: Supramolecular organization of heterooligomers and the mechanism of filament assembly

Aurelie Bertin*†, Michael A. McMurray*†, Patricia Grob†‡, Sang-Shin Park*§, Galo Garcia III*, Insiyyah Patanwala*, Ho-leung Ng*, Tom Alber*, Jeremy Thorner*¶, and Eva Nogales*‡¶ʈ

*Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; ‡Howard Hughes Medical Institute; and ʈLife Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720

Communicated by Howard K. Schachman, University of California, Berkeley, CA, April 7, 2008 (received for review February 27, 2008) Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, native and recombinant complexes resist dissociation at high ionic and Shs1/Sep7). Only Shs1 is nonessential. The four essential strength (0.5–1 M KCl) but polymerize into filaments when the salt septins form a complex containing two copies of each, but their concentration is reduced (Յ50 mM KCl) (5, 13). Various strategies arrangement was not known. Single-particle analysis by EM con- have been used to ascertain the interactions within the core firmed that the heterooligomer is octameric and revealed that the complex. Two-hybrid (16) is fraught with potential problems be- subunits are arrayed in a linear rod. Identity of each subunit was cause septin complexes do not normally assemble in the nucleus and determined by examining complexes lacking a given septin, by because apparent associations might arise indirectly via ‘‘bridging’’ antibody decoration, and by fusion to marker (GFP or through other members of the complex or other septin-associated maltose binding ). The rod has the order Cdc11–Cdc12– proteins (15). By using purified septins for in vitro binding to assess Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 and, hence, lacks polarity. their capacity for pairwise interactions, apparent self-association At low ionic strength, rods assemble end-to-end to form filaments could occur in the absence of a preferred partner, and ability to but not when Cdc11 is absent or its N terminus is altered. Filaments associate with another septin could be influenced by the presence invariably pair into long parallel ‘‘railroad tracks.’’ Lateral associ- of a third (5). The predicted coiled coil might account for such ation seems to be mediated by heterotetrameric coiled coils be- nonphysiological associations. An analogy is c-Jun, which by itself tween the paired C-terminal extensions of Cdc3 and Cdc12 pro- forms homodimers but yields c-Fos-c-Jun heterodimers preferen- jecting orthogonally from each filament. Shs1 may be able to tially when both are present (17). replace Cdc11 at the end of the rod. Our findings provide insights Coexpression in bacteria of various combinations of yeast septins into the molecular mechanisms underlying the function and reg- gave a more consistent picture of the organization of the complex ulation of cellular septin structures. (5). When Cdc10, Cdc11, and (His)6–Cdc12 were coexpressed, ͉ ͉ ͉ stoichiometric (His)6–Cdc12–Cdc11 binary complexes were recov- electron microscopy yeast complexes GTP ered, but no significant amount of Cdc10 was incorporated, sug- gesting that Cdc11 interacts directly with Cdc12 and that Cdc3 is eptins comprise a discrete family of GTP-binding proteins needed to recruit Cdc10. However, when (His)6–Cdc3, Cdc10, and Saccha- Sconserved from fungi to humans (1). Budding yeast ( Cdc11 were coexpressed, no protein other than (His)6–Cdc3 was romyces cerevisiae) encodes seven paralogous septins. Five (Cdc3, recovered. This result suggested that, in the absence of Cdc12, Cdc3 Cdc10, Cdc11, Cdc12, and Shs1) are expressed in mitosis, found in is not competent to associate with Cdc10. Indeed, when coex- filaments at the mother-bud neck, and participate in cytokinesis (2). pressed, Cdc3 and Cdc12 associate avidly, and Cdc3, Cdc10 and Cdc3, Cdc10, Cdc11, and Cdc12 (but not Shs1) are essential for (His)6–Cdc12 form stoichiometric ternary complexes. Collectively, viability and were first identified in the collection of temperature- these findings indicated that assembly of the octameric complex has sensitive cell division cycle (cdc) mutants isolated by Hartwell (3). Ϸ an intrinsic order, with Cdc12 serving as the linchpin because it All contain a 300-residue GTP-binding domain (G domain, associates directly with both Cdc11 and Cdc3. However, significant 30–40% pairwise identity) but are distinguished by more variable ambiguities remained. In binding studies in vitro, Cdc10 displayed N- and C-terminal extensions [supporting information (SI) Fig. S1]. separate interactions with both Cdc3 and Cdc12. Thus, incorpora- Cdc3 has the longest N-terminal extension, whereas Shs1 has the tion of Cdc10 into ternary complexes with Cdc3 and Cdc12 could longest C-terminal extension (CTE). At their distal end, the CTEs reflect simultaneous contact of Cdc10 with both Cdc3 and Cdc12, of Cdc3, Cdc11, Cdc12, and Shs1 (but not Cdc10) contain a segment as initially proposed (5) Alternatively, association of Cdc12 with of 40–50 residues with coiled coil-forming heptad repeats (4). The Cdc3 may induce a conformational change in Cdc3 that allows it to CTEs are essential for Cdc3 and Cdc12 function in vivo but not for interact with Cdc10, as suggested above. Hence, the structural Cdc11 (5). Available data indicate that guanine nucleotide binding promotes septin folding and stability, but mutations that block

