Increased penetrance and severity of septin defects in cdc42 hemizygotes
To address the role of Cdc42p in septin ring assembly, we have focused on two cdc42
mutants that cause defects in septin localization without overt effects on actin organization. In an earlier study, we described a mutant, cdc42V36T,K94E
, that displayed relatively mild septin and cell morphology phenotypes (Gladfelter et al., 2001a
). We also noted that the severity of many other cdc42
mutants was greatly affected by gene dosage so that elevated copy number could partially rescue mutant phenotypes (Gladfelter et al., 2001a
). Conversely, we reasoned that lowering the gene copy number might reveal more severe phenotypes, which could be useful for characterizing mutants with mild defects. To that end, we examined the effects of reducing mutant gene dosage by generating hemizygous cdc42V36T,K94E
Δ mutant diploids. This reduction of gene copy number by a factor of two made the mutant phenotype significantly more penetrant and more severe ( B and ). In contrast, control hemizygous CDC42
Δ diploids were indistinguishable from homozygous CDC42
Figure 1. Septin defects in cdc42 mutants. (A) Strains DLY5461 (CDC42/GAL1p-CDC42), DLY4223 (cdc42V36T,K94E/GAL1p-CDC42), DLY4224 (cdc42Y32H/GAL1p-CDC42), and DLY5470 (cdc42Y32H/cdc42Y32H) were processed to visualize septins. Control Western blots confirmed that (more ...)
Examination of the phenotypes of hemizygous cdc42/cdc42Δ mutant strains further allowed us to identify septin defects associated with a novel allele, cdc42Y32H ( A and ). In contrast to the hemizygous cdc42Y32H/cdc42Δ strain, septin staining appeared completely normal in haploid cdc42Y32H and homozygous cdc42Y32H/cdc42Y32H or heterozygous cdc42Y32H/CDC42 diploids containing this allele. However, cells from each of these strains displayed a mild elongated bud morphology ( B), indicating that cdc42Y32H has a slight dominant effect on bud morphology in addition to a recessive defect in septin organization that is only detectable at low gene dosage. For the purposes of this report, we have concentrated on the septin defect.
The septin localization defects in hemizygous cdc42V36T,K94E
Δ and cdc42Y32H
Δ strains appeared quite distinct. cdc42V36T,K94E
Δ cells frequently showed faint or even undetectable septin staining at the neck and prominent mislocalized septin rings within the bud ( A and ). In contrast, septin staining in cdc42Y32H
Δ cells was generally localized to the neck but with aberrant patterns of staining including septin “bars” running along the mother bud axis (similar to those observed in gin4
mutants [Longtine et al., 1998
]) or irregular septin zones ( A and ). Occasional cdc42Y32H
Δ cells displayed additional septin-containing patches in the bud, particularly when cells were grown in minimal medium. Septin staining in heterozygous cdc42V36T,K94E
cells was indistinguishable from that of wild-type cells (unpublished data). Thus, these two cdc42
alleles displayed distinct recessive defects in septin localization that were ameliorated upon raising the gene dosage.
Initial assembly of the septin ring in cdc42 mutants
Examination of the septin rings that initially formed in unbudded cells revealed further differences between the cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants. The cdc42V36T,K94E/cdc42Δ septin rings were quite similar to those in wild-type cells, although occasional cells displayed fainter or wider rings ( A). However, the initial rings formed in cdc42Y32H/cdc42Δ cells had strikingly larger diameters than those in wild-type cells ( A). Interestingly, cdc42V36T,K94E/cdc42Δ cells subsequently developed unusually broad necks, whereas cdc42Y32H/cdc42Δ cells generally had narrow and sometimes “stretched”-appearing necks ( B). Thus, the diameter of the initial septin ring was not correlated with the width of the subsequent neck in these mutants.
