Cdc10 and Cdc12 are active GTPases
Cdc3, Cdc10, and Cdc11 were purified to near homogeneity from bacterial cells using an NH2-terminal tandem-affinity tag (His6-GST). After detaching the tag by tobacco etch virus (TEV) protease digestion, and removal of protease and uncleaved fusion, soluble untagged septins were obtained ( A). Nearly homogenous preparations of soluble Cdc12-His6 were also produced. GTPase activity of these proteins was measured by release of free phosphate from γ[32P]GTP ( B). Only Cdc10 and Cdc12 displayed detectable GTPase activity above the background control (purified His6-GST).
Figure 1. Purified Cdc10 and Cdc12 bind and hydrolyze GTP. (A) The indicated septin preparations (1–5 μg) were analyzed by SDS-PAGE and stained with Coomassie dye. (B) GTPase activity was measured by phosphate release from γ[32P]GTP for (more ...)
Cdc10, the only septin lacking a COOH-terminal coiled-coil, reproducibly displayed more robust GTPase activity than Cdc12, suggesting that catalytic function of Cdc12 might be restrained by its COOH-terminal coiled-coil. Hence, we purified a truncated version, Cdc12(Δ339–407)-His6 (hereafter Cdc12ΔC; A). Cdc12ΔC displayed a rate of GTP hydrolysis four- to fivefold higher than that of full-length Cdc12 and comparable to Cdc10 ( C), consistent with the idea that its COOH-terminal coiled-coil is a negative regulatory domain. However, Cdc11 lacking its coiled-coil, Cdc11(Δ357–416), did not display any GTPase activity above the nonspecific background (unpublished data). As expected for an authentic GTPase, chelation of Mg2+ with excess EDTA reduced Cdc12ΔC activity to the background level (unpublished data). The apparent Km of Cdc12ΔC for GTP was ~0.6 μM (unpublished data).
To further confirm the intrinsic GTPase activity of Cdc12, we introduced mutations into this septin at positions analogous to those that prevent GTP binding and/or GTP hydrolysis by a canonical GTPase, Ras. Cdc12(S43V) alters a residue equivalent to that in H-Ras(G12V). H-Ras(G12V) binds GTP, but is deficient in GTP hydrolysis (McGrath et al., 1984
). Cdc12(T48N) alters a residue equivalent to that in H-Ras(S17N), which is unable to bind any nucleotide (Feig, 1999
). As expected, both mutations largely abrogated the GTPase activity of both full-length Cdc12 ( C) and Cdc12ΔC ( D). To verify that the phosphate release assay reflects conversion of GTP to GDP, we used α[32
P]GTP as substrate and a TLC-based assay to examine product formation. Indeed, GDP was formed with time as the amount of GTP diminished; moreover, the S43V mutation drastically reduced the rate of product formation, to the same extent as was observed using the phosphate release assay (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200312070/DC1
Mutation T48N prevents GTP binding to Cdc12
Binding of [35S]GTPγS to Cdc12, Cdc12ΔC, and the derived site-directed mutants was assessed as described in the Materials and methods. Both full-length Cdc12 and Cdc12ΔC formed stable complexes with GTPγS over time (). The S43V mutation did not prevent GTP binding or alter the kinetics of GTP binding to either Cdc12 or Cdc12ΔC (), even though it did prevent GTP hydrolysis (, C–E). In contrast, the T48N mutation abolished the ability of Cdc12 and Cdc12ΔC to bind GTP (). Thus, both mutations caused effects in vitro consonant with those of analogous mutations in other GTPases, and thus provided tools for discriminating the roles of GTP binding and GTP hydrolysis by Cdc12 in vivo.
GTP binding to Cdc12 is required for normal bud morphology and cytokinesis
Cdc12 is indispensable for viability (Cvrckova et al., 1995
). To examine the effect of the cdc12
) and cdc12
) mutations, each was integrated at the CDC12
locus. At 30°C, cells expressing Cdc12(S43V) or cells expressing Cdc12(T48N) showed no obvious defect. At elevated temperature (37°C), the GTP binding-defective mutant, cdc12
), displayed abnormalities in both morphology () and cytokinesis ( A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200312070/DC1
). The GTP hydrolysis-defective mutant, cdc12
), showed no detectable aberration, even at 37°C (), and even when overexpressed from a multi-copy plasmid (unpublished data). At 37°C, expression of Cdc12(T48N) was lower than that of normal Cdc12 (Fig. S3), which might have accounted for its phenotype. However, even when overexpressed from a multi-copy (2 μm DNA) plasmid (Fig. S3), the same morphological and cytokinesis defects were observed at 37°C ( A) Presence of wild-type CDC12
on a CEN
plasmid restored a normal phenotype to cdc12
) cells ( A); thus, the defect is recessive.
Figure 2. GTP binding-dependent functions of Cdc12 and Cdc10 overlap. (A) Wild-type (BY4741) transformed with empty vector (YCplac33), cdc12(S43V) (YMVB2), cdc12(T48N) (YMVB1), cdc12(T48N) (YMVB1) carrying an episomal vector overexpressing cdc12(T48N) (pMVB49), (more ...)
GTP binding-dependent functions of Cdc10 and Cdc12 overlap
The fact that Cdc12(T48N) lacked a phenotype at 30°C raised the possibility that GTP binding to other septins might partially mask its effects. The septin with the greatest homology to Cdc12 is Cdc10 (40% identity). Moreover, Cdc10 was the only other active GTPase in vitro ( B). To test for functional overlap, the equivalent mutation to prevent GTP binding, cdc10(S46N), was constructed and integrated at the CDC10 locus in a cdc10Δ mutant. As observed for cdc12(T48N), cdc10(S46N) cells had no phenotype at 30°C, but displayed aberrant bud morphology ( B) and cytokinesis defects (Fig. S2) at 37°C. A cdc10(S46N) cdc12(T48N) double mutant showed dramatic morphology defects even at 30°C, and was inviable at 37°C (see also A), whereas both single mutants to grow at 37°C. This additive phenotype indicates redundancy in the function of GTP binding to Cdc10 and Cdc12, and indicates that lack of GTP binding causes a defect in septin function even at 30°C, but that defect is exacerbated at higher temperature. In marked contrast, a double GTP hydrolysis-defective mutant, cdc10(S41V) cdc12(S43V), did not display any aberration at any temperature tested ( B).
Figure 6. Synergistic roles for Cla4 phosphorylation and GTP binding in septin collar assembly. (A) A cla4Δ mutant (YMVB12), cdc12(T48N) (YMVB3), cla4Δ cdc12(T48N) (YMVB14), cdc12(T48N) cdc10(S46N) (YMVB50), and cla4Δ cdc12(T48N) cdc10( (more ...)
Formation of heteromeric septin complexes does not require GTP binding
An inherent property of septins is the ability to form heteromeric complexes with other septins (Frazier et al., 1998
; Kinoshita et al., 2002
; Sheffield et al., 2003
). We used three independent approaches, all of which showed that GTP binding to Cdc12 is not necessary for its association with other septins. First, when immunoprecipitated with anti-Cdc12ΔC antibody, Cdc12 and Cdc12(T48N) coimmunoprecipitated equivalent amounts of Cdc3-GFP, Cdc10-GFP, and Cdc11-GFP ( A), even from extracts of yeast cultures that had been shifted to a temperature (37°C) at which the cdc12
) mutant cells display a pronounced phenotype. Second, purified Cdc12 and Cdc12(T48N) interacted equally well with the three other septins (Cdc3, Cdc10, and Cdc11), with Cdc12 itself, and with the septin-binding fragment of Hsl1 (Barral et al., 1999
; Shulewitz et al., 1999
), which were all purified from bacteria as GST fusions and immobilized on glutathione-agarose beads, but did not bind to immobilized GST ( B). Incubation at different temperatures (4, 25, or 37°C), or preincubation with 0.1 mM GDP, GTP, or GTPγS, did not affect the affinity of Cdc12 or Cdc12(T48N) for any of the other septins, itself, or Hsl1(ΔN) (unpublished data). Thus, GTP binding to Cdc12 is not required for its ability to associate with other septin partners and does not require any other yeast proteins to do so. In contrast, Cdc12ΔC showed greatly reduced binding to GST-Cdc3 ( B), indicating that its coiled-coil contributes to this interaction.
