Abstract
Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3-Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11-Cdc12-Cdc3 or Shs1-Cdc12-Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.
Highlights
If NC dimerization by yeast septins depends upon GTP hydrolysis, as predicted by the ‘NC priming’ model, preventing GTP binding should perturb both NC and G dimerization
Previous studies of the role of nucleotide in yeast septin assembly mutated residues that are predicted to contact bound nucleotide and examined effects on septin-nucleotide interactions in vitro and septin-septin interactions via affinity co-purification (Nagaraj et al, 2008; Sirajuddin et al, 2009; Versele et al, 2004; Versele and Thorner, 2004; Farkasovsky et al, 2005). These methods are limited by the fact that the nucleotide-binding pocket (NBP) itself is a major part of the G interface, and so these mutations might directly perturb septin-septin interactions in ways that do not necessarily involve roles for nucleotide state
A complete nucleotide hydrolysis/exchange cycle is critical for actin and tubulin NTPases because their cellular functions rely on rapid dynamics of polymer assembly and disassembly
Summary
The septin family of cytoskeletal proteins is highly conserved between humans, yeast, and other non-plant eukaryotes, both in structure and function (Oh and Bi, 2011; Mostowy and Cossart, 2012; Dolat et al, 2014; Fung et al, 2014). Septin dysfunction is linked to a wide variety of human diseases, including cancer, male infertility, and hereditary neuralgic amyotrophy (Oh and Bi, 2011; Mostowy and Cossart, 2012; Dolat et al, 2014; Fung et al, 2014). The majority of human septins are constitutively expressed across all cell types (Mostowy and Cossart, 2012). It remains unclear how cells assemble only the oligomer species that are functionally appropriate
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