The CTE Is Necessary for Cdc12 Function
Cdc12 contains an apparent coiled coil-forming segment at its C terminus (), as predicted by algorithms like MultiCoil (Wolf et al., 1997
) that recognize sequence covariation in 4-3 hydrophobic repeats compatible with the formation of coiled coils. In Cdc12, the predicted 40-residue coiled coil extends from Glu368 to the C-terminal residue (Lys407). In addition, programs for secondary structure prediction indicate that the immediately preceding sequence (Arg341-Trp367) has a striking propensity to form an extended α-helix. Both of these elements lie downstream of the septin-unique domain, which flanks the GTP-binding domain and is present in all septins (). The predicted α-helix is broken at Asn339-Pro340. Therefore, we consider the region from residue 339 to the C terminus to be the CTE.
Cdc12, Cdc3, and Cdc11 are required for viability, whereas Cdc10 is essential only at 37°C, and Shs1 is dispensable even at elevated temperature. Therefore, we first used a genetic approach to examine whether the CTE is necessary for Cdc12 function in vivo. For this purpose, we constructed a truncated allele, CDC12(
) (hereafter Cdc12ΔC) and tested whether it was able to support growth when present as the sole source of Cdc12. Either full-length CDC12
or the truncated allele, under control of the authentic CDC12
promoter, was introduced on a low-copy-number (CEN
) plasmid into a heterozygous cdc12
diploid. After sporulation and tetrad dissection, the ability of normal Cdc12 and Cdc12ΔC to rescue the inviability of the cdc12
Δ spores was compared (). For diploids transformed with the plasmid expressing normal Cdc12, all of the tetrads (≥20 analyzed) displayed three or four viable spores, showing that plasmid-borne CDC12
readily rescued the inviability of cdc12
Δ spores (which also were marked by the insertion of a resistance marker, kanMX
, to the antibiotic G418). By contrast, for the diploids transformed with the plasmid expressing Cdc12ΔC, only two viable spores were obtained in every tetrad (≥20 analyzed), and those spores were always G418 sensitive. In the heterozygous diploids, and when produced from a CEN
plasmid, the truncated protein was stably expressed at a level equivalent to full-length Cdc12 (, far right). Hence, Cdc12ΔC could not support cell viability. Cdc12ΔC was unable to rescue viability of cdc12
Δ cells even when overexpressed from a multicopy (2 μm DNA) plasmid or from the GAL1
promoter (unpublished data). Hence, the inability of Cdc12ΔC to rescue Cdc12-deficient cells was not due to its lack of expression or instability. Also, the truncated protein is folded because Cdc12ΔC binds GTP as tightly as normal Cdc12 and has a GTPase activity that is at least 5 times higher than wild-type Cdc12 (Versele and Thorner, 2004
). These properties of the mutant protein suggested that perhaps its elevated GTPase activity might account for its deleterious function in vivo. Consistent with this possibility, full-length Cdc12 mutants compromised for either GTP binding or GTP hydrolysis are able to rescue the lethality of cdc12
Δ cells (Versele and Thorner, 2004
). However, when mutations (T48N and S43V, respectively) that abrogate, respectively, GTP binding and GTP hydrolysis were introduced into Cdc12ΔC (Versele and Thorner, 2004
), neither of the corresponding Cdc12ΔC derivatives could support the growth of cdc12
Δ spores (). Thus, the CTE of Cdc12 is necessary for the essential function of Cdc12 in vivo and that function is independent of the GTP-dependent activities of Cdc12.
Figure 2. The essential function of Cdc12 requires its C-terminal extension. (A) A cdc12Δ/CDC12 heterozygous diploid (Y21935) was transformed with a CEN plasmid carrying either wild-type CDC12 (pMVB45), CDC12ΔC (pMVB48), CDC12(S43V)ΔC (pMVB52), (more ...)
Another potential explanation for the inability of Cdc12ΔC to substitute for normal Cdc12 is that it may not localize properly. Because Cdc12 is essential and Cdc12ΔC is nonfunctional, we could only test whether the CTE is required to target Cdc12 to the bud neck by expressing full-length Cdc12-GFP or Cdc12ΔC-GFP in otherwise wild-type cells. Both Cdc12-GFP and Cdc12ΔC-GFP were expressed at the same level and were recruited to the bud neck (). However, we consistently noted that Cdc12ΔC-GFP was incorporated less efficiently into the collar than its wild-type counterpart, despite its equivalent expression, as judged by the reproducibly dimmer signal at the bud neck in every cell (). Moreover, heterozygous cdc12/CDC12 diploids transformed with the plasmid expressing CDC12ΔC displayed a mild elongated-bud morphology. This dominant effect suggested that Cdc12ΔC interferes with the function of Cdc12 in the same cell.