Figure 3. Septin–septin interactions do not require GTP binding. (A) Wild-type (BY4741) or cdc12(T48N) cells (YMVB3) transformed with CEN plasmids containing Cdc3-GFP, Cdc11-GFP (pSB5), or Cdc10-GFP (pLA-10) were grown to mid-exponential phase at 30°C (more ...)
Third, to further confirm that GTP binding is not required for formation of multi-septin complexes, we coexpressed His6-Cdc12 with one, two, or three other septins (Cdc3, Cdc3 + Cdc10, and Cdc3 + Cdc10 + Cdc11) in Escherichia coli at 37°C using two compatible, bicistronic vectors. His6-tagged Cdc12 and any associated proteins then were purified on Ni2+-nitrilotriacetate (NTA) beads. As judged by Coomassie staining, stoichiometric complexes of His6-Cdc12 with the other coexpressed septins were observed in every case ( C). Inclusion of 0.1 mM GDP, GTP, or GTPγS in the buffers during purification did not affect the stoichiometry or yield of these complexes (unpublished data). When His6-Cdc12(T48N) and Cdc10(S46N) were used instead of their wild-type counterparts, equivalent complexes were observed in every case ( C). Examination of nucleotide binding in vitro showed that eliminating GTP binding to Cdc10 and Cdc12 was sufficient to abrogate GTP binding to the heterotetrameric complex ( D). Thus, even in the context of a heterotetrameric complex, Cdc10 and Cdc12 seem to be the only septins capable of GTP binding.
GTP binding is required for septin filament assembly in vitro
Dialysis into low salt buffer (50 mM KCl) promotes assembly of multi-septin complexes into filaments (Frazier et al., 1998
; Kinoshita et al., 2002
). We used two different assays to examine filament formation by our preparations of recombinant heterotetrameric complexes before and after dialysis into low salt. First, purified heterotetrameric complexes were applied in high salt elution buffer or after dialysis into low salt to polylysine-coated glass slides, stained with anti-Cdc12ΔC antibody and visualized using TRITC-conjugated secondary antibody. Wild-type complexes displayed prominent filament-like structures even in elution buffer, and the quantity, size, and brightness of these structures dramatically increased upon dialysis into low salt buffer (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200312070/DC1
). No such structures formed when Cdc10 or Cdc11 was absent, such as in His6
Cdc12–Cdc3–Cdc10, and His6
Cdc12–Cdc3–Cdc11 complexes (unpublished data), consistent with the reported inability of septin complexes lacking either Cdc10 or Cdc11 isolated from yeast cells to form filaments in vitro (Frazier et al., 1998
). By this method, His6
-Cdc12(T48N)–Cdc3–Cdc10(S46N)–Cdc11 heterotetramers failed to form filament-like structures, either before or after dialysis (Fig. S4). Heterotetrameric complexes containing just Cdc12(T48N), but wild-type Cdc3, Cdc10, and Cdc11, did form filament-like forms, but much shorter in length than those produced by wholly wild-type heterotetrameric complexes (unpublished data).
These conclusions were confirmed at the ultrastructural level by EM. In elution buffer, wild-type heterotetramers contained filaments; individual filaments were ~10 nm wide and 200 nm–2 μm in length. The filaments were often paired or formed hairpins, and sometimes had lateral striations (). The dimensions and appearance of the filaments reconstituted from recombinant proteins are quite similar to those reported for filaments prepared from septin complexes isolated from yeast (Frazier et al., 1998
). Our results show that the above-mentioned characteristics are intrinsic to the septins and do not require any other yeast protein. After dialysis into low salt buffer, wild-type heterotetramers assembled into very long paired filaments, many with numerous apparent branches (). His6
Cdc12 (T48N)–Cdc3–Cdc10(S46N)–Cdc11 heterotetramers never formed any filaments observable by EM under any condition examined (). These findings suggest that GTP binding to Cdc10 and Cdc12 is necessary to induce (or stabilize) a conformation of the heterotetrameric complex that supports its ability to self-associate into filaments.
Septin filament assembly in vitro requires GTP binding. Filament formation of the indicated complexes (see C) in elution buffer or after dialysis against low salt buffer was visualized by negative-stain transmission EM. Bars, 200 nm.
