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Saccharomyces Cerevisiae Septins: Supramolecular Organization of Heterooligomers and the Mechanism of Filament Assembly

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Saccharomyces Cerevisiae Septins: Supramolecular Organization of Heterooligomers and the Mechanism of Filament Assembly Saccharomyces cerevisiae septins: 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 septin 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 proteins (GFP or through other members of the complex or other septin-associated maltose binding protein). 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 43 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.
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