Figure 7. Cdc10 is essential for normal septin collar organization at the bud neck. (A) Wild-type strain (BY4741) and a cdc10Δ mutant (YMVB5) were transformed with plasmids expressing Cdc12-GFP (pLP17) or Cdc12ΔC-GFP (pMVB33), grown at 26°C (more ...)
To confirm this conclusion, a haploid strain was constructed that contained a single copy of the normal CDC12 gene and a single copy of the truncated allele (also under control of a CDC12 promoter) integrated into the genome at the CDC12 locus. We found that even one copy of Cdc12ΔC in otherwise wild-type cells was sufficient to disrupt normal cell morphology, as indicated by the markedly elongated buds (). When the ratio of Cdc12ΔC to Cdc12 was further elevated by expression of CDC12ΔC from a multicopy plasmid, morphological perturbation of the cells was even more pronounced, even though the steady-state level of Cdc12ΔC was still somewhat less than that of Cdc12 (). Expression of Cdc12ΔC (from its own promoter) on either a CEN or 2 μm DNA plasmid was sufficient to noticeably perturb the bud neck localization of C-terminally GFP-tagged versions of Cdc3, Cdc10, Cdc11, and Cdc12 itself (unpublished data). Prolonged overexpression of Cdc12ΔC driven by the GAL1-promoter caused even more severe defects in cell morphology and grossly disrupted the organization of the septin collar, as judged by the mislocalization and aberrant deposition of three other septins, Cdc3, Cdc10, and Cdc11 (each marked with a C-terminal GFP tag) or of Cdc12ΔC-GFP itself (). Overexpression of normal Cdc12 in the same manner had no such effects. Thus, the CTE of Cdc12 is critically important for the proper organization of septins in vivo.
Cdc12 Interacts with Cdc3 via the CTE and with Cdc10 Independently of the CTE
To determine whether the CTE of Cdc12 participates in direct septin–septin interactions, an in vitro assay was used to test the binding of any given septin (with or without its CTE) to every other septin (with or without its CTE). First, glutathione-agarose beads were coated with GST fusions either to full-length septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) or to derivatives of each septin lacking its cognate CTE, namely, Cdc3(Δ427–520), Cdc11(Δ357–415), Cdc12(Δ339–407), and Shs1(Δ418–551). Cdc10 is the only yeast septin that lacks a CTE (). Second, each septin to be tested (with or without its CTE) was tagged with a (His)6 tract (at either its N or C terminus), expressed in bacteria, purified by metal-chelate affinity chromatography, and then incubated in solution with the bead-bound GST-fusions. After washing the beads, bound proteins were eluted and analyzed by SDS-PAGE. Neither Cdc12-His6 nor Cdc12ΔC-His6 bound to beads coated with GST alone (). Full-length Cdc12-His6 interacted strongly with three other septins (GST-Cdc3, GST-Cdc10 and GST-Cdc11) and also with itself (GST-Cdc12), but not with GST-Shs1 (). By comparison, binding of Cdc12ΔC-His6 to GST-Cdc3 and GST-Cdc11, and to GST-Cdc12 itself, was markedly reduced (). In contrast, Cdc12 and Cdc12ΔC interacted with GST-Cdc10 with apparently equal affinity. Conversely, a GST-fusion to Cdc3ΔC interacted poorly with either Cdc12-His6 or Cdc12ΔC-His6 (). Also, the interactions between Cdc12-His6 and Cdc12ΔC-His6 with either GST-Cdc11ΔC or GST-Cdc12ΔC were reduced compared with their interactions with full-length GST-Cdc11 and GST-Cdc12 ().
Figure 3. Role of septin CTEs in pairwise septin–septin interactions. (A) Equivalent amounts of the indicated GST-septin fusions [Cdc3, Cdc3(Δ427–521), Cdc10, Cdc11, Cdc11(Δ357–416), Cdc12, and Cdc12(Δ339–407)] (more ...)
To corroborate and extend these conclusions, the ability of His6-Cdc3, His6-Cdc10, and Cdc11-His6 to associate with the panel of GST-fusions was assessed. His6-Cdc3 did not bind detectably to GST alone (, left). His6-Cdc3 bound well to GST-Cdc12, yet not to GST-Cdc12ΔC, confirming that the CTE of Cdc12 is essential for Cdc3–Cdc12 association (). His6-Cdc3 also bound to Cdc10, but not to GST-Cdc11 () or GST-Shs1 (). Like Cdc12-His6, His6-Cdc3 displayed some self-association that was dependent on its CTE.
Consistent with the preceding results, His6-Cdc10 bound well to both GST-Cdc12 and GST-Cdc12ΔC (, middle). Likewise, in accord with the binding of His6-Cdc3 to GST-Cdc10, His6-Cdc10 bound to both GST-Cdc3 and GST-Cdc3ΔC. His6-Cdc10 did not bind detectably to either GST-Cdc11 (, middle) or to GST-Shs1 (). Like Cdc12 and Cdc3, Cdc10 displayed a modest degree of self-association.
In agreement with the lack of interaction between His6-Cdc10 and GST-Cdc11, Cdc11-His6 did not interact detectably with GST-Cdc10 (, right). The strongest interaction observed for Cdc11-His6 was with GST-Shs1 (). In fact, the only septin able to associate with Shs1 was Cdc11. The binding of Cdc11-His6 to GST-Shs1 was markedly and reproducibly reduced when the CTE of Shs1 was absent. Cdc11-His6 also interacted with GST-Cdc3, GST-Cdc3ΔC, GST-Cdc12, GST-Cdc12ΔC, and with itself (either GST-Cdc11 or GST-Cdc11ΔC) (, right). Thus, all of these interactions of Cdc11 were largely independent of the CTEs of any of the septins.
The results of these binding interactions are compiled in . Briefly, Cdc3–Cdc12 interaction requires the CTE of each protein; both Cdc3 and Cdc12 associate with Cdc10, and do so independently of their CTEs. Cdc10 does not associate at all with Cdc11 or Shs1. The only septin that binds Shs1 is Cdc11, and this interaction is largely dependent on the CTE of Shs1. Finally, Cdc3, Cdc10, Cdc11, and Cdc12, all self-associate to at least some degree (Shs1 was not tested). However, this analysis of the direct pairwise interactions between the septins, although very informative, left a few ambiguities. Cdc11 bound to Cdc12, but it was unclear from these particular experiments whether this interaction is influenced by the CTE of either protein. It also was unresolved whether Cdc11 binds to Cdc3.
Summary of direct septin–septin interactions
Requirements for Septin Complex Formation
In the cell, septins are all present together, and multivalent contacts among them may contribute to higher order interactions that mediate formation of the multiseptin complexes found in vivo. Therefore, we coexpressed the core septins Cdc3, Cdc10, Cdc11, and Cdc12 (with or without their CTE) in all possible binary, tertiary, and quaternary combinations in E. coli by using compatible bicistronic vectors. (Shs1 was not included in this analysis because, unlike the core septins, it is not an essential protein in vivo and because our binding assays indicated that it only associates with Cdc11 and does so via its CTE.) In each case, only a single septin (usually Cdc12) carried a (His)6 tag and was the septin expressed from the vector and promoter that ensured that it would be the protein expressed in the most limiting amount (unpublished data). The His6-tagged protein and any associated polypeptides were purified by Ni2+-chelate affinity chromatography and the composition of the resulting complexes was analyzed by SDS-PAGE.
As judged by densitometry of the Coomassie Blue-stained protein bands, formation of stoichiometric complexes between Cdc3 and His6-Cdc12 and between His6-Cdc12 and Cdc11 was observed reproducibly (). No complexes between His6-Cdc3 and Cdc11, or between His6-Cdc10 and Cdc11, were detected (unpublished data). Revealingly, the amount of Cdc3 bound in complexes with His6-Cdc10, and the amount of Cdc10 bound in complexes with His6-Cdc12, was strikingly substoichiometric (, right). In marked contrast, Cdc10 was incorporated stoichiometrically into complexes that contained both Cdc3 and His6-Cdc12 (, left), suggesting that efficient incorporation of Cdc10 into multiseptin complexes requires its simultaneous association with both Cdc3 and Cdc12.