cycles of GTP binding and hydrolysis cause no overt phenotypes in Author contributions: A.B., M.A.M., J.T., and E.N. designed research; A.B., M.A.M., P.G., vivo (6, 7). The collar of filaments at the bud neck is apposed to the G.G., and I.P. performed research; M.A.M., S.-S.P., and H.-l.N. contributed new reagents/ plasma membrane, an interaction attributed to a polybasic segment analytic tools; A.B., M.A.M., P.G., T.A., J.T., and E.N. analyzed data; and A.B., M.A.M., P.G., situated proximal to the G domain (8, 9). The collar filaments T.A., J.T., and E.N. wrote the paper. impose a barrier to diffusion of integral membrane proteins be- The authors declare no conflict of interest. tween mother and bud (10, 11) and act as a scaffold to recruit Freely available online through the PNAS open access option. proteins required for bud-site selection and a morphogenesis check- †A.B., M.A.M., and P.G. contributed equally to this work. point (12). §Current address: Department of Biotechnology, College for Natural Sciences, Dongguk Native yeast septin complexes purified by immunoaffinity con- University, Gyeongju, 780-714, South Korea. tain near-equimolar Cdc3, Cdc10, Cdc11, and Cdc12 and have an ¶To whom correspondence may be addressed: [email protected]. or [email protected]. apparent molecular mass compatible with 2:2:2:2 stoichiometry (13, This article contains supporting information online at www.pnas.org/cgi/content/full/ 14). Likewise, Cdc3, Cdc10, Cdc11, and Cdc12 coexpressed in and 0803330105/DCSupplemental. purified from bacterial cells form octameric complexes (5, 15). The © 2008 by The National Academy of Sciences of the USA

8274–8279 ͉ PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803330105 Downloaded by guest on October 1, 2021 A B C Anti-Cdc11 IgG A 82.2 Cdc3 63.2 Cdc11

