The Regulatory Budding Yeast Septin Shs1 Promotes Ring and Gauze Formation in a Phosphorylation-Dependent Manner

Septins are a cytoskeletal family of proteins that exhibit a remarkable degree of structural plasticity. In the budding yeast Saccharomyces cerevisiae, septins form filaments, gauzes and rings, and their self-assembly is dependent on the cell cycle stage. At the molecular level, a complex of four mitotic septins (Cdc3, Cdc10, Cdc11, and Cdc12) forms an octameric, rod-shaped, complex with Cdc11 at either end. In low salt the Cdc11-containing octamer assembles into filaments that pair. Using biochemical methods and electron microscopy, we have found that the regulatory septin Shs1 substitutes for Cdc11 at the terminal position in septin octameric rods. Such substitution results in the formation of septin rings instead of paired filaments. Ring formation requires the N-terminal 18 amino acids, including the a0 helix, as well as the C-terminal extension of Shs1. Using fluorescence microscopy and electron tomography we see that deletion of SHS1 in budding yeast results in the formation of an incomplete septin collar and disorganization of the 10-nm filaments at the bud neck. In vitro, phosphomimetic sites on the C-terminal extension of Shs1 inhibit ring formation, whereas a phosphomimetic at Shs1(S259), positioned at both end-most surfaces of the octameric rod, promotes formation of septin gauzes. Our studies show that substitution of a septin subunit can dictate a change in ultrastructure, while phosphorylation can either promote an alternative assembly or inhibit septin self-assembly altogether.