GTP binding to Cdc10 and Cdc12 is required for septin collar assembly
To determine what aspect of septin organization fails in vivo in the absence of GTP binding, Cdc12-GFP and Cdc12(T48N)-GFP were expressed in either wild-type or cdc12(T48N) cdc10(S46N) cells. At 26°C, both the normal and mutant fusions localized properly to the bud neck in either normal or mutant cells. After a shift to 37°C, localization of both fusions was not perturbed in the normal cells. However, by 4 h after shift to 37°C, Cdc12(T48N)-GFP in the cdc12(T48N) cdc10(S46N) strain (or GFP-tagged versions of any of the other three normal septins) was disorganized or absent altogether from the bud neck in ≥95% of the cells, even though the mutant protein was stably expressed, as judged by immunoblotting (unpublished data). In contrast, localization of septins in a GTP hydrolysis-defective double mutant, cdc10(S41V) cdc12(S43V), at 37°C was indistinguishable from the wild type (unpublished data). One explanation for absence of proper septin organization at the neck at 37°C in the GTP binding-defective septin mutants is that lack of GTP binding to Cdc10 and Cdc12 makes the septin collar unstable at that temperature. Alternatively, lack of GTP binding could prevent efficient assembly of the septin collar at elevated temperature.
To distinguish these possibilities, we examined septin collar assembly in synchronized cells. Cells were arrested in G1 by treatment with α-factor at 26°C and released from the block at 37°C ( A). We compared Cdc12-GFP in wild-type cells to Cdc12(T48N)-GFP in cdc12(T48N) cdc10(S46N) cells. By 20 min after release, both Cdc12 and Cdc12(T48N) assembled into a patch at the incipient bud site. In wild-type cells, by 40 min after release, Cdc12 formed into a circular structure around the neck of the emerging bud and, by 60 min had formed a prominent collar at the neck. By 80 min after release, the collar split into two rings and the cells underwent cytokinesis. In the mutant cells, Cdc12(T48N) remained as a patch at the bud tip throughout the time course of the experiment, even though the bud elongated and the cell continued to grow during this period ( A). In the absence of the collar, the mutant cells failed to undergo cytokinesis. An otherwise identical experiment was performed in cdc12(T48N) cdc10(S46N) cells expressing Cdc3-GFP with the same result (unpublished data). Thus, reorganization of the patch into the collar is dependent on GTP binding, whereas recruitment of septins to the presumptive bud site is not. Consistent with this view, we observed in mutant cells that, whereas the old patch of Cdc12(T48N) persisted at the previous bud site, new Cdc12(T48N) fluorescence appeared at the new presumptive bud site by 80 min after release ( A).
Figure 5. GTP binding is necessary for septin collar assembly. (A) Wild-type cells (BY4741) carrying CDC12-GFP (pLP29), and cdc10(S46N) cdc12(T48N) (YMVB8) expressing cdc12(T48N)-GFP (pMVB91) were grown to mid-exponential phase on SCD(-His) at 26°C, synchronized (more ...)
Shifting cdc12(T48N) cdc10(S46N) cells to 37°C upon release from G1 arrest could bias the block observed to the first point in the cell cycle where GTP binding is required for septin function, but might not reveal other stages where GTP binding is needed. For this reason, the same strains were arrested in S phase at 26°C by treatment with hydroxyurea and released from the block at 37°C ( B). At 26°C, normal septin collars assembled in wild-type cells expressing Cdc12-GFP and in mutant cells expressing Cdc12(T48N) during the S phase block (0-min time point). Upon release at 37°C, both strains proceeded normally through the cell cycle (20-min time point) and underwent cytokinesis (40-min time point), indicating that GTP binding is not required for the stability or function of the collar once formed. In the next cycle, the wild-type cells assembled new patches at the presumptive bud site (60 min) and reorganized them into new collars in budded cells (80 min). In the ensuing cycle, the mutant cells also assembled new septin patches at the bud site (60 min). However, these patches persisted at the bud tip throughout the time course of the experiment (80 min), and were never able to form a collar at the neck of the emerging bud ( B). Thus, GTP binding to septins is critical for their reorganization from the patch into the collar (rather than in maintaining stability of the collar once assembled).