Figure 4. Reconstitution of stoichiometric septin complexes requires the CTE of Cdc12. (A) His6-Cdc12 or His6-Cdc10, as indicated, was coexpressed in E. coli with the indicated untagged septin(s), and any resulting complexes were purified using Ni2+-NTA-agarose. (more ...)
The only other ternary complex observed was between Cdc3, Cdc11, and His6-Cdc12 (, left), in keeping with the fact that Cdc12 associated in stoichiometric binary complexes with both Cdc3 and Cdc11. Presence of all three of these septins in stoichiometric amounts in the ternary complex suggests that Cdc3 and Cdc11 associate with different sites on Cdc12. This conclusion is also in accord with the GST-pull down assays, in which strong Cdc3–Cdc12 association required the CTE of each protein (), whereas Cdc11 was able to associate with either full-length Cdc12 or Cdc12 lacking its CTE ().
When Cdc10, Cdc11, and His6-Cdc12 were coexpressed, His6-Cdc12-Cdc11 binary complexes were recovered, but no significant amount of Cdc10 was incorporated (unpublished data). Moreover, when His6-Cdc3, Cdc10, and Cdc11 were coexpressed, no protein other than His6-Cdc3 was recovered efficiently; likewise, when Cdc3, His6-Cdc10, and Cdc11 were coexpressed, no other protein copurified with Cdc10 (unpublished data). However, when all four proteins were coexpressed, stoichiometric heterotetrameric complexes of Cdc3, Cdc10, Cdc11, and His6-Cdc12 were readily and reproducibly recovered (). These results reinforce the conclusion that presence of Cdc12 is critical to assembly of heteromeric septin complexes.
Finally, this coexpression approach was used as an independent means to confirm the role of the Cdc12 CTE in Cdc3–Cdc12 interaction, but not in Cdc12–Cdc11 interaction. Indeed, as expected, Cdc11 was copurified in stoichiometric complexes with either His6-Cdc12 or His6-Cdc12ΔC, whereas Cdc3 formed stoichiometric complexes with His6-Cdc12, but copurified in only trace amounts with His6-Cdc12ΔC (, left). Likewise, stoichiometric ternary complexes containing Cdc3, Cdc10, and His6-Cdc12 were readily purified, but neither Cdc3 nor Cdc10 were efficiently recovered with His6-Cdc12ΔC (, middle), in agreement with the fact that the CTE of Cdc12 is required for Cdc3–Cdc12 association and that incorporation of Cdc10 into complexes requires the presence of both Cdc3 and Cdc12 ().
Similarly, stoichiometric ternary complexes containing Cdc3, Cdc11, and His6-Cdc12 can be readily purified, but Cdc3 is very inefficiently recovered with His6-Cdc12ΔC, even though Cdc11 binding to His6-Cdc12ΔC is undiminished (, middle). Finally, heterotetrameric complexes prepared with His6-Cdc12 contain stoichiometric amounts of each of the other three core septins; but, in the absence of the Cdc12 CTE, only Cdc11 is recovered in a stoichiometric amount with His6-Cdc12Δ and both Cdc3 and Cdc10 are greatly underrepresented (, right). These results confirm that the CTE of Cdc12 is required for its association with Cdc3, but not with Cdc11.
The Cdc12 CTE, but Not Its Coiled Coil Element, Is Sufficient for Interaction with Cdc3
Because Cdc12 and Cdc3 association requires their CTEs (and self-association of each of these two septins is also dependent on their CTEs), the simplest interpretation of these findings is that the CTEs physically interact. Moreover, the presence of predicted coiled coil sequences within the CTEs raised the possibility that CTE–CTE interaction is mediated by coiled coil formation. To determine whether the predicted coiled coil sequences are, by themselves, sufficient to interact, synthetic peptides corresponding to Cdc12(369–407), Cdc3(459–503) and, as a control, Cdc11(369–415), were prepared and their circular dichroism signal was monitored at 222 nm (diagnostic of helix formation) as a function of temperature (unpublished data). No individual peptide adopted a stable α-helical conformation at physiological pH, temperature, and salt concentration. At high peptide concentration (400 μM), Cdc12(369–407) formed an unstable homo-oligomer under physiological conditions. Unexpectedly, mixing Cdc12(369–407) and Cdc3(459–503) produced no synergistic effects, and no stable interactions were detected in any two-way or three-way combinations of the three peptides. Thus, the predictions of MultiCoil (Wolf et al., 1997
) notwithstanding, these isolated polypeptide segments were unable to form stable autonomous coiled coils.
Given that the predicted coiled coil sequences in both Cdc3 and Cdc12 are preceded by an additional segment predicted to be α-helical that could buttress the helix-forming propensity of a juxtaposed coiled coil, we examined whether a longer portion of the Cdc12 CTE that included the additional predicted α-helix would be sufficient to mediate association of Cdc12 CTE with other septins. Using purified bacterially expressed proteins, only His6
-Cdc3 and His6
-Cdc12, and not His6
-Cdc10 or Cdc11-His6
, bound to beads coated with purified GST-CTECdc12
(residues 339–407) (). Thus, the entire CTE of Cdc12 is both necessary and sufficient for mediating the interaction between Cdc12 and Cdc3 and also contributes to Cdc12–Cdc12 self-association. In vivo a GFP-CTECdc12
chimera was not stably recruited to the bud neck (unpublished data); however, when extracts of such cells were passed over glutathione-agarose beads coated with the panel of GST-septin fusions, GFP-CTECdc12
bound avidly to GST-Cdc3, but not to GST-Cdc3ΔC (unpublished data), consistent with the binding observed between the highly purified proteins (). The fact that GFP-CTECdc12
was not stably incorporated into the septin collar, whereas Cdc12ΔC-GFP is (), indicates that stable filament assembly requires the globular, N-terminal GTP-binding domain of Cdc12. This conclusion is in accord with our finding that GTP binding to Cdc12 is indeed essential for septin collar formation in vivo and for septin filament polymerization in vitro (Versele and Thorner, 2004
Figure 5. The Cdc12 CTE is sufficient for interaction with Cdc3. GST-Cdc12(339–407) [GST-CT-ECdc12] expressed in bacteria from pMVB172 was immobilized on glutathione-agarose and incubated with the indicated purified His6-tagged septins. After washing, bound (more ...)
Roles for the CTE of Cdc3 and Cdc11
The pairwise interaction assays, the analysis of multiseptin complex formation, and the binding properties of the isolated CTECdc12 described above all demonstrate that Cdc12 interacts via its CTE with Cdc3, and vice versa. These two core septins are both essential proteins in yeast. By contrast, the only septin that requires its CTE to interact with Cdc11 is the nonessential septin Shs1. If the reason that the CTE of Cdc12 is required for viability () is that it is essential for mediating association with the CTE of Cdc3, the Cdc3 CTE should therefore also be necessary for cell viability. By the same logic, if the only function of the CTE of Cdc11 is to recruit the nonessential septin Shs1, a Cdc11ΔC mutant should be viable.
To test these ideas, truncations that removed the CTEs of Cdc3 and Cdc11, namely, Cdc3(Δ440–520) and Cdc11(Δ372–415), respectively, were constructed and introduced on plasmids into the corresponding heterozygous cdc3
diploids. After sporulation, the resulting tetrads were analyzed (in each case, at least 20 tetrads were examined). In our hands, deletion of CDC11
is lethal in BY4741 (a derivative of S288c), as has been observed for other S288c derivatives (Casamayor and Snyder, 2003
), and in W303 (unpublished data), in contrast to what has been reported for one strain background (YEF473), in which viable cdc11
Δ haploids purportedly were recovered (Frazier et al., 1998
). As anticipated, Cdc3ΔC was unable to rescue the viability of cdc3
Δ spores (), whereas Cdc11ΔC was able to maintain the viability of cdc11
Δ spores (). Even when overexpressed from a multi-copy plasmid, Cdc3ΔC was unable to support growth when present as the sole source of Cdc3 (unpublished data). Likewise, cdc3
Δ haploids carrying a URA3
-marked plasmid CDC3
(to maintain viability) and LEU2
-based plasmids (either centromeric or multi-copy) expressing CDC3
were unable to grow when plated on medium containing 5-fluoro-orotic acid, which selects for cells that have lost the URA3
plasmid. As observed for overexpression of Cdc12ΔC (), overexpression of Cdc3ΔC in otherwise wild-type cells caused elongated bud morphology and cytokinesis defects, and perturbed the localization of Cdc3-GFP, as well as that of Cdc10-GFP, Cdc11-GFP, and Cdc12-GFP (unpublished data). Thus, like the Cdc12 CTE, the Cdc3 CTE is critically important for proper organization of septins in vivo.
