confusion assay requires both Far1 and Far1–Cdc24 binding (Dorer et al., 1995
; Valtz et al., 1995
; Nern and Arkowitz, 1998
), and it accurately reflects the ability of cells to establish a new polarization axis along pheromone gradients (Valtz et al., 1995
; Nern and Arkowitz, 1998
). Using this assay, we found that cells expressing Ste4 were proficient at chemotropism, whereas those lacking Ste4 were defective (A, left). Among the different bypass methods, the membrane-targeted Ste5 reagents promoted the most efficient mating, but a consistent behavior emerged regardless of the bypass method or the absolute signaling level; namely, removal of Ste4 or addition of excess pheromone disrupted chemotropic mating without altering MAP kinase pathway signaling.
Gβγ activates MAP kinase cascade signaling via interactions with Ste5 and Ste20 (Whiteway et al., 1995
; Inouye et al., 1997
; Feng et al., 1998
; Leeuw et al., 1998
; Pryciak and Huntress, 1998
). To unequivocally determine whether these interactions are dispensable for the chemotropic role of Gβγ, we performed quantitative assays of chemotropic mating in ste5
Δ cells, by using activated Ste11 (Ste11ΔN) or excess Ste12 to induce signaling or transcription. Indeed, these cells were proficient at chemotropism, whereas strains that also lacked Ste4 (ste4
Δ) were not (B). Thus, the chemotropism role of Gβγ does not require it to interact with Ste5 or Ste20. These findings provided a framework from which to further probe the chemotropism and polarity functions of the receptor–Gαβγ module without concern for their effects on MAP kinase pathway signaling.
The Pheromone Receptor and All Three Gαβγ Subunits Are Required for Chemotropism
The ability of Gβγ to mediate chemotropism without regulating MAP kinase cascade signaling is consistent with the fact that Gβγ interacts directly with polarity proteins via Far1 (Butty et al., 1998
; Nern and Arkowitz, 1998
). However, because chemotropism is a directional phenomenon, it would be logical that Gβγ–Far1 binding could help guide polarization in the proper direction only if Gβγ was activated in a spatially asymmetric manner, congruent with the pheromone gradient. Because Gβγ activation is regulated by the receptor and Gα subunit, we directly compared the requirement for the receptor and all three G protein subunits in chemotropic mating assays. As mentioned above, MAP kinase signaling, transcription, or both were activated independent of Gβγ, so that genetic perturbation of the receptor–Gαβγ module would affect only chemotropism. We found that only the cells with an intact receptor–Gαβγ module could use pheromone gradients to increase their mating success, whereas cells lacking the receptor (ste5
Δ) or any one of the G protein subunits (ste5
Δ, or ste5
Δ) could not (C). It is important to note that under these signaling bypass conditions the receptor–Gαβγ module can only contribute to mating success when pheromone gradients are intact, whereas it performs no detectable role when gradients are absent (i.e., the wild-type and null alleles of each component are indistinguishable).
Microscopic analysis (D) confirmed that the intact receptor–Gαβγ module allowed cells to locate and fuse with mating partners, as judged by the formation of dumbbell-shaped zygotes, although the mating-defective cells could still form polarized mating projections. Therefore, under these conditions the receptor-Gαβγ module is not required for polarization per se, but for properly guiding cell polarization toward a mating partner. Note that these findings are consistent with the expectation that polarization in the “correct” direction (i.e., toward the source of pheromone) should require spatial regulation of Gβγ activity, and so they do not necessarily imply that the receptor and/or Gα perform separate polarization functions.
