The step-wise pathway of septin hetero-octamer assembly in budding yeast

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.DOI: http://dx.doi.org/10.7554/eLife.23689.001

Architecture, remodeling, and functions of the septin cytoskeleton

“…This indicates that when the supposed optimal pathway for octamer formation is no longer available, the cells have developed secondary pathways for octamer formation to avoid detrimental effects. This explains the lack of a strong phenotype at low temperatures for the cdc10 mutation (Weems and McMurray 2017). This also defines an adaptive mechanism for the organism’s survival under different cellular GTP levels.…”

“…In fact, the conserved lysine in the GTP-binding P-loop of septins is an arginine in yeast Cdc11, calling into question the necessity of GTP binding and subsequently GTP hydrolysis of yeast septins in filament assembly. To address this, a recent study used Bimolecular Fluorescence Complementation (BiFC) in combination with the drug mycophenolic acid (MPA) to lower the cytosolic GDP and GTP levels to assess the step-wise association of septin subunits (Weems and McMurray 2017). It was discovered that under normal conditions, Cdc10 forms a homodimer before interacting with two Cdc11-Cdc12-Cdc3 trimers in high GTP conditions and Shs1-Cdc12-Cdc3 trimers in low GTP conditions (Weems and McMurray 2017).…”

“…To address this, a recent study used Bimolecular Fluorescence Complementation (BiFC) in combination with the drug mycophenolic acid (MPA) to lower the cytosolic GDP and GTP levels to assess the step-wise association of septin subunits (Weems and McMurray 2017