Figure 6. The Cdc3 CTE is essential, but the Cdc11 CTE is not. (A) A cdc3Δ/CDC3 heterozygous diploid (Y25223) was transformed with empty vector, or a CEN plasmid carrying either CDC3 (pMVB100) or CDC3ΔC (pMVB102), sporulated and the meiotic products (more ...)
Although absence of the Cdc11 CTE is not lethal, cells containing Cdc11ΔC as the only source of this septin displayed severe cytokinesis defects and elongated buds, indicating that Cdc11 CTE is required for proper septin collar function. These results with regard to the CTE of Cdc11 are in agreement with a previous report (Casamayor and Snyder, 2003
), but at odds with another prior study (Lee et al., 2002
). We found that Cdc11ΔC is more unstable than full-length Cdc11 (unpublished data), which suggested that the observed phenotypes of cells expressing Cdc11ΔC as the sole source of this septin might simply be due to the lower level of the mutant protein. However, overproduction of CDC11
using the GAL1
promoter exacerbated, rather than ameliorated, the aberrant morphology of cells expressing Cdc11ΔC (). In contrast to Cdc12ΔC-GFP and Cdc3ΔC-GFP, Cdc11ΔC still localized normally to the bud neck, even in cells lacking normal Cdc11 () and did not interfere with localization of Cdc12-GFP at the bud neck (). Thus, unlike the CTE of Cdc3 and Cdc12, the CTE of Cdc11 does not seem to play a major role in septin collar formation, but rather in septin collar function.
Roles of Cdc10 in Septin Collar Architecture
Cdc12 associates with Cdc3 via its CTE and with Cdc10 (and Cdc11) in a CTE-independent manner. To determine whether both of these modes of binding are necessary for incorporation of Cdc12 into the septin collar in vivo and to assess the specific role that Cdc10 may play in localizing Cdc12 to the bud neck, we examined the subcellular localization of Cdc12-GFP and Cdc12ΔC-GFP in normal and in cdc10Δ cells, which are viable at or below normal growth temperature (30°C). In wild-type cells, both Cdc12-GFP and Cdc12ΔC-GFP decorated the bud neck, but Cdc12ΔC seemed to be incorporated less efficiently, as expected, based on the reproducibly dimmer staining seen in every cell (Figures and ). In cells lacking Cdc10, Cdc12-GFP still localized at the bud neck, but at a greatly reduced level. However, Cdc12-GFP is significantly less stable in cells lacking Cdc10 than in wild-type cells (, right), which presumably accounts for the reduction in signal observed. In marked contrast, no detectable Cdc12ΔC-GFP was present at the bud neck in cells lacking Cdc10, even though Cdc12ΔC-GFP is no less stable under these conditions than Cdc12-GFP (). Thus, in the absence of the Cdc3-Cdc12 interaction mediated by its CTE, recruitment of Cdc12 to the septin collar in vivo requires Cdc10. These data suggest that, in the absence of Cdc10, the residual interactions between Cdc12ΔC and other septins observed in vitro are not sufficient to permit its stable incorporation into the septin collar at the bud neck.
Close inspection revealed that, in cells lacking Cdc10, even normal Cdc12-GFP never localized to both sides of the septin collar, as it does in wild-type cells (). To mark the cell boundary so that we could discriminate septin recruitment to the mother and bud sides of the septin collar, the chitin deposited at the bud neck was stained with calcofluor white. Whether expressed in wild-type cells at a near normal level (from the CDC12
promoter on a CEN
plasmid) or highly overexpressed (from a 2 μm DNA vector), Cdc12-GFP localization is centered around the chitin ring and seems to be symmetrically distributed on the mother and daughter sides of the septin collar (). In cdc10
Δ cells, however, Cdc12-GFP localized only to the daughter-side of the bud neck, even when copiously overproduced (). Likewise, both Cdc3-GFP and Cdc11-GFP also localize asymmetrically to the bud side of the neck in cdc10
Δ cells [unpublished data; see also Supplemental Material cited in Castillon et al
)]. Thus, lack of Cdc10 grossly perturbs septin organization; therefore, Cdc10 plays an important role in dictating the arrangement of the septin filaments in the septin collar at the bud neck.
