Nucleotide binding and filament assembly of recombinant yeast septin complexes.
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 gamma-phosphate.
Farkasovsky
Marian
M
Department of Structural Biology, Max-Planck-Institute for Molecular Physiology, D-44227 Dortmund, Germany.
Herter
Peter
P
Voss
Beate
B
Wittinghofer
Alfred
A
D001483:Base Sequence; D003124:Colorimetry; D017931:DNA Primers; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate