RESUMO
Budding yeast septins are essential for cell division and polarity. Septins assemble as palindromic linear octameric complexes. The function and ultra-structural organization of septins are finely governed by their molecular polymorphism. In particular, in budding yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for the first time, the behavior of the Shs1 protomer bound to membranes at nanometer resolution, in complex with the other septins. Using electron microscopy, we have shown that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or two-dimensional gauzes. Using a set of specific mutants we have demonstrated a synergistic role of both nucleotides and lipids for the organization and oligomerization of budding yeast septins. Besides, cryo-electron tomography assays show that vesicles are deformed by the interaction between Shs1 oligomers and lipids. The Shs1-Shs1 interface is stabilized by the presence of phosphoinositides, allowing the visualization of micrometric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Lipídeos/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Lipossomos/química , Lipossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mutação , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tomografia/métodosRESUMO
Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.