Cdc3, Cdc11, and Cdc12 Are Necessary and Sufficient for Septin Filament Formation
To gain insight about the contribution of individual septins to septin polymer assembly and the properties of septin filaments, each of the purified stoichiometric complexes reconstituted by coexpression of recombinant proteins was examined for its ability to form polymers by using both an immunostaining assay with anti-Cdc12ΔC antibodies and examination in the EM after negative staining (). Because high ionic strength decreases the ability of heteromeric septin complexes to form septin filaments and low ionic strength promotes filament formation (Frazier et al., 1998
; Kinoshita et al., 2002
; Versele and Thorner, 2004
), the filament-forming capacity of each complex was examined both in the high-salt (HS) elution buffer (containing 250 mM NaCl and 300 mM imidazole) and after dialysis into a low-salt (LS) buffer (50 mM KCl).
Figure 8. Ultrastructural analysis of reconstituted septin filaments. (A–E) The ability of the indicated purified, recombinant, heteromeric septin complexes (left panel) to form filaments in the high-salt elution buffer used for purification (HS) or after (more ...)
The His6Cdc12-Cdc3-Cdc10-Cdc11 heterotetrameric complexes formed filaments, even in HS buffer (). In the EM, the filaments are typically straight and almost invariably paired. The range of widths measured for individual filaments (n = 30) was 7–10 nm, and for paired filaments (n = 30) was 16–22 nm. Lengths varied greatly, from 50 nm to >1 μm. Occasionally, lateral striations occurring with a repeat length of 22–33 nm were visible (as measured on four different filament pairs, each containing >15 striations). In LS buffer, the filaments often associated laterally into bundles of up to 20 filaments. These bundles could be observed as straight, strikingly bright objects visible by light microscopy after immunostaining ().
In LS buffer (but not in HS), the His6Cdc12-Cdc3-Cdc11 ternary complex also formed filaments (). However, the appearance of these filaments was rather different from those generated from the heterotetrameric His6Cdc12–Cdc3–Cdc10–Cdc11 complexes. At the light microscope level, the filament bundles composed of the His6Cdc12–Cdc3–Cdc11 heterotrimeric complexes seemed shorter, more curved, and less bright than those formed from the His6Cdc12–Cdc3–Cdc10–Cdc11 heterotetrameric complexes. EM analysis confirmed that short, curved filaments were formed. Moreover, unlike the filaments made from the His6Cdc12–Cdc3-Cdc10–Cdc11 complex, these filaments were not paired in register, although many of the curved forms comprise apparently unorganized bundles of multiple filaments ().
His6Cdc12 alone did not polymerize into filaments, but sometimes small rings or disks (50–200 nm in diameter) could be observed by immunostaining and by EM (unpublished data). The His6Cdc12–Cdc11 complex nearly always formed rings or disks (200–500 nm in diameter) in either HS or LS buffers (). By contrast, the His6Cdc12–Cdc3 complex formed rather amorphous-looking blobs (400–500 nm in diameter) at the light microscope level, which, at the EM level, sometimes had a honeycomb-like appearance with knobby protrusions (). The His6Cdc12–Cdc3–Cdc10 ternary complex failed to form any regular structure that could be discerned by either immunostaining or EM (). Finally, the complexes that contain stoichiometric amounts of His6Cdc12ΔC and Cdc11, but substoichiometric amounts of Cdc3 and Cdc10, did not form filaments, but instead displayed rings (unpublished data), similar to those formed by the His6Cdc12–Cdc11 heterodimer complex ().
These data demonstrate that the three essential septins— Cdc3, Cdc11, and Cdc12—are both necessary and sufficient for polymerization of heteromeric septin complexes into filaments, whereas Cdc10 is required to permit those polymers to organize into straighter and more regular filament pairs. The simplest model compatible with all of these data (see Discussion) is that Cdc3–Cdc12–Cdc1l complexes polymerize end to end. We entertained the notion that the Cdc3–Cdc11 association observed under some circumstances () might play a pivotal role in this polymerization. Therefore, and because polymerization is enhanced at low salt, we tested whether Cdc3–Cdc11 association is salt-sensitive. Indeed, binding of Cdc11-His6 to GST-Cdc3 was robust at low salt and decreased at high salt, whereas binding of Cdc11-His6 to GST-Shs1 (which is mediated by their CTEs) was not salt dependent ().