Free Gβγ Is Insufficient for De Novo Polarization
To study the polarity function of the receptor–Gαβγ module in a setting where cells do not have to detect the direction of a localized stimulus, we assayed de novo polarization in response to a uniform field of pheromone. Polarization was restricted to the de novo pathway by using rsr1
Δ mutant strains, in which the default pathway is inactivated (Nern and Arkowitz, 1999
). First, we tested whether pheromone had a role in de novo polarization beyond activating the MAP kinase cascade. Pathway signaling was activated by expressing either Ste5ΔN-CTM (PGAL1-STE5
) or an activated form of Ste11 fused to Ste7 (PGAL1-STE11
), which permits normal mating morphology by reducing cross-activation of other signaling pathways (Harris et al., 2001
). Despite being able to trigger default polarization (i.e., in RSR1
cells), pathway activation by Ste5ΔN-CTM or Ste11ΔN-Ste7 could not trigger de novo polarization (i.e., in rsr1
Δ cells) (A). This inability was not due to interference from excess MAP kinase pathway signaling, as cells harboring these activators (ste5
Δ + PGAL1-STE5
) could undergo de novo polarization when pheromone was added (B). Importantly, we found that de novo polarization requires Gβγ activity, as cells lacking the Gβ subunit (ste4
Δ + PGAL1-STE11
) did not polarize even when pheromone was added (B). Notably, however, communication between Gβγ and Ste5 was not required, because pheromone could stimulate polarization in cells lacking Ste5 (ste5
Δ + PGAL1-STE11
) (B). As expected, transcriptional induction levels and kinetics in these strains were uninfluenced by the factors that governed polarization, such as RSR1
, or pheromone (, A and D). Therefore, as with chemotropism, the ability of pheromone and Gβγ to regulate de novo polarization is separable from any regulatory effects on the MAP kinase cascade.
Figure 2. De novo polarization requires pheromone and Gα, in addition to Gβγ. (A) Polarization was examined in RSR1 or rsr1Δ cells, after signaling was activated by α factor (strains PPY398 and PPY1259) or by galactose induction (more ...)
Because de novo polarization does not require cells to sense the direction from which pheromone emanates, and because Gβγ interacts directly with Far1 and Cdc24, (Butty et al., 1998
; Nern and Arkowitz, 1998
), it seemed possible that Gβγ alone would be sufficient to promote de novo polarization, with pheromone serving only to generate free Gβγ by dissociating the Gαβγ heterotrimer. To test this view, Gβγ was activated without using pheromone, by deletion of GPA1
or by overexpression of STE4
). To avoid persistent growth arrest due to constitutive MAP kinase pathway signaling, GPA1
was deleted in a ste5
Δ strain harboring PGAL1-STE11
(B) or in a ste4
Δ strain harboring PGAL1-STE4
(C). Remarkably, although each method of Gβγ activation (i.e., gpa1
Δ or PGAL1-STE4
) could induce cell cycle arrest and cell polarization by the default pathway (i.e., in RSR1
cells), neither method could induce de novo polarization (i.e., in rsr1
Δ cells) (, B and C). Furthermore, the ability of pheromone to trigger de novo polarization was actually eliminated by the gpa1
Δ mutation (, B and C, right columns), and thus it requires Gα in addition to Gβγ. Therefore, although Gβγ can directly communicate with polarization proteins, free Gβγ is not sufficient for de novo polarization. This deficiency might reflect a separate role for ligand-bound receptors or GTP-loaded Gα, or it might indicate that receptors and Gα can promote an asymmetric distribution of Gβγ activity even when external pheromone is distributed uniformly.
To follow the pattern of cell surface growth, we used fluorophore-conjugated ConA to differentially label cell wall formed before versus during the period of mating pathway activation. It was previously shown that when de novo polarization failed due to disruption of Far1–Cdc24 interaction, cells could initiate polarized growth, but the polarization axis could not be maintained; consequently, it wandered about a broad region of the cell periphery (Nern and Arkowitz, 2000
). Similarly, we observed that when cells failed de novo polarization due to absence of pheromone, Gpa1, or Ste4, they still showed asymmetric cell surface growth, but it was broadly distributed across one hemisphere (, B and C, right). Thus, consistent with the requirement for Fus3 (Matheos et al., 2004
), our results suggest that activation of the MAP kinase cascade is sufficient to initiate asymmetric growth, even in the absence of Gα or Gβγ. Nevertheless, organization of a well-focused and persistent polarization axis requires both Far1–Cdc24 interaction and an intact receptor–Gαβγ module.