48.8 (His)6-Cdc12 10 nm

37.1 Cdc10 MW Markers (kDa) Anti-His6 IgG (His6-Cdc12) B 1 2 D E F 34 5 6 78 E 10 nm 32 nm

10nm 10nm 4 nm 10nm 10nm Fig. 2. Location of Cdc11 and (His)6–Cdc12 determined by antibody decoration. (A) A sample of the preparation of purified Cdc3–Cdc10–Cdc11–(His)6–Cdc12 Full-length Cdc11-less Cdc10-less heterooctamers shown in Fig. 1 A–D was incubated on ice in high-salt buffer with rabbit polyclonal anti-Cdc11 antibodies. Three representative raw (unaveraged) Fig. 1. Characterization of yeast heterooligomeric septin complexes. (A)A images of the resulting complexes observed are shown. (B)AsinA, except ␮ sample (50 g) of the purified Cdc3–Cdc10–Cdc11–(His)6–Cdc12 heterooctam- incubation was with a mouse monoclonal anti-(His)6 antibody. Although binding ers used in most of these studies, prepared as described in Materials and of this antibody was substoichiometric under the high-salt conditions, image Methods, was resolved by electrophoresis on a 10% SDS/PAGE gel and stained processing readily identified particles with antibody bound. Left, three represen- with Coomassie Brilliant blue. MW, molecular weight. (B) Low magnification tative raw (unaveraged) images of the resulting complexes observed are shown, view of the preparation shown in (A) suspended in high-salt (300 mM) buffer, wherein the extra density (horizontal arrows) is most often positioned closest to adsorbed on a carbon grid, stained, and viewed in the EM as described in the second subunit in from one or both ends of the rod. Right, one of the class Materials and Methods.(C) Four representative class averages (Ϸ150 particles averages obtained after multiple rounds of multireference alignment and clas- each) derived from processing a total of 11,000 particles from micrographs of sification, in which the antibody is seen bound symmetrically around the center Cdc3–Cdc10–Cdc11–(His)6–Cdc12 complexes, as in (B). (D) Higher magnifica- of the rod, with at least one of its Fab arms in contact with the second subunit tion view of an average of 1,986 particles of the Cdc3–Cdc10–Cdc11–(His)6– from the end (most clearly seen for the contact indicated by the arrow). Cdc12 (full-length) complex. (E) Four representative class averages (100 par- ticles each) (Left) and a higher magnification view of the average of the whole data set of 2,093 particles (Right) observed in a preparation of Cdc3–Cdc10– beaded rod with a long dimension of 32–35 nm (Fig. 1B). Averaging (His)6–Cdc12 (Cdc11-less) complexes, examined as in B.(F) Four representative images of large numbers of classes of individual rods (Fig. 1C) class averages (10 particles each) (Left) and a higher magnification view of the afforded enhanced resolution that revealed that each rod is com- average of the whole data set of 824 particles (Right) observed in a prepara- Ϸ tion of Cdc3–Cdc11–(His) –Cdc12 (Cdc10-less) complexes, examined as in B. posed of eight globular densities of roughly equivalent size ( 4– 6 5-nm diameter) (Fig. 1D). This latter dimension corresponds well to other small GTP-binding proteins (20), suggesting that the organization of the rod-like native (13) and recombinant (18) yeast globular densities reflect the conserved G domain common to septin complexes seen by negative staining in the EM was unknown. Cdc3, Cdc10, Cdc11, and Cdc12 (Fig. S1). The calculated molecular We developed reliable recombinant methods for producing and mass for eight protein spheres of such dimension agrees well with purifying yeast septin complexes. By using single-particle EM that determined for native and recombinant septin complexes in analysis, we confirm that the complex of the four essential septins which the apparent stoichiometry of the four septins is 2:2:2:2. Thus, is a linear octameric rod. We then applied various tactics to pinpoint by its appearance in the EM, each rod is a heterooctamer, presum- the position of each septin within the rod. Our results show that the ably containing two molecules of each of the four different septins. rod has two-fold rotational symmetry about an axis running or- Human hexameric septin rods often displayed a sharp kink in EM thogonally through its center. Recently, a septin complex isolated images, suggesting that they can flex at their center (18). Although the yeast octameric rod is longer, we did not observe any kinked from the nematode Caenorhabditis elegans, which encodes only two rods, although some had a slight smooth curvature (Fig. 1 B and C). septins (UNC-59 and UNC-61), was shown to form symmetric rods containing two copies of each subunit (19). Likewise, a crystal Arrangement of Subunits Within the Octameric Rod. We applied structure and EM analysis of a complex comprising three human several strategies to delineate the position of each septin in the rod. BIOPHYSICS septins (SEPT2, SEPT6, and SEPT7) revealed a symmetrical First, we examined images of complexes prepared in the absence of heterohexameric rod (18). These findings suggest that all eukaryotic a given septin and ascertained the effect on rod structure. Second, septin complexes will be organized as linear symmetrical rods. The we decorated rods with an antibody directed against a septin or human heterohexamer forms filaments through the interface sur- epitope-tagged septin and located the bound IgG. Third, we rounding the guanine nucleotide-binding site in SEPT7 (18), incorporated into complexes septins tagged at their N- or C- whereas the yeast complex forms filaments through a salt-sensitive terminal end by fusion to a small monomeric marker protein, and interface involving the N and C termini of Cdc11. Moreover, the located the extra nearby density. yeast heterooctamer cooperatively assembles into parallel filaments When coexpressed in bacteria in the absence of Cdc11, stoichi- paired through the predicted C-terminal coiled-coil domains of ometric complexes of Cdc3, Cdc10, and (His)6–Cdc12 were ob- Cdc3 and Cdc12. Thus, septins in different organisms not only form tained, as before (5, 15). These complexes appeared as rods with six complexes with different numbers of subunits but also can revers- clear-cut globular densities (Fig. 1E). Thus, Cdc3, Cdc10, and ibly form filaments via different interfaces. Cdc12 formed a stable hexameric rod when no Cdc11 was present, suggesting that Cdc11 occupies a terminal position in the rod, with Results and Discussion a Cdc11–Cdc11 dimer at one end or one Cdc11 at each end. When Yeast Core Septin Complex Is a Linear Rod. In negatively stained EM Cdc3–Cdc10–Cdc11–(His)6–Cdc12 octamers were incubated with images, the purified recombinant Cdc3–Cdc10–Cdc11–(His)6– purified rabbit polyclonal anti-Cdc11 antibodies, the IgG bound to Cdc12 complex prepared at high ionic strength (Fig. 1A) was a both termini of an individual octameric rod (Fig. 2A). Moreover,