Cdc42p polarizes to a tight patch at the presumptive bud site (Ziman et al., 1993
), and it is presumably this localized pool of Cdc42p that triggers the assembly of the concentric septin ring. Thus, one possible explanation for the increased diameter of the septin ring in cdc42Y32H
Δ cells would be that Cdc42pY32H
polarizes to a larger diameter patch, which assembles septins correspondingly farther away from the center of the patch. However, we found that the localization of Cdc42pY32H
was indistinguishable from that of wild-type Cdc42p as judged by immunofluorescence microscopy ( C). This result suggests that the defect in the initial assembly of the septin ring arises from impaired function, rather than impaired localization, of Cdc42pY32H
In contrast to cdc42Y32H/cdc42Δ cells, the apparently normal initial rings in most cdc42V36T,K94E/cdc42Δ cells suggest that cdc42V36T,K94E/cdc42Δ mutants are primarily defective in maintaining septins at the neck during bud growth and not in assembling a septin ring before bud formation. This surprising observation raised the possibility that Cdc42p may act after bud emergence to stabilize the septin collar at the neck, as well as promoting initial septin ring assembly.
Cdc42p-independent maintenance of septin organization
To ask directly whether Cdc42p and its exchange factor, Cdc24p, were required to maintain septin localization after bud emergence, we inactivated conditional cdc24 and cdc42 alleles in budded cells. The Ts alleles of these genes analyzed to date were identified based on their homogeneous unbudded terminal phenotype, which may have biased the screen in favor of alleles that were still capable of contributing to septin maintenance in budded cells even at restrictive temperature. To avoid this problem, we used two new Ts alleles (cdc42-6 and cdc42-27) that were selected only for lethality rather than for any particular terminal phenotype (see Materials and methods). To provide as much time as possible for the inactivation of mutant gene products after bud formation, we arrested mutant cells in G2 by overexpressing Swe1p at the permissive temperature. We then shifted the cells to 37°C and maintained the G2 arrest for 4 h to allow ample time for Cdc42p inactivation. In both cdc42 and cdc24 mutants, septins remained localized to the neck with no observable diminishment in the intensity of staining ( A). In contrast, the mutants failed to maintain a polarized actin distribution under these conditions ( B), indicating that long-term maintenance of actin polarity does in fact require continued Cdc42p function. These results suggest that Cdc42p was effectively inactivated after shift to the restrictive temperature but that this did not affect maintenance of septins at the neck. Thus, Cdc42p appears not to be required for maintenance of septin organization.
Figure 2. Cdc42p is required for maintenance of actin polarization but not septin organization in budded cells. Strains RSY136 (GAL1p-SWE1), DLY5079 (cdc42–6 GAL1p-SWE1), DLY4849 (cdc42–17 GAL1p-SWE1), or DLY5078 (cdc24–4 GAL1p-SWE1) were (more ...)
These results appear to rule out the possibility that the mislocalization of the septin rings in budded cdc42V36T,K94E/cdc42Δ mutants is due to a defect in a “septin maintenance” function of Cdc42p. Rather, it appears that subtle defects in the initial assembly of the septin ring in cdc42V36T,K94E/cdc42Δ mutants cause septins to disassemble gradually from the neck after a bud has formed, subsequently reassembling at ectopic locations (this phenotype will be described in more detail elsewhere). In summary, the two septin-specific cdc42 alleles that we examined display distinct defects in the initial assembly of the septin ring. The cdc42V36T,K94E/cdc42Δ mutant forms an unstable ring, whereas the cdc42Y32H/cdc42Δ mutant forms a more stable but much larger diameter septin ring.
Molecular pathways underlying the cdc42 septin defects
One way to identify the molecular pathways that are impaired in the septin-defective cdc42
mutants is to identify suppressors that restore septin organization. We began by asking whether overexpression of known Cdc42p effectors could rectify the defect in our mutants. As reported previously for haploid strains (Gladfelter et al., 2001a
), overexpression of the Cdc42p-activated kinase Cla4p or the scaffold protein Bem1p but not of Ste20p or other effectors suppressed the septin misorganization phenotype of hemizygous cdc42V36T,K94E
Δ mutants, and we observed a similar pattern for cdc42Y32H
Δ mutants () . Cla4p and Bem1p participate in a feedback loop that regulates the phosphorylation state of Cdc24p (Gulli et al., 2000
; Bose et al., 2001
), and moderate overexpression of these proteins suppresses many cdc42
mutants with varied defects (Gladfelter et al., 2001a
). Thus, suppression in these cases may reflect a global enhancement of Cdc42p function rather than a specific compensation of impaired septin organization pathways.
Figure 3. Effect of overexpressing Cdc42p effectors and GAPs on the septin defects of cdc42 mutants. Strains DLY4223 (cdc42V36T,K94E/GAL1p-CDC42) and DLY4224 (cdc42Y32H/GAL1p-CDC42) were transformed with pDLB722 (CLA4), pDLB723 (STE20), pDLB678 (BEM1), pMOSB229 (more ...)
Like effectors, GTPase-activating proteins (GAPs) recognize specifically the GTP-bound form of small G proteins. There are three proteins currently thought to act as Cdc42p-directed GAPs in yeast: Rga1p, Rga2p, and Bem3p (Zheng et al., 1993
; Stevenson et al., 1995
). Strikingly, we found that overexpression of Rga1p (though not of Rga2p or Bem3p) effectively suppressed the septin defects of both cdc42V36T,K94E
Δ and cdc42Y32H
Δ mutants (; see C).
Figure 5. Rga1p GAP activity and cdc42 suppression. (A) Cdc42p prebound to [γ-32P]GTP was incubated with GST (•), GST-Rga1p GAP domain (○), or the same domain containing the K872A change (□), and radioactivity remaining bound to (more ...)
GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H
The finding that overproduction of a Cdc42p GAP could ameliorate the septin defects of cdc42V36T,K94E/cdc42Δ and cdc42Y32H/cdc42Δ mutants raised the possibility that the septin misorganization arose due to a defect in the ability of Cdc42pV36T,K94E and Cdc42pY32H to hydrolyze GTP. To test directly whether such a defect existed, mutant and wild-type versions of Cdc42p were produced as recombinant GST fusion proteins in E. coli, purified using glutathione-sepharose, and loaded with [γ-32P]GTP. GTP hydrolysis was followed by monitoring loss of the 32P associated with Cdc42p using a filtration assay. As shown in A, GTP hydrolysis by Cdc42pV36T,K94E was ~40% slower than GTP hydrolysis by wild-type Cdc42p, whereas GTP hydrolysis by Cdc42pY32H was indistinguishable from wild-type.
Figure 4. GTP hydrolysis by Cdc42pV36T,K94E and Cdc42pY32H. GST-Cdc42p (•), GST-Cdc42pY32H (), and GST-Cdc42pV36T,K94E (□) were prebound to [γ-32P]GTP and incubated with buffer alone (A) or with a 1:10 molar ratio of recombinant (more ...)
To investigate whether GTP hydrolysis by the mutants was appropriately stimulated by GAPs, we produced recombinant Rga1p GAP homology domain (comprising the COOH-terminal 224 residues) as a GST fusion protein in E. coli. After purification on glutathione-sepharose beads, this domain effectively stimulated GTP hydrolysis by Cdc42p and by Cdc42pV36T,K94E in vitro ( B). However, Cdc42pY32H was almost completely insensitive to the Rga1p GAP domain ( B). Thus, both of these mutants affect Cdc42p GTP hydrolysis but in different ways: Cdc42pV36T,K94E displays a slower intrinsic GTPase activity that is still responsive to the Rga1p GAP, whereas Cdc42pY32H intrinsic GTPase activity is normal but cannot be greatly stimulated by the Rga1p GAP.
It is curious that overexpression of Rga1p was able to suppress the septin defects of the cdc42Y32H/cdc42Δ mutant despite the fact that the Rga1p GAP domain was unable to stimulate GTP hydrolysis by Cdc42pY32H in vitro. It is possible that full-length Rga1p retains significant GAP activity for Cdc42pY32H in vivo and that suppression occurs by enhancing this residual activity. However, these findings could also indicate that Rga1p can somehow improve septin organization in vivo without acting as a GAP for Cdc42p.
Suppression of cdc42 septin defects by Rga1p requires a functional GAP domain
To address whether the effect of Rga1p on septin organization depends on its GAP activity, we generated a point mutant form of Rga1p that lacked GAP activity. Previous studies identified a lysine residue conserved among Rho-GAPs (Lys 872 in Rga1p) that is essential for activation of the Rho A GTPase by mammalian Rho-GAP (Li et al., 1997
). Mutation of Lys 872 to Ala in the GST-Rga1p-GAP domain construct similarly eliminated GAP activity ( A). In addition, interaction of the Rga1pK872A
GAP domain with Cdc42p was greatly diminished and no longer sensitive to GTP/GDP status ( B). When this mutation was introduced into full-length RGA1
, overexpression of rga1K872A
no longer suppressed the septin defects of either of our cdc42
mutants ( C), suggesting that Rga1p GAP function, and by extension Cdc42p GTP hydrolysis, is in fact important for septin organization.
Dominant effect of GTPase-defective Cdc42p on septin organization
The results described above establish a correlation between the effects of certain cdc42
alleles on septin organization and alterations in GTP hydrolysis by the encoded mutant proteins. However, it remained possible that the correlation was entirely coincidental and that the septin organization defects of these mutants were unrelated to their altered GTP hydrolysis. To test whether preventing Cdc42p GTP hydrolysis itself would affect septin organization, we turned to the CDC42Q61L
allele that was generated by homology to the corresponding oncogenic allele of ras
and has been characterized extensively as showing an essentially complete defect in GTP hydrolysis. Previous studies showed that moderate or high level expression of CDC42Q61L
in yeast is lethal (Ziman et al., 1991
), whereas low level expression can be tolerated (Mosch et al., 1996
). We constructed strains expressing CDC42Q61L
from a crippled version of the GAL1
promoter in addition to wild-type CDC42
expressed from its own promoter. These cells were able to proliferate well on galactose-containing medium, and the level of Cdc42pQ61L
expression was roughly comparable to that of endogenous wild-type Cdc42p ( B). However, there were striking defects in septin organization in these cells: unbudded cells displayed large and faint initial rings similar to those observed in cdc42Y32H
Δ mutants, and budded cells displayed misorganized and diffuse septin staining at the neck ( A and ). In addition, cells expressing Cdc42pQ61L
frequently had broad necks similar to those observed in cdc42V36T,K94E
Δ mutants. Thus, preventing GTP hydrolysis by Cdc42p leads to dominant effects on septin localization and neck morphology.
Figure 6. Effect of blocking or accelerating Cdc42p GTP hydrolysis on septin organization. (A) Strains DLY5237 (CDC42 EG43p-CDC42) and DLY5240 (CDC42 EG43p-CDC42Q61L) were grown to exponential phase in either YEPD or YEP with galactose to induce CDC42 or CDC42 (more ...)
Effect of increasing the intrinsic GTPase activity of Cdc42p
Yeast Cdc42p has an unusually slow intrinsic GTPase activity compared with several homologues from other organisms. This appears to be due to an “arginine finger” motif present in fly or mammalian Cdc42p but absent from yeast Cdc42p that accelerates GTP hydrolysis. Mutation of Lys 186 to Arg introduces a similar arginine finger into yeast Cdc42p and correspondingly increases its intrinsic rate of GTP hydrolysis (Zhang et al., 1999
). We found that cdc42K186R
strains displayed dramatic defects in septin organization ( C and ), suggesting that overly rapid GTP hydrolysis also impairs septin assembly.
A role for Cdc42p GAPs in septin organization
The association between defects in Cdc42p GTP hydrolysis and defects in septin organization suggests that proper assembly of the septin ring requires proper regulation of GTP hydrolysis. If this is true, then mutational inactivation of Cdc42p GAPs Rga1p, Rga2p, and Bem3p might be expected to perturb septin organization also. Although none of the single mutants displayed striking septin defects, double mutants (particularly rga1Δ rga2Δ) were somewhat defective, and the triple mutants showed a strong defect comparable to that of cdc42V36T,K94E/cdc42Δ mutants () , indicating that these proteins share a role in septin organization.
Figure 7. Cdc42p GAPs and septin organization. (A) Strains DLY1 (WT), DLY3344 (rga1Δ), DLY3353 (rga2Δ), DLY3346 (bem3Δ), DLY3341 (rga1Δ bem3Δ), DLY3361 (rga2Δ bem3Δ), DLY3347 (rga1Δ rga2Δ), (more ...)