Gβ Mutants Reveal Distinct Roles for Two Gα–Gβ Binding Interfaces
To further investigate the requirement for the intact receptor–Gαβγ module in chemotropism and de novo polarization, we used a series of Gβ mutants to disrupt regulation of Gβγ by the Gα subunit. Crystal structures of mammalian Gαβγ heterotrimers reveal two contact surfaces between Gα and Gβ, termed the “switch interface” and the “N-terminal interface” (Wall et al., 1995
; Lambright et al., 1996
). The switch (Sw) interface involves a region of Gα that undergoes a conformational switch upon GTP binding, whereas the N-terminal (Nt) interface involves an N-terminal helix of Gα that protrudes away from the globular GTPase domain (A). The Gα and Gβ residues contacting one another in each interface are well conserved between mammalian and yeast counterparts (Lambright et al., 1996
; Sondek et al., 1996
); in Gβ these include 16 residues in nine regions. Here, we studied a series of Ste4 (Gβ) mutations that collectively affect all positions predicted to contact Gα. These mutants were derived from multiple sources; briefly, mutations in six of the nine Gα-contact regions were identified in three different genetic screens, and the remaining regions were changed by site-directed mutagenesis (see Materials and Methods
Figure 3. Ste4 mutations in Gα–Gβ binding interfaces. (A) Model for orientation of the G protein-coupled receptor rhodopsin, the heterotrimeric G protein transducin, and the membrane. Adapted from Hamm, 2001 ; copyright Proceedings of the (more ...)
By a two-hybrid assay (B), the Ste4 mutations identified in genetic screens reduced Gpa1 binding to levels that were either undetectable (L117R, W136R L138F, and L154R N156K) or extremely faint (K126E, D224E, and D272A), whereas site-directed mutations at the remaining positions (N92G K94E S96A and W411R) had only mild effects (suggesting why they were not identified in screens). A similar rank order of defects was seen using a coprecipitation assay of these AD–Ste4 fusions with Gpa1-GST (unpublished observations). In comparison, these mutations had minimal effects on interaction with Ste5 and Far1 (B); this is consistent with our findings (to be described in detail elsewhere) that binding to Ste5 and Far1 requires Ste4 (Gβ) and Ste18 (Gγ) residues in the coiled-coil region of the Gβγ dimer (Lamson and Pryciak, unpublished observations), which has been previously implicated in downstream signaling (Leberer et al., 1992
; Grishin et al., 1994a
; Leeuw et al., 1998
; Winters et al., 2005
). As expected for mutants released from repression by Gpa1, these Ste4 mutants caused constitutively active pathway signaling (C), although some mutants that retained detectable Gpa1 binding also showed incomplete deregulation, because signaling could still be increased by α factor. Therefore, we focused most of our subsequent studies on four Ste4 mutants with the strongest Gpa1-binding defects and the most deregulated signaling, which include two in the Nt interface (L117R and K126E) and two in the Sw interface (W136R L138F and L154R N156K, hereafter termed WL/RF and LN/RK, respectively). These mutations did not affect Ste4 protein levels but they eliminated detectable coimmunoprecipitation with Gpa1 (D).
Despite similar behavior in binding and signaling assays, mutations in the two Gα–Gβ interfaces had opposite effects on polarity control. Mutations in the Nt interface disrupted chemotropism, because the cells were largely insensitive to the presence or absence of pheromone gradients (A). This phenotype is consistent with deregulated Gβγ activity. Surprisingly, however, the Sw interface mutants were chemotropism proficient (A), even though by signaling and binding criteria they seemed to be as strongly dissociated from Gpa1 as the Nt interface mutants. Although it was possible that the individual Sw mutations have a less disruptive effect on Gpa1 interaction than the Nt mutations, two further observations suggested that the explanation was not this simple. First, chemotropism remained intact even when multiple Sw interface mutations were combined, such as W136R L138F L154R N156K (WL/RF + LN/RK; A), as well as D224V D272A, L154S D272A, and W136R L138F D272A (unpublished observations). Second, by making less drastic substitutions at Nt interface residues (i.e., K126A, K126N, L117A, and L117G), we could detect residual Gpa1 binding for several of these new mutants (as well as the original K126E); yet, they all showed a stronger defect in chemotropic mating than the Sw mutants WL/RF and LN/RK (B).
Figure 4. Ste4 mutations in the Nt interface, but not the Sw interface, disrupt polarity control. (A) Chemotropism proficiency was assessed by patch mating assays performed in the absence (−) or presence (+) of exogenous α factor. PPY867 (ste4Δ (more ...)
The Ste4 mutants also segregated into two phenotypic classes in de novo polarization assays, in which the Sw interface mutants remained competent, whereas the Nt interface mutants were defective (C, rsr1Δ). Notably, although the Sw interface mutants could mediate de novo polarization, they still required the addition of pheromone, as with wild-type Ste4. Thus, the Sw interface mutants remain capable of mediating a pheromone-dependent step, despite their strong dissociation from Gpa1 in binding and signaling assays. This raised the possibility that the Sw interface mutants can maintain a weak interaction between Gα and Gβ at the Nt interface (perhaps only when membrane-associated) and thus remain in regulatory communication with the pheromone receptor. This scenario, and others, was addressed by further experiments described below.
Suppression of an Nt Interface Mutant by a Compensatory Mutation in Gα
First, we wanted to determine whether the stronger phenotype of the Nt mutants was truly a consequence of disrupted interaction between Gβ and Gα, rather than between Gβ and some other protein involved in cell polarity. Although these Ste4 mutants could still bind Far1 (B), in principle we could not rule out effects on binding to other, unknown partners. Therefore, we attempted to restore Gα–Gβ binding via a compensatory mutation in Gpa1. One of the Nt interface mutations (Ste4-K126E) involves a residue that, based on mammalian Gαβγ structures, is expected to form an ion pair between Lys126 in Ste4 and Glu28 in Gpa1 (A). The Ste4 mutation changes Lys126 to Glu (K126E), thereby reversing the charge. To make a compensatory charge-reversal mutation in Gpa1, we changed Glu28 to Lys (E28K). This Gpa1–E28K mutation, but not a control mutation (Gpa1-E28A), restored measurable binding with Ste4-K126E (B), although not to wild-type levels. Also, the Gpa1 E28K and E28A mutations each reduced binding to wild-type Ste4, although by a mild degree that was most noticeable when Ste4 was expressed at lower levels from a weak promoter (B). It was not entirely surprising that Gpa1-E28K only partially restored binding to Ste4-K126E and that Ste4-K126E caused a stronger binding defect than Gpa1-E28K, because the mammalian Gβ residue homologous to Ste4 Lys126 contacts Gα not only through this ion pair but also through hydrogen bonding and van der Waals interactions (Lambright et al., 1996
). Nevertheless, these binding effects were enough to confer informative phenotypes in mating assays.
Figure 5. Allele-specific suppression of a Ste4 Nt interface mutant. (A) Red side chains highlight an ion pair predicted to form between Ste4(Gβ)-K126 and Gpa1(Gα)-E28 in the Nt interface, based on homologous residues (Gβ-K89 and Gα-E16) (more ...)
Indeed, the chemotropism defect of the Ste4-K126E mutant was at least partially suppressed by the Gpa1-E28K mutant, because mating of cells harboring Ste4-K126E was more efficient when coexpressed with Gpa1-E28K than with Gpa1-wild type (WT) (, C and D). Whereas the Gpa1–E28K mutation reduced mating when combined with Ste4-WT, it increased mating by an average of 4.6-fold when combined with Ste4-K126E (D). Although mating was not restored to wild-type levels, it would be unreasonable to expect this given the incomplete restoration of Gpa1–Ste4 binding. Notably, the observed suppression was allele-specific, because Gpa1-E28A did not suppress Ste4-K126E, and neither Gpa1-E28K nor Gpa1-E28A could suppress the other Nt mutant, Ste4-L117R (, C and D). Furthermore, although the Gpa1–E28K mutation improved mating by Ste4-K126E, it reduced mating by Ste4-WL/RF and Ste4-LN/RK, such that these Sw mutants were actually more defective than Ste4-K126E in cells expressing Gpa1-E28K (C). This finding makes it highly unlikely that the phenotypic differences between Ste4 Nt and Sw mutations can be explained by their different impact on binding between Gβγ and an unknown factor. Instead, the pattern of allele-specific suppression and enhancement found with the Gpa1–E28K mutation supports a special role for Gα–Gβ binding via the Nt interface in chemotropism and cell polarization, and it suggests that this interface can remain functional when the Sw interface is dissociated by mutation. Because the Gpa1-E28K mutant is sensitized to disruption of the Sw interface, an intact Sw interface may help maintain Gα–Gβ association when the Nt interface is mildly disrupted.
Differences between Nt and Sw Interface Mutants Are Not Due to Altered Gpa1–Fus3 Interaction
Gpa1 can interact with the MAPK Fus3 via a docking motif in its N terminus, and mutations in this motif (Gpa1-K21E R22E, denoted Gpa1-EE) disrupt Fus3 binding and reduce mating (Metodiev et al., 2002
). This raised the possibility that an intact Nt interface is required mainly to allow proper interaction between the Gpa1 N terminus and Fus3. To test this notion, we used the Gpa1-EE mutant to disrupt interaction between Gpa1 and Fus3, and we found that the Nt and Sw mutations in Ste4 still showed their characteristic phenotypic differences (A). We also tested the Ste4 mutants in cells lacking Fus3 (ste4
Δ). Here, mating by the Sw interface mutants was not as efficient as that of wild-type Ste4, but it was still more efficient than that of the Nt interface mutants (A). Thus, both approaches suggest that the role of the Nt interface, and the different behavior of the Nt versus Sw mutants, cannot be explained by indirect effects on the Gpa1–Fus3 interaction.
Figure 6. Qualitative differences between Ste4 Nt and Sw mutants are independent of Gpa1–Fus3 interaction. (A) Patch mating assays used the following strain and plasmid combinations. Left, strain PPY1228 (ste4Δ gpa1Δ) harbored the indicated (more ...)
It is notable that we did not detect a strong mating defect for the Gpa1-EE mutant alone, suggesting that the Gpa1–Fus3 interaction may not be required for chemotropism. To address this issue further, we compared the roles of Fus3 and Far1. Loss of either protein caused a mating defect, but a much greater defect occurred when both Fus3 and Far1 were absent (B), indicating that although both proteins are required for maximum mating efficiency, each protein can perform its function without the other. Moreover, an important distinction between the roles of Fus3 and Far1 was apparent: the fus3
Δ cells were proficient at chemotropism (i.e., sensitive to whether pheromone gradients were present or absent), whereas the far1
Δ cells were defective (B). To rule out the possibility that chemotropic proficiency in fus3
Δ cells reflects redundancy between Fus3 and Kss1, we performed additional mating assays using fus3
Δ cells (in which sterility was suppressed by PGAL1-STE12
). Despite low overall mating efficiency, the fus3
Δ cells could still use pheromone gradients, and this behavior required Far1 (C). The simplest overall interpretation is that detecting gradients and using gradient information to locate mating partners does not require Fus3, whereas the role of Fus3 in polarized morphogenesis (Matheos et al., 2004
) is distinct from gradient sensing per se. These results also imply that, unlike cell cycle arrest (Gartner et al., 1998
), phosphorylation of Far1 by Fus3 is dispensable for chemotropism.
Fusion of Gβ to Gα Suggests a Role for the Nt Interface in Receptor Coupling
Finally, we addressed whether maintenance of the Nt interface was necessary for coupling of the heterotrimer (Gαβγ) to the receptor. Existing models for the coupling between GPCRs and heterotrimeric G proteins predict that the Nt interface lies tangential to the membrane (Hamm, 1998
). In addition, the N terminus of Gα has been implicated in receptor recognition (Taylor et al., 1994
; Itoh et al., 2001
; Cabrera-Vera et al., 2003
). These facts suggested that the Nt interface mutations may not only disrupt interaction between Gβ and Gα, but they may also disrupt the way Gαβγ interacts with the receptor. However, the ability to test this notion was hindered by the fact that the Ste4 mutations caused constitutive signaling, which obscured whether the receptor might still exert some regulatory control over the G protein. To circumvent this difficulty, we took advantage of a previously described Ste4–Gpa1 fusion protein (Klein et al., 2000
), with the rationale that forced association to Gpa1 may inhibit constitutive signaling by the Ste4 mutants and thus allow us to assay receptor coupling.
Starting with the prior Ste4–Gpa1 fusion construct, we replaced the original GAL1 promoter with the native STE4 promoter, and then compared its function with wild-type (unfused) polypeptides. By multiple assays, we found this Gβ–Gα fusion (Ste4-Gpa1) to function in a manner that was virtually indistinguishable from separate Gβ and Gα polypeptides. This included growth arrest (A, left), regulation by the RGS-family protein Sst2 (A, right), and pheromone-induced transcription (B). Furthermore, the Gβ–Gα fusion was able to mediate total mating levels and chemotropic mating behavior that was similar to the wild-type heterotrimer (, C and D). Thus, fusion of Gβ to Gα does not interfere with Gαβγ function in either signaling or gradient detection.
Figure 7. Phenotypes of Ste4–Gpa1 fusion proteins. (A) A Ste4–Gpa1 fusion expressed from the native STE4 promoter functions indistinguishably from separate Ste4 and Gpa1 polypeptides. Halo assays show growth arrest of ste4Δ gpa1Δ (more ...)
Next, we incorporated the Nt and Sw interface mutations into the STE4 portion of the STE4-GPA1 fusion gene (in both the GAL1 promoter and native STE4 promoter contexts), and we used FUS1-lacZ assays to determine whether forced association with Gpa1 could suppress the constitutive signaling activity of the Ste4 mutants. Again, the Nt interface and Sw interface mutants showed distinct phenotypes, but here it was the Sw interface mutants that were more strongly deregulated; that is, fusion to Gpa1 could squelch the constitutive signaling of the Nt interface mutants but not that of the Sw interface mutants (E, left and middle). This suggests that dissociation of the Sw interface is the primary regulator of downstream signaling. There was a slight difference between the two Sw interface mutants in the native promoter context, because fusion to Gpa1 partially reduced signaling by Ste4-LN/RK but not Ste4-WL/RF (E, top). Protein levels were unaffected by these mutations (F).
Further analysis of these mutant fusion proteins showed that although constitutive signaling by the Nt interface mutants was suppressed by fusion to Gpa1, signaling could not be efficiently reactivated by the addition of pheromone, in contrast to the fusion containing wild-type Ste4 (E, right). The feeble pheromone response suggests that mutations in the Nt interface disrupt coupling between Gαβγ and the receptor, which may explain their defective behavior in both chemotropism and de novo polarization assays. Thus, although both Sw and Nt mutants show constitutive signaling when not fused to Gpa1, their different behaviors when fused to Gpa1 suggest the possibility that their different chemotropism/polarity phenotypes are a consequence of disrupted receptor–Gαβγ coupling in the Nt mutants, and by inference that this coupling can still occur in Sw mutants despite their constitutive signaling.
GTP Hydrolysis by Gα Is Required for De Novo Polarization and Chemotropism
The findings described above suggest that proper communication between Gαβγ and the receptor is necessary for directionally persistent polarization even when the pheromone stimulus is provided uniformly. In theory, this receptor-Gαβγ communication could be required solely to promote GTP–GDP nucleotide exchange on Gα, perhaps allowing GTP-bound Gα to perform a polarization role that acts synergistically with Gβγ. Alternatively, receptor–Gαβγ communication might be required to generate asymmetry in the distribution of Gβγ activity (and/or Gα-GTP), which otherwise would remain symmetric (e.g., in gpa1
Δ cells or with constitutively active Ste4 mutants). To address these possibilities, we used an activated mutant form of the Gα subunit, Gpa1-Q323L (herein referred to as Gpa1-QL), which is defective at GTP hydrolysis (Dohlman et al., 1996
; Apanovitch et al., 1998
). We found that simultaneous activation of both Gα and Gβγ, by coexpressing Gpa1-QL with Ste4, was still not sufficient for de novo polarization in the absence of pheromone (, A and B). This was true regardless of whether Gpa1-QL was expressed with Ste4-WT or with the constitutively active Sw interface mutant, Ste4-WL/RF. In fact, we found that trapping Gpa1 in the GTP-bound state was detrimental, because cells expressing Gpa1-QL could not polarize even after exposure to pheromone (, A and B, + α factor). Therefore, polarization requires more than just the acquisition of both GTP-bound Gα and active Gβγ.
Figure 8. Polarity control requires GTP hydrolysis by Gpa1. (A and B) De novo polarization was monitored using ste4Δ gpa1Δ rsr1Δ cells (PPY1380) expressing Ste4 and Gpa1 variants as either separate or fused polypeptides, after 4-h induction (more ...)
It seemed possible that activated Gα and Gβγ subunits might have to remain in close mutual proximity and that this might be accomplished during receptor-mediated activation but not by coexpression of mutationally activated subunits. Therefore, to force GTP-bound Gα to remain associated with Gβγ, we incorporated the Gpa1-QL mutation into the Ste4–Gpa1 fusion. In signaling assays, either the Ste4-WL/RF or Gpa1-QL mutations (or both) caused constitutive activity (C); yet, none of these fusions could induce de novo polarization in the absence of pheromone (, A and B, −α factor). Notably, the fusions containing the Gpa1-QL mutation did promote a detectable increase in elongation (and more so than when the same subunits were expressed as separate polypeptides), but these elongated cells did not form the pointed, pear-shaped shmoos seen during pheromone treatment. Thus, the cells were still missing some aspect of pheromone-induced polarization that can focus morphogenesis to a restricted portion of the cell perimeter.
Furthermore, the ability of pheromone to trigger de novo polarization remained intact when Ste4 was fused to Gpa1, but this was disrupted by the Gpa1-QL mutation (, A and B, +α factor). Consistent with these findings, the fusions containing the Gpa1-QL mutation were also defective in chemotropic mating assays (D), and the Gpa1-QL mutant (expressed as a separate polypeptide) eliminated the mating advantage of Sw interface mutants over Nt interface mutants (E). Thus, interfering with GTP hydrolysis activates Gβγ signaling, but it disrupts cell polarization and chemotropism, regardless of whether Gα is fused to Gβ or kept separate. Finally, it should be noted that the chemotropism and polarity defects shown by the Gpa1-QL mutant are recessive to wild-type Gpa1 (unpublished observations). Together, these findings suggest that the ability of pheromone-bound receptor molecules to guide cell polarization, either along a de novo polarization axis or along pheromone gradients, requires normal coupling to the Gα GTP hydrolysis cycle.