Budding yeast Bud3 and Bud4 cooperatively organize septin architecture and support cytokinetic spatial memory.

During cytokinesis, budding yeast assemble a double septin ring composed of circumferential filaments that compartmentalize the membrane at the bud neck. This structure's integrity depends on two proteins, Bud3 and Bud4. Using in vitro reconstitution assays, we demonstrate that Bud3 and Bud4 organize septin filaments in distinct ways and cooperate to assemble complex, higher order networks. They bind to different septins and require septins to associate with each other, suggesting that they modulate septin architecture independently but synergistically. Furthermore, both proteins possess lipid-binding domains that interact directly with membranes and are involved in their recruitment to the bud neck and in septin organization. In vivo experiments using bud3 bud4 double mutants reveal that the double ring is essential for concentrating at the division site proteins that mark cytokinetic remnants, while other proteins landing at the bud neck at cytokinesis are unaffected. We propose that the septin double ring serves as a selective spatial memory, preserving a molecular track of cytokinesis across subsequent cell divisions.