Bertin et al. PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8275 Downloaded by guest on October 1, 2021 given their bivalent Y-shaped nature, the anti-Cdc11 IgG often was A found holding two octameric rods together side-by-side (Fig. 2A). MBP-Cdc3 These results established that Cdc11 occupies the outermost posi- tion at both termini of the octameric rod. The only direct interaction with another core septin detected for Cdc11 is with Cdc12, and Cdc12 (but not Cdc11) interacts with Cdc3, and Cdc3, in turn, interacts with Cdc10 (even when Cdc11 is 10 nm absent, as long as Cdc12 is present) (5, 15). These data suggested that the arrangement in the rod must include the order Cdc11– Cdc12–Cdc3–Cdc10. Because we established that Cdc11 is at each B Cdc10-GFP end, Cdc12 was likely the second subunit in from each end. To test whether Cdc12 occupies this position, we first tried polyclonal anti-Cdc12 IgG directed against its G domain. However, the Cdc3–Cdc10–Cdc11–(His)6–Cdc12 complexes were only ineffi- ciently decorated, presumably because the main antigenic deter- 10 nm minants are buried in the rod [consistent with rod formation requiring contacts made by Cdc12 via its globular G domain (18)]. As an alternative, we incubated the Cdc3–Cdc10–Cdc11–(His)6– Fig. 3. Location of MBP–Cdc3 and Cdc10–GFP. (A) Four representative class Cdc12 complexes with a commercial anti-(His)6 mAb. To examine averages of purified MBP–Cdc3–Cdc10–Cdc11–(His)6–Cdc12 heterooctamers re- particles only at their periphery, a mask was used (see Materials and sulting from three rounds of multireference alignment and classification (2,620 Methods), and a total of 49 particles displayed density with an particles total). The extra density (horizontal arrows) is most often positioned antibody-like profile in this region. As shown in the class average closest to the third subunit in from one or both ends of the rod. (B) Four from those 49 particles (Fig. 2B), the antibody density was juxta- representative class averages of purified Cdc3–Cdc10–GFP–Cdc11–(His)6–Cdc12 heterooctamers resulting from three rounds of multireference alignment and posed to the second subunit from both ends. These findings classification (3,377 particles total). The extra density (horizontal arrows) is most confirmed that Cdc12 is the septin that occupies the penultimate often positioned closest to one or both of the two central subunits in the rod. In position at each end of the octameric rod. A and B, the majority of the averages yielded only one extra density per octamer, Based on biochemical data, the subunit adjacent to Cdc12 on the most likely because of flexibility in the joint between the marker protein and side opposite that contacting Cdc11 was likely Cdc3. To establish septin, making detection of the second marker improbable. the position of Cdc3, we first tried a polyclonal antibody directed against its 14 C-terminal residues. Approximately 14% of the treated rods displayed a bound IgG-like profile (data not shown); cells are viable at 20–25°C, they manifest gross morphological however, the extra density was found in diverse positions with abnormalities at 30° and are inviable at 37° (5, 13, 22). Thus, the respect to the rod (36% close to the middle, 36% near the ends, and inability to form the full heterooctameric complex (and presumably 28% at intermediate positions). If in ␣-helical conformation, the the absence of normal filaments) is not well tolerated. Cdc3 CTE (113 residues) could be more than 160 Å long and, if Based on the molecular organization (UNC-59–UNC-61– flexible, capable of sampling from the center to the ends of the rod, UNC61–UNC59) of the minimal tetrameric septin rod from C. explaining our observation. As an alternative means to locate Cdc3, elegans (and the presumption that UNC-59 and UNC-61 are likely we prepared complexes containing an MBP–Cdc3 fusion as the sole orthologs of Cdc12 and Cdc3, respectively), it was proposed by source of this septin. The maltose binding protein (MBP) sequence others (19) that the more elaborate yeast septin complex would be was fused in-frame to the N terminus of Cdc3, which was expected symmetrically organized around a central Cdc12–Cdc3–Cdc3– to be more structured than the CTE and hold the 42-kDa MBP Cdc12 core. Our data show that this prediction was incorrect. In appendage more rigidly. Indeed, MBP–Cdc3–Cdc10–Cdc11– addition, a model based on prior findings (5) suggesting that yeast (His)6–Cdc12 complexes yielded stable octameric rods with the septin complexes might form polar filaments also requires revision. same overall structure observed for complexes with untagged Cdc3, Our results show that the yeast septin complex has the order except that extra density was often juxtaposed to the third subunit Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11. Thus, from one or both ends (Fig. 3A). like the worm (19) and human (18) septin rods, the yeast rod lacks We used similar strategies to confirm that the subunit adjacent polarity because it has an axis of two-fold rotational symmetry to Cdc3 at the center of the rod is Cdc10. First, when Cdc3, Cdc11, situated between and perpendicular to the two central Cdc10 and (His)6–Cdc12 were coexpressed, stable ternary complexes were subunits. formed, as seen before (5, 15). These Cdc10-less complexes ap- peared as short rods composed of three distinct densities (Fig. 1F), Filament Formation by Salt-Dependent End-to-End Rod Polymeriza- consistent with the idea that a central Cdc10 dimer is needed to join tion. At Ն250 mM salt, Cdc3–Cdc10–Cdc11–(His)6–Cdc12 com- two outer trimeric arms (Cdc11–Cdc12–Cdc3) together to form the plexes are octameric rods (Fig. 1B). When ionic strength was octameric rod. The fact that two of the densities in the Cdc11– lowered (Յ50 mM salt), we observed very long paired filaments Cdc12–Cdc3 trimers seem distinctly less globular than the subunits (Fig. 4A), each with the same diameter as that of the rod (5, 15), in octameric rods suggests that, in the absence of Cdc10, the suggesting that electrostatic interactions drive filament assembly. remaining subunits undergo conformational transitions that affect When complexes were deposited onto EM grids immediately after their packing and/or relative orientation. Second, we prepared lowering the salt, we saw very occasionally, in addition to filaments, complexes containing a Cdc10–GFP fusion as the sole source of this possible intermediates that are twice the length of the rod, or even septin. This same chimera (21) fully complements a cdc10⌬ muta- less frequently, three or four times the rod length (Fig. S2). tion, indicating that the 27-kDa GFP appendage does not compro- Filament formation seems a highly cooperative process, for two mise Cdc10 folding or function. The Cdc3–Cdc10–GFP–Cdc11– reasons. First, filaments are very long and invariably align pairwise (His)6–Cdc12 complexes yielded stable octameric rods in which (like railroad tracks), separated by a 15- to 25-nm gap. Paired extra density was juxtaposed to the center of the rod, projecting filaments are observed whether visualized by negative staining (Fig. from the fourth subunit from one or both ends (Fig. 3B), thus 4A) or viewed in vitreous ice by cryo-EM (Fig. 4B). Thus, pairing demonstrating that a pair of Cdc10 subunits is at the center of the is not an artifact of staining or the support used. Second, paired rod. It has been reported that haploid cells lacking Cdc10 can be filaments appear to be in register because the ends of each filament isolated and appear to lack neck filaments (13, 22). Although these are flush with each other, not staggered (Fig. 4A). These observa-

8276 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803330105 Bertin et al. Downloaded by guest on October 1, 2021 Cdc10 doublet may induce conformational effects propagated to A B C the end of the rod or, more likely, the presence of Cdc10 may affect the orientation or conformation of the Cdc3 CTE, thereby pro- moting optimal filament pairing, which is coupled to filament elongation. To define the interfaces that mediate rod and filament formation, we made mutations in contacts predicted from the crystal structures 100 nm of human SEPT2 and the SEPT2–SEPT6–SEPT7 heterohexamer (18). In a linear array, a septin must contact each of its neighboring subunits asymmetrically. At its so-called G interface, a septin D E interacts via its G domain with the G domain in the adjacent septin, an association involving contacts around the guanine nucleotide- binding site in each monomer (18). On its other side, a septin binds its neighbor via a so-called N–C interface (18), which involves the 33 nm N-terminal ␣-helix (␣0) and the more distal ␣-helix (␣6) (Fig. S1); the latter was at the C-terminal end of the visible electron density 4 nm (18). These ␣0 and ␣6 helices distinguish septins from Ras-like 20 nm 50 nm GTPases. At an N–C interface, the paired septin CTEs (which were not visible in the human heterohexamer electron density map) Fig. 4. End-on-end polymerization of the rods forms paired filaments. (A)A project from the same face of the rod and are juxtaposed (18), permitting interaction of complementary sequences. sample of the purified Cdc3–Cdc10–Cdc11–(His)6–Cdc12 heterooctamers shown in Fig. 1 A–D were diluted from high salt (300 mM) to low salt (50 mM), To confirm that Cdc11–Cdc11 association mediates filament incubated for 2 h, and then examined in the EM after negative staining. (B)As formation and delineate what interface mediates this interaction, in A, except that the sample was viewed via cryo-EM after vitrification in liquid we generated two Cdc11 mutants, coexpressed them with Cdc3, ethane, as described in Materials and Methods.(C) Purified Cdc3–Cdc10– Cdc10, and (His)6–Cdc12, and examined the efficiency of their ⌬␣ Cdc11( 0)–(His)6–Cdc12 heterooctamers were examined after dilution and incorporation into the resulting complexes and the ability of those incubation in low salt, as in A.(Inset) Three representative class averages and complexes to form filaments. We found, first, that Cdc11(⌬357- the average of the most common class (Bottom Right) of the same sample of 415), in which the CTE (but not ␣6) was removed, was efficiently purified Cdc3–Cdc10–Cdc11(⌬␣0)–(His)6–Cdc12 heterooctamers examined in high salt. (D) Paired filaments displaying periodic densities between the incorporated into octameric rods, which were indistinguishable in filaments (Left) and at higher magnification (Right). (E) Example of a bundle the EM from the rods containing full-length Cdc11, and that these of paired filaments formed after prolonged incubation under low-salt condi- rods polymerized efficiently into filaments in vitro at low salt (data tions at relatively high protein concentration (Ͼ1 ␮gmlϪ1) and above neutral not shown). These findings are consistent with our previous obser- pH (Left) and a representative power spectrum (Right) derived from the vations that CTE-less Cdc11 is capable of homotypic interaction in Fourier transform of the image of such a bundle of paired filaments. vitro and that yeast cells expressing Cdc11(⌬357-415) as the sole source of this septin, although displaying far from normal mor- phology, are nonetheless viable, whereas those expressing CTE-less tions suggest further that filament elongation and filament pairing Cdc3 or Cdc12 are inviable (5). Thus, EM observation confirmed are coupled. Moreover, the filaments have no intrinsic polarity that the Cdc11 CTE does not have an essential role in either because the rod of which they are composed is symmetric. octamer or filament assembly, and rather, may help recruit other Under some conditions, still not well defined (but influenced by proteins to rods or filaments. By contrast, deletion of the predicted pH and protein concentration), the filament doublets coalesced Cdc11 ␣0 element [Cdc11(⌬2-18) or Cdc11(⌬␣0)] also produced into larger bundles, which are likely nonphysiological aggregates octameric rods (Fig. 5A) indistinguishable in the EM from the rods (Fig. 4E Left). These bundles appeared to be organized in register containing full-length Cdc11 (Fig. 4C Inset), but these were unable (Fig. 4E Left) and diffracted reasonably well. The power spectrum to polymerize into filaments (Fig. 4C). These data corroborate that of the bundles (Fig. 4E Right) showed both a 4-nm axial repeat Cdc11–Cdc11 association is required for rod polymerization and corresponding to each globular subunit and a Ϸ33-nm repeat indicate that this interaction is an N–C interface. corresponding to the length of the octameric rod. No clear re- If so, the Cdc11–Cdc12 interaction isaGinterface. To test this peat was found in the direction perpendicular to the bundle, prediction, Cdc3, Cdc10, and Cdc11 were coexpressed with (His)6– suggesting lack of crystalline order in that direction. In low salt, Cdc12(W267A), which carries a point mutation in a conserved anti-Cdc11 antibodies decorated these bundles with a periodicity residue equivalent to a side chain that mediates an important

compatible with the length of an octamer and with a banding contact for G dimer association of human SEPT2 (18). This BIOPHYSICS pattern indicating that homotypic lateral interactions align like mutation in Cdc12 greatly reduced Cdc11 incorporation onto the subunits across the bundle (Fig. S3). Furthermore, in agreement ends of the rod and greatly diminished the frequency of formation with our findings that Cdc11 is the terminal subunit at each end of of full-length octamers (Fig. 5B), consistent with the conclusion that a rod and that filaments form by end-to-end polymerization of the the Cdc11–Cdc12 interaction surface isaGinterface. In agreement rods, when decorated with anti-Cdc11 IgG, filaments in bundles with the general need for guanine nucleotide binding for octamer always had antibody bound at their free ends (Fig. S3). formation and stability, coexpression of Cdc3 and Cdc11 with two mutants, Cdc10(S46N) and (His)6–Cdc12(T48N), previously dem- Cdc11–Cdc11 Interaction Is Critical for Rod Polymerization. Several onstrated to be GTP binding-defective (7), markedly reduced lines of evidence demonstrated that direct Cdc11–Cdc11 interac- formation of octameric rods (Fig. 5C). tion is the mechanism of filament assembly. First, Cdc11-less The next interface in the rod, that between Cdc12 and Cdc3, hexameric rods (Fig. 1E) never formed filaments detectable in the should be an N–C interface given the prior finding that the Cdc3 EM when the salt concentration was lowered (data not shown), in and Cdc12 CTEs are critical for their association and for rod and agreement with our prior finding that Cdc3–Cdc10–(His)6–Cdc12 filament formation both in vivo and in vitro (5). In some EM images, complexes did not polymerize (5). Theoretically, Cdc10-less trim- cross-bridges between the paired filaments were seen (Fig. 4D Left). eric rods might be able to form ‘‘inside-out’’ Cdc3–Cdc12–Cdc11– At higher magnification (Fig. 4D Right), these structures had a Cdc11–Cdc12–Cdc3 hexamers. However, we have not detected periodicity consistent with their arising from interaction of the such forms in our EM studies, suggesting that the central Cdc10– paired parallel CTEs of Cdc3 and Cdc12 from one filament

Bertin et al. PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8277 Downloaded by guest on October 1, 2021 100 100 100 ABCCdc3-Cdc10-Cdc11-(His) -Cdc12 Cdc3-Cdc10-Cdc11-(His) -Cdc12 6 Cdc3-Cdc10-Cdc11-(His)6-Cdc12 6 Cdc3-Cdc10-Cdc11(∆α0)- Cdc3-Cdc10-Cdc11- Cdc3-Cdc10(S46N)-Cdc11- 75 (His) -Cdc12 75 75 6 (His)6-Cdc12(W267A) (His)6-Cdc12(T48N)

50 50 50

25 25 25 Relative frequency (%)

0 0 0 87654 87654 87654 Number of subunits in hetero-oligomer Number of subunits in hetero-oligomer Number of subunits in hetero-oligomer

Fig. 5. Effect of site-directed mutations on the efficiency of stable heterooctameric rod assembly. Purified preparations of the wild-type Cdc3–Cdc10–Cdc11– (His)6–Cdc12 complex (black bars) and the three mutant complexes indicated in each panel (white bars), Cdc3–Cdc10–Cdc11(⌬␣0)–(His)6–Cdc12 (A), Cdc3–Cdc10– Cdc11–(His)6–Cdc12(W267A) (B), and Cdc3–Cdc10(S46N)–Cdc11–(His)6–Cdc12(T48N) (C), were examined under high-salt conditions in the EM after negative staining, and the number of subunits in the rods formed were scored; 3,043 particles of the Cdc3–Cdc10–Cdc11(⌬␣0)–(His)6–Cdc12 complexes, 2,130 total particles for the Cdc3–Cdc10–Cdc11–(His)6–Cdc12(W267A) complexes, and 1,267 particles for the Cdc3–Cdc10(S46N)–Cdc11–(His)6–Cdc12(T48N) complexes were selected, aligned, and classified, first without any reference before a second round of alignment and classification by using references resulting from the first round of alignment, and classification was performed.

