BUDDING YEAST SEPTIN FILAMENTS: SAXS STUDIES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Septin proteins assemble into a cytoskeletal polymer. Septin cytoskeleton is essential for cytokinesis, the last step in cell division. In budding yeast, filamentous rings of septins have been visualized at the bud neck and constrict it in order to separate the mother and daughter cells. We focus on the essential four species of septins that can be co-expressed in E.coli: Cdc3, Cdc10, Cdc12 and Cdc11. We have recently shown in our negative-stain cryoEM study that septin self-assembly is salt-dependent and forms a 400 kDa octameric rod with two copies of each septin per octamer at high salt concentrations (above 200 mM monovalent salt) while septin octamers polymerize into a paired filament in low salt conditions. We have also demonstrated that the octameric rod is anti-parallel and dispalys the following sequence of subunit interactions: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11. The polymerized filament is therefore non polar, consistent with the recent 4 A resolution crystal structure of a subset of three human septins. The coiled-coil domains of septins are invisible in the crystal structure and we believe these domains are involved in pairing the filament at low ionic strengths. 3D EM reconstruction is under way, and currently shows a rod shape at 26 A resolution, which however is missing ~20% of the total protein mass. The missing mass can be attributed to the flexible coiled-coil domains since the crystallographically determined globular domains can be fit in the EM model. We have begun solution x-ray scattering studies at SSRL and obtained a high quality. At 0.5 M monovalent salt, we observe no concentration- or time- dependent change in the scattering data in the protein concentration range 0.75-3 mg/ml. The radius of gyration and the maximum dimension of the septing assembly have been determined as 100 and 350 A, respectively. Our 3D reconstruction using the SAXS data resulted in a rod shape with a gentle saw-tooth like modulation along the long axis.