Nucleotide binding and filament assembly of recombinant yeast septin complexes
-
Marian Farkasovsky
Abstract
Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the γ-phosphate.
References
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1995). Current Protocols in Molecular Biology (New York, USA: John Wiley and Sons).Suche in Google Scholar
Barral, Y., Mermall, V., Mooseker, M.S., and Snyder, M. (2000). Compartmentalization of the cell cortex by septins is required for maintenance of cell polarity in yeast. Mol. Cell5, 841–851.10.1016/S1097-2765(00)80324-XSuche in Google Scholar
Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996). Ras/Rap effector specificity determined by charge reversal. Nature Struct. Biol.3, 244–251.10.1038/nsb0396-244Suche in Google Scholar PubMed
Bouquin, N., Barral, Y., Courbeyrette, R., Blondel, M., Snyder, M., and Mann, C. (2000). Regulation of cytokinesis by the Elm1 protein kinase in Saccharomyces cerevisiae. J. Cell Sci.113, 1435–1445.10.1242/jcs.113.8.1435Suche in Google Scholar PubMed
Byers, B. and Goetsch, L. (1976). A highly ordered ring of membrane-associated filaments in budding yeast. J. Cell Biol.69, 717–721.10.1083/jcb.69.3.717Suche in Google Scholar PubMed PubMed Central
Carroll, C.W., Altman, R., Schieltz, D., Yates, J.R., and Kellogg, D. (1998). The septins are required for the mitosis-specific activation of the Gin4 kinase. J. Cell Biol.143, 709–717.10.1083/jcb.143.3.709Suche in Google Scholar PubMed PubMed Central
Casamayor, A. and Snyder M. (2003). Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function. Mol. Cell. Biol.23, 2762–2777.10.1128/MCB.23.8.2762-2777.2003Suche in Google Scholar PubMed PubMed Central
Caviston, J.P., Longtine, M.S., Pringle, J.R., and Bi, E. (2003). The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast. Mol. Biol. Cell14, 4051–4066.10.1091/mbc.e03-04-0247Suche in Google Scholar PubMed PubMed Central
Cvrckova, F., De Virgilio, C., Manser, E., Pringle, J.R., and Nasmyth, K. (1995). Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev.9, 1817–1830.10.1101/gad.9.15.1817Suche in Google Scholar PubMed
Desai, A. and Mitchison, T.J. (1997). Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol.13, 83-117.10.1146/annurev.cellbio.13.1.83Suche in Google Scholar PubMed
De Virgilio, C., DeMarini, D.J., and Pringle, J.R. (1996). SPR28, a sixth member of the septin gene family in Saccharomyces cerevisiae that is expressed specifically in sporulating cells. Microbiology142, 2897–2905.10.1099/13500872-142-10-2897Suche in Google Scholar PubMed
Dobbelaere, J. and Barral, Y. (2004). Spatial coordination of cytokinetic events by compartmentalization of the cell cortex. Science305, 393–396.10.1126/science.1099892Suche in Google Scholar
Dobbelaere, J., Gentry, M.S., Hallberg, R.L., and Barral, Y. (2003). Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Dev Cell4, 345–357.10.1016/S1534-5807(03)00061-3Suche in Google Scholar
Fares, H., Goetsch, L., and Pringle, J.R. (1996). Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae. J. Cell Biol.132, 399–411.10.1083/jcb.132.3.399Suche in Google Scholar
Field, C.M., Al-Awar, O., Rosenblatt, J., Wong, M.L., Alberts, B., and Mitchison, T.J. (1996). A purified Drosophila septin complex forms filaments and exhibits GTPase activity. J. Cell Biol.133, 605–616.10.1083/jcb.133.3.605Suche in Google Scholar
Frazier, J.A., Wong, M.L., Longtine, M.S., Pringle, J.R., Mann, M., Mitchison, T.J., and Field, C. (1998). Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function. J. Cell Biol.143, 737–749.10.1083/jcb.143.3.737Suche in Google Scholar
Gladfelter, A.S., Pringle J.R., and Lew, D.J. (2001). The septin cortex at the yeast mother-bud neck. Curr. Opin. Microbiol.4, 681–689.10.1016/S1369-5274(01)00269-7Suche in Google Scholar
Gladfelter, A.S., Bose, I., Zyla, T.R., Bardes, E.S., and Lew, D.J. (2002). Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p. J. Cell Biol.156, 315–326.10.1083/jcb.200109062Suche in Google Scholar
Haarer, B.K. and Pringle, J.R. (1987). Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck. Mol. Cell. Biol.7, 3678–3687.Suche in Google Scholar
Hartwell, L.H. (1971). Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp. Cell Res.69, 265–276.10.1016/0022-2836(71)90420-7Suche in Google Scholar
Joberty, G., Perlungher, R.R., Sheffield, P.J., Kinoshita, M., Noda, M., Haystead, T., and Macara, I.G. (2001). Borg proteins control septin organization and are negatively regulated by Cdc42. Nat. Cell Biol.3, 861–866.10.1038/ncb1001-861Suche in Google Scholar PubMed
Kholod, N. and Mustelin, T. (2001). Novel vectors for co-expression of two proteins in E. coli. Biotechniques31, 322–328.10.2144/01312st03Suche in Google Scholar PubMed
Kim, H.B., Haarer, B.K., and Pringle, J.R. (1991). Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site. J. Cell Biol.112, 535–544.10.1083/jcb.112.4.535Suche in Google Scholar
Kinoshita, M., Kumar, S., Mizoguchi, A., Ide, C., Kinoshita, A., Haraguchi, T., Hiraoka, Y., and Noda, M. (1997). Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures. Genes Dev.11, 1535–1547.10.1101/gad.11.12.1535Suche in Google Scholar
Kinoshita, M., Field, C.M., Coughlin, M.L., Straight, A.F., and Mitchison, T.J. (2002). Self- and actin-templated assembly of mammalian septins. Dev. Cell3, 791–802.10.1016/S1534-5807(02)00366-0Suche in Google Scholar
Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Nasmyth, K., and Schiebel, E. (1999). Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast15, 963–972.10.1002/(SICI)1097-0061(199907)15:10B<963::AID-YEA399>3.0.CO;2-WSuche in Google Scholar
Lee, P.R., Song, S., Ro, H.S., Park, C.J., Lippincott, J., Li, R., Pringle, J.R., De Virgilio, C., Longtine, M.S., and Lee, K.S. (2002). Bni5p, a septin-interacting protein, is required for normal septin function and cytokinesis in Saccharomyces cerevisiae. Mol. Cell. Biol.22, 6906–6920.10.1128/MCB.22.19.6906-6920.2002Suche in Google Scholar
Longtine, M.S., DeMarini, D.J., Valencik, M.L., Al-Awar, O.S., Fares, H., De Virgilio, C., and Pringle, J.R. (1996). The septins: roles in cytokinesis and other processes. Curr. Opin. Cell Biol.8, 106–119.10.1016/S0955-0674(96)80054-8Suche in Google Scholar
Longtine, M.S., Fares, H., and Pringle, J.R. (1998). Role of the yeast Gin4p protein kinase in septin assembly and the relationship between septin assembly and septin function. J. Cell Biol.143, 719–736.10.1083/jcb.143.3.719Suche in Google Scholar
Mendoza, M., Hyman, A.A., and Glotzer, M. (2002). GTP binding induces filament assembly of a recombinant septin. Curr. Biol.12, 1858–1863.10.1016/S0960-9822(02)01258-7Suche in Google Scholar
Mino, A., Tanaka, K., Kamei, T., Umikawa, M., Fujiwara, T., and Takai, Y. (1998). Shs1p: a novel member of septin that interacts with Spa2p, involved in polarized growth in Saccharomyces cerevisiae.Biochem. Biophys. Res. Commun.251, 732–736.10.1006/bbrc.1998.9541Suche in Google Scholar
Mortensen, E.M., McDonald, H., Yates, J.R. III, and Kellogg, D.R. (2002). Cell cycle-dependent assembly of a Gin4-septin complex. Mol. Biol. Cell13, 2091–2105.10.1091/mbc.01-10-0500Suche in Google Scholar
Nguyen, T.Q., Sawa, H., Okano, H., and White, J.G. (2000). The C. elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokinesis and morphogenesis but have no essential function in embryogenesis. J. Cell Sci.113, 3825–3837.Suche in Google Scholar
Praefcke, G.J.K., Geyer, M., Schwemmle, M., Kalbitzer, H.R., and Herrmann, C. (1999). Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. J. Mol. Biol.292, 321–332.10.1006/jmbi.1999.3062Suche in Google Scholar
Rudolph, M.G., Linnemann, T., Grünewald, P., Wittinghofer, A., Vetter, I.R., and Herrmann, C. (2001). Thermodynamics of Ras/effector and Cdc42/effector interactions probed byisothermal titration calorimetry. J. Biol. Chem.276, 23914–23921.10.1074/jbc.M011600200Suche in Google Scholar