associating with the paired parallel CTEs of Cdc3 and Cdc12 from coupling between rod polymerization and filament pairing is that another filament, presumably forming an antiparallel four-helix polymerization of octamers places the CTEs of Cdc3 and Cdc12 bundle (Fig. 6). The distance between filaments in a pair (15–25 in a position to promote interaction between filaments and vice nm) is compatible with the calculated lengths of the CTEs of these versa. A more speculative possibility is that, at high salt, contact two proteins, assuming that they are ␣-helical and associate pri- of the Cdc12 CTE with the surface of Cdc11 may be favored over marily via overlap of their distal coiled coil-forming elements. its pairing with the Cdc3 CTE. If so, the Cdc12 CTE may mask These results provide further evidence that filaments pair in register the interface of Cdc11 required for its homotypic interaction. If via homotypic contacts. this intraoctamer contact is alleviated at low salt, the surface of Given this model, a possible explanation for the observed Cdc11 necessary for rod polymerization would be freed simul- taneously with release and pairing of the Cdc12 CTE with the Cdc3 CTE, which then can engage in lateral contacts between elongating filaments. This simple masking idea predicts that at low salt, the Cdc3 and Cdc12 CTEs in Cdc11-less hexamers (which do not polymerize into filaments) should be paired and available to mediate side-by-side association of the hexamers, which we do not observe. Maybe binding of the Cdc11 G domain to Cdc12 influences pairing of its CTE with that of Cdc3 or the orientation or conformation of the resulting coiled coil. Whether the Cdc3 and Cdc12 CTEs are essential for filament pairing or elongation cannot be tested easily because no stable octamers form in their absence (5).

Shs1 May Serve as an Alternative Terminal Subunit. Shs1 (23, 24) is often considered an accessory septin because null alleles (shs1⌬) have a mild phenotype (22, 24) and because it is recovered in substoichiometric amounts in native septin complexes purified under certain conditions (14). However, Shs1 is the mitotic septin that seems the most heavily modified by both phosphorylation and SUMOylation (reviewed in ref. 6), and its state of modification is correlated with the dynamic behavior of the septin filaments at the mother-bud neck (25). Cdc11 and Shs1 occupy a distinct phyloge- netic subfamily (1) and are, among the five mitotic septins, the most closely related in primary structure both within their G domains and Fig. 6. Schematic diagram of the yeast septin rod, filament, and paired filament over the first half of their CTEs. Moreover, various assays to assess assemblies. The yeast heterooctameric septin complex is a linear rod with the Shs1 interaction with other septins demonstrated binding to Cdc12 subunits arrayed in the order and with the interfaces indicated. Each septin forms associations with its neighbors through either a G interface or an N–C interface. (15, 24). Thus, Shs1 may be able to replace Cdc11 at the end of the The rod is nonpolar because it has a two-fold axis of rotational symmetry running rod. If so, the resulting Shs1-containing octamers may generate left-to-right between and orthogonal to the central pair of Cdc10 septins. The septin filaments with special features or the ability to recruit unique CTEs of the two Cdc3 and Cdc12 pairs project from the same face of the rod and proteins, thus fine-tuning yeast cell morphogenesis (22), although presumably associate to form parallel coiled coils that are essential for rod Shs1 is not essential for viability. Hence, it will be interesting to stability because no rod assembly or filament formation occurs in their absence. examine whether these predictions about Shs1 are correct. Filaments form via end-on-end assembly of the rods mediated by an ionic In conclusion, our results suggest that the subunit that mediates strength-dependent Cdc11–Cdc11 interaction through an N–C interface. The CTE end-on-end interactions between heterooligomers may exert con- of Cdc11 is not required either for rod assembly or for filament formation. Filament formation and pairing in register are coupled, with the pairing presum- trol over septin polymerization into filaments. In contrast to the ably mediated by lateral association between the parallel Cdc3–Cdc12 coiled coil human SEPT7–SEPT6–SEPT2–SEPT2–SEPT6–SEPT7 rod, on one filament with a corresponding parallel Cdc3–Cdc12 coiled coil on the which can form filaments through the G interface of SEPT7, the neighboring filament, thereby forming an antiparallel four-helix bundle. yeast Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 rod

8278 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803330105 Bertin et al. Downloaded by guest on October 1, 2021 achieves filament formation through the N–C interface of Cdc11 Several rounds of multireference alignment and classification were then per- (and perhaps, as argued above, Shs1 may be able to mediate similar formed, and new references were selected from the class averages until no end-on-end contacts). Thus, the yeast heterooctamer displays an further improvement was obtained. The following numbers of particles were analyzed for each of the indicated complexes [tagged subunit(s) in italics]. Cdc3– assembly interface distinct from that of the mammalian septin Cdc10–Cdc11–(His)6–Cdc12 complexes: 4,929 particles were selected and pro- heterohexamers (18, 26). This difference suggests that although cessed into 202 classes. Cdc3–Cdc10(S46N)–Cdc11–(His)6–Cdc12(T48N) complexes: assembled filament structures share alternating G- and N–C inter- 1,267 particles were selected and classified into 10 classes. Cdc3–Cdc11–(His)6– faces (Fig. 6), discrete mechanisms may regulate assembly of Cdc12 (Cdc10-less) complexes: 824 particles were selected and classified into 10 different septin filaments in the same or different organisms. Of classes. Cdc3–Cdc10–(His)6–Cdc12 (Cdc11-less) complexes: 2,093 particles were course, the SEPT7–SEPT6–SEPT2–SEPT2–SEPT6–SEPT7 com- selected and processed into 20 classes. Cdc3–Cdc10–GFP–Cdc11–(His)6–Cdc12 (Cdc10-tagged) complexes: 3,377 particles were windowed out and classified into plex used for structural studies may not be the native form in any 108 classes. MBP–Cdc3–Cdc10–Cdc11–(His)6–Cdc12 (Cdc3-tagged) complexes: mammalian cell type. If mammalian heterohexamers are capped in 2,630 particles were windowed out and classified into 117 classes. Cdc3–Cdc10– vivo at each end by an additional Cdc11-like subunit, they would Cdc11–(His)6–Cdc12 complexes stained with anti-(His)6 mAb: 2,796 particles were closely resemble the yeast heterooctamer in overall organization selected and classified into 32 classes, corresponding to the main shapes and and in polymerizing via an N–C mode of interaction. orientations of the septin complex in the data set. An additional subclassification of each of these classes was obtained by using a mask selecting only for the data Materials and Methods at the periphery of the particles. The resulting data were then segregated into 150 final classes, five of which (total of 49 particles; 2% of the total data set) Septin Complex Expression and Purification. Complexes containing Cdc3, Cdc10, showed a clear density with an antibody-like profile. Cdc3–Cdc10–Cdc11–(His)6– Cdc11, and (His)6-tagged Cdc12 were expressed in Escherichia coli as described Cdc12 complexes stained with polyclonal anti-Cdc12 antibodies: 3,969 particles before (5) and then purified by immobilized metal affinity, size exclusion, and ion were selected and classified into 150 classes. Cdc3–Cdc10–Cdc11–(His)6–Cdc12 exchange chromatography (see SI Methods). Cdc3–Cdc11–(His)6–Cdc12 (Cdc10- complexes stained with polyclonal anti-Cdc3 C-terminal peptide antibodies: less), Cdc3–Cdc10-(His)6–Cdc12 (Cdc11-less), MBP–Cdc3–Cdc10–Cdc11–Cdc12, 11,575 particles were selected and classified into 773 classes. For Cdc3–Cdc10– and Cdc10–GFP–Cdc3–Cdc11–(His)6–Cdc12 complexes were prepared the same Cdc11–(His)6–Cdc12 complexes stained with polyclonal anti-Cdc11 antibodies, way by using the appropriate vectors to express the indicated combinations of the IgG was sufficiently well immobilized that it was readily visualized in raw proteins. images. Filaments formed under low-salt conditions were also vitrified in liquid ethane EM and Image Processing. Purified septin complexes were dialyzed against by using a Vitrobot (FEI) in a 100% humidity environment on a Quantifoil grid (Structure Probes, Inc.). The specimen was transferred to a cryo-specimen holder buffer [2 mM MgCl2, 50 mM Tris⅐HCl (pH 8)] containing either 300 mM or 10 mM KCl (or NaCl) by using an 10- to 50-␮l capacity Spindialyzer (Harvard Apparatus). (Gatan) and observed on a Tecnai F20 (FEI) by using 200 kV. Micrographs were recorded under low-dose conditions at a magnification of ϫ30,000 and under- For the high-salt samples, the septin complexes were diluted to 0.01 mg/ml, focus of Ϸ3 ␮m. adsorbed onto carbon-coated grids, and stained with 2% uranyl formate. See SI Methods for details on antibodies used for EM studies. ACKNOWLEDGMENTS. We thank Mark Longtine, Douglas Kellogg, and Mi- Samples were examined by using a Tecnai T12 microscope (FEI) operated at 120 chael Knop for the communication of unpublished information, useful con- kV, and images ertr collected by using film (cat. no. S0163; Kodak) at a magnifi- versations, and/or the gift of reagents. This work was supported by Postdoc- cation of ϫ30,000 and Ϸ1-␮m underfocus. Micrographs were digitized on a toral Research Fellowships 61-1357 (to A.B.) and 61-1295 (to M.A.M.) from the Nikon Coolscan 8000 scanner with a pixel size of 4.23 Å at the sample level. Jane Coffin Childs Memorial Fund for Medical Research, National Institutes of Particles were classified and aligned to generate class averages by using SPIDER Health Predoctoral Traineeship GM07232 (to G.G.), National Institutes of (27). Particles were windowed out into 135 ϫ135 pixels images by using the Boxer Health–National Research Service Award Kirschstein Postdoctoral Fellowship GM69165 (to H.-l.N.), National Institutes of Health Research Grant GM48958 interface of EMAN (28) and appended into a single SPIDER file, then normalized (to T.A.), National Institutes of Health Research Grant GM21841 (to J.T.), and against the background. One round of reference-free alignment and classifica- the Agouron Foundation, Department of Energy Office of Biological and tion was performed before references were selected from the first class averages. Environmental Research, and the Howard Hughes Medical Institute (to E.N.).

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Bertin et al. PNAS ͉ June 17, 2008 ͉ vol. 105 ͉ no. 24 ͉ 8279 Downloaded by guest on October 1, 2021