Cla4 protein kinase promotes GTP-dependent septin collar assembly
We were struck by the fact that cla4
mutants accumulate septins at the bud tip (Cvrckova et al., 1995
; Weiss et al., 2000
; Schmidt et al., 2003
), similar to the phenotype of septins defective in GTP binding. If Cla4 action, like GTP binding, contributes to septin reorganization necessary for collar assembly, loss of Cla4 function should aggravate the phenotype of septin mutants defective in GTP binding. Indeed, unlike cla4
Δ or cdc12
) single mutants, which grow at all temperatures tested (26, 30, and 37°C) and only have a mild phenotype at 37°C, a cla4
) double mutant has dramatically aberrant morphology at 30°C, even more striking than cdc12
) cells, and is inviable at 37°C ( A). Moreover, a cla4
) triple mutant has pronounced morphological defects even at 26°C, grows in a remarkably hyphal-like fashion at 30°C, and is inviable at 37°C ( A).
If Cla4 and septins function in the same process, then overproduction of Cla4 might rescue the defects caused by GTP binding-deficient septins. Indeed, overexpression of CLA4
driven by the GAL
promoter on a CEN
plasmid largely suppressed the temperature-induced morphological aberrations and cytokinesis defects of cdc12
) cells ( B). This effect required the catalytic activity of Cla4 because overexpression of a kinase-dead allele, cla4
) (Tjandra et al., 1998
), did not rescue the phenotype of cdc12
) cells. This effect was highly specific to Cla4 because overexpression to the same extent of each of the two other S. cerevisiae
PAKs, Ste20 and Skm1 (as judged by immunoblotting [unpublished data] of NH2
-terminally Myc epitope-tagged proteins), was unable to ameliorate the defects of cdc12
) cells ( B).
We noted that prolonged overexpression of CLA4
is detrimental to cell growth (but only well after 8 h of Gal induction). To verify that CLA4
overexpression suppresses the effects of the GTP binding-defective septin mutations by restoring septin collar assembly at bud emergence (rather than appearing to do so by simply inhibiting growth), three strains were arrested with α-factor on Gal-containing medium for 3 h at 26°C and released into Gal-containing medium at 37°C (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200312070/DC1
). These were cdc12
) cells expressing Cdc12(T48N)-GFP, as before; cla4
Δ cells expressing Cdc12-GFP; and cdc12
) cells expressing Cdc12(T48N)-GFP, but also overexpressing CLA4
. As expected, the GTP binding-defective septin assembled into a patch at the bud tip, but failed to form a collar. Likewise, in cells lacking Cla4, the bulk of Cdc12-GFP remained at the bud tips, as observed previously (Weiss et al., 2000
). By contrast, in the cdc12
) mutant overexpressing CLA4
, the patch formed, the cells proceeded through the cell cycle, and then the collar assembled efficiently (Fig. S5). Additional evidence corroborated that rescue of collar formation is specific to Cla4; for example, overexpression of two other putative septin regulators, Gin4 protein kinase (Longtine et al., 1998
) or the bud neck-localized protein, Bni5 (Lee et al., 2002
), did not suppress the septin collar assembly defect of the GTP binding-defective septin mutants (unpublished data).
Cla4 physically associates with and directly phosphorylates septins
Our finding that catalytically inactive Cla4 did not suppress the phenotype of the GTP binding-defective septins ( B) suggested that septins might be substrates of Cla4, a possibility not heretofore explored. To test this idea, NH2-terminally Myc-tagged versions of Cla4 and Cla4(K594A) were immunopurified from yeast extracts and incubated with γ[32P]ATP and His6-tagged recombinant septins. Cla4 recovered from yeast showed potent autophosphorylation that was abrogated by the K594A mutation ( A). Likewise, Cla4, but not Cla4(K594A), efficiently phosphorylated Cdc3, Cdc10, Cdc11, and to a much lesser extent, Cdc12. Cla4 did not phosphorylate GST ( A), GFP (unpublished data), or even another yeast septin (either full-length [GST-Shs1] or COOH-terminally-truncated [GST-Shs1ΔC)]; A). Cdc3, Cdc10, Cdc11, or Cdc12 were not phosphorylated by immunoprecipitated HA-tagged Elm1 or Gin4, although both kinases were active, as judged by their autophosphorylation (unpublished data).
