Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of hetero-octamers, Cdc11/Shs1–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11/Shs1. Slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1–Cdc12–Cdc3–Cdc3–Cdc12–Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium occupies the site of a ‘missing’ Arg side chain found in other fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers natively co-exist with octamers. We propose that guanidinium reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to octamers. Since homodimerization by a GTPase-active human septin also creates hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply throughout phylogeny.
Septins self-assemble into heteromeric rods and filaments to act as scaffolds and modulate membrane properties. How cells tune the biophysical properties of septin filaments to control filament flexibility and length, and in turn the size, shape, and position of higher-order septin structures, is not well understood. We examined how rod composition and nucleotide availability influence physical properties of septins such as annealing, fragmentation, bundling, and bending. We found that septin complexes have symmetric termini, even when both Shs1 and Cdc11 are coexpressed. The relative proportion of Cdc11/Shs1 septin complexes controls the biophysical properties of filaments and influences the rate of annealing, fragmentation, and filament flexibility. Additionally, the presence and apparent exchange of guanine nucleotide also alters filament length and bundling. An Shs1 mutant that is predicted to alter nucleotide hydrolysis has altered filament length and dynamics in cells and impacts cell morphogenesis. These data show that modulating filament properties through rod composition and nucleotide binding can control formation of septin assemblies that have distinct physical properties and functions.
Polarized growth is critical for the development and maintenance of diverse organisms and tissues, but particularly central to fungi where nutrient uptake, communication, and reproduction all rely on cell asymmetries. To achieve polarized growth, fungi spatially organize both their cytosol and cortical membranes. The septins, a family of GTP-binding proteins, have emerged as key regulators of spatial compartmentalization in fungi and other eukaryotes. By forming higher-order structures on fungal plasma membranes, septins are thought to contribute to the generation of cell asymmetries by acting as molecular scaffolds and forming diffusional barriers. Here we discuss the links between septins and polarized growth and consider molecular models for how septins contribute to cellular asymmetry in fungi.
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