Sheffield, P.J., Oliver, C.J., Kremer, B.E., Sheng, S., Shao, Z., and Macara, I.G. (2003). Borg/septin interactions and the assembly of mammalian septin heterodimers, trimers, and filaments. J. Biol. Chem.278, 3483–3488.10.1074/jbc.M209701200Suche in Google Scholar
Spiegelman, B.M., Penningroth, S.M., and Kirschner, M.W. (1977). Turnover of tubulin and the N site GTP in Chinese hamster ovary cells. Cell12, 587–600.10.1016/0092-8674(77)90259-8Suche in Google Scholar
Stieglitz, B. (2002). Diploma thesis, Ruhr-University, Bochum, Germany.Suche in Google Scholar
Takizawa, P.A., DeRisi, J.L., Wilhelm, J.E., and Vale, R.D. (2000). Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science290, 341–344.10.1126/science.290.5490.341Suche in Google Scholar PubMed
Tucker, J., Sczakiel, G., Feuerstein, J., John, J., Goody, R.S., and Wittinghofer, A. (1986). Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J.5, 1351–1358.10.1002/j.1460-2075.1986.tb04366.xSuche in Google Scholar PubMed PubMed Central
Versele, M. and Thorner J. (2004). Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. J. Cell Biol.164, 701–715.10.1083/jcb.200312070Suche in Google Scholar PubMed PubMed Central
Versele, M., Gullbrand, B., Shulewitz, M.J., Cid, V.J., Bahmanyar, S., Chen, R.E., Barth, P., Alber, T., and Thorner, J. (2004). Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae. Mol. Biol. Cell15, 4568–4583.10.1091/mbc.e04-04-0330Suche in Google Scholar PubMed PubMed Central
Vrabioiu, A.M., Gerber, S.A., Gygi, S.P., Field, C.M., and Mitchison, T.J. (2004). The majority of the Saccharomyces cerevisiae septin complexes do not exchange guanine nucleotides. J. Biol. Chem.279, 3111–3118.10.1074/jbc.M310941200Suche in Google Scholar PubMed
Wach, A., Brachat, A., Poehlmann, R., and Philippsen, P. (1994). New heterologous modules for classical or PCR-basedgene disruptions in Saccharomyces cerevisiae. Yeast10, 1793–1808.10.1002/yea.320101310Suche in Google Scholar PubMed
©2005 by Walter de Gruyter Berlin New York
Artikel in diesem Heft
- Digestive versus regulatory proteases: on calpain action in vivo
- Psc3 cohesin of Schizosaccharomyces pombe: cell cycle analysis and identification of three distinct isoforms
- Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA
- Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p
- Nucleotide binding and filament assembly of recombinant yeast septin complexes
- Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases
- Inhibition of aldosterone biosynthesis by staurosporine
- Rab6 interacts with the mint3 adaptor protein
- Antidiabetic potential of two novel fatty acid derivatised, N-terminally modified analogues of glucose-dependent insulinotropic polypeptide (GIP): N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)
- Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation
- A possible alternative mechanism of kinin generation in vivo by cathepsin L
- hDLG/SAP97, a member of the MAGUK protein family, is a novel caspase target during cell-cell detachment in apoptosis
Artikel in diesem Heft
- Digestive versus regulatory proteases: on calpain action in vivo
- Psc3 cohesin of Schizosaccharomyces pombe: cell cycle analysis and identification of three distinct isoforms
- Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA
- Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p
- Nucleotide binding and filament assembly of recombinant yeast septin complexes
- Identification and partial characterization of two eukaryotic UDP-galactopyranose mutases
- Inhibition of aldosterone biosynthesis by staurosporine
- Rab6 interacts with the mint3 adaptor protein
- Antidiabetic potential of two novel fatty acid derivatised, N-terminally modified analogues of glucose-dependent insulinotropic polypeptide (GIP): N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)
- Kinetics of autocatalytic zymogen activation measured by a coupled reaction: pepsinogen autoactivation
- A possible alternative mechanism of kinin generation in vivo by cathepsin L
- hDLG/SAP97, a member of the MAGUK protein family, is a novel caspase target during cell-cell detachment in apoptosis