Figure 7. Cla4 phosphorylates septins. (A) NH2-terminally myc-tagged Cla4 (pMVB113) or Cla4(K495A) (pMVB112) were expressed in cla4Δ cells (YMVB12) and recovered by immunoprecipitation, and equal amounts, as verified by immunoblotting (outer right panel), (more ...)
To verify that Cla4 directly phosphorylates septins in vitro, GST-Cla4 and GST-Cla4(K594A) were expressed and purified from E. coli
and incubated with His6
-tagged septins. Again, both Cdc3 and Cdc10 served as phosphoacceptors for Cla4 and were not detectably phosphorylated by Cla4(K594A) (Fig. S6 A, available at http://www.jcb.org/cgi/content/full/jcb.200312070/DC1
). However, Cdc11 (Fig. S6 A) and Cdc12 (unpublished data) did not serve as substrates for recombinant Cla4. Moreover, the isolated Cla4 kinase domain, GST-Cla4(524–842) (hereafter called Cla4ΔN), did not phosphorylate septins at all, although it was very active, as judged by its robust autophosphorylation (Fig. S6 A) and by its ability to phosphorylate a nonspecific substrate, myelin basic protein (unpublished data).
These observations suggested that a region in Cla4 upstream of its COOH-terminal kinase domain is required for its ability to recognize and efficiently phosphorylate Cdc3 and Cdc10. In agreement with the presence of a docking site(s) for Cdc3 and Cdc10 in the NH2-terminal regulatory region of Cla4, we found that full-length Cla4 interacts very tightly with Cdc10 and Cdc3 in an in vitro pull-down assay (and only weakly, if at all with Cdc11 or Cdc12), whereas Cla4ΔN did not associate with either Cdc3 or Cdc10 above the nonspecific background level (Fig. S6 B). The fact that recombinant Cla4 interacts poorly, if at all, with Cdc11 and Cdc12 provides a straightforward explanation for why these septins were not in vitro substrates for the bacterially expressed enzyme. Recruitment of Cdc11 and Cdc12 via their binding to Cla4-bound Cdc3 and Cdc10 present in Cla4 recovered from yeast likely explains phosphorylation of these septins in the immune complex kinase assay ( A).
To confirm that septins are phosphorylated in vivo in a Cla4-dependent manner, we examined Cdc10, the best substrate for Cla4 in vitro. We expressed Cdc10-GFP in wild-type cells or an isogenic cla4
Δ mutant, labeled the cells with [32
, and recovered Cdc10-GFP by immunoprecipitation with anti-GFP mAb. In several independent experiments, absence of Cla4 reduced incorporation of radioactivity into Cdc10-GFP to 50% or lower than that seen in control cells ( B). Thus, Cdc10 is a phosphoprotein in vivo and Cla4 is responsible for at least half of its endogenous phosphorylation. Residual phosphorylation on Cdc10 in a cla4
Δ mutant might be due to one or both of the other yeast PAKs, Ste20 and Skm1. Ste20 can contribute to septin localization (Cvrckova et al., 1995
) and overproduction of Skm1 causes morphological aberrations (Martin et al., 1997
). However, Ste20 and Skm1 phosphorylate Cdc10 in immune complex kinase assays, but much less efficiently than Cla4 (unpublished data). We mapped two primary sites for Cla4 in Cdc10 (Ser256 and Ser312) both in vitro and in vivo (see online supplemental Materials and methods). Preventing phosphorylation of the major site by a cdc10
) mutation results in an elongated bud phenotype at 37°C ( C), which is not significantly worse in a cdc10
) double mutant (unpublished data). Strikingly, preventing both GTP binding to Cdc10 and phosphorylation at Ser256 in the double mutant cdc10-
) synergistically caused a severe morphological defect at 37°C ( C). Moreover, in these cells, Cdc12-GFP localized exclusively to the tips and lateral aspects of buds and did not form a collar at the neck ( C). First, these data confirm the physiological importance of Cla4 phosphorylation of Cdc10 in vivo, and second, demonstrate that Cla4-mediated phosphorylation of septins acts in concert with GTP binding to regulate the septin reorganization necessary for transition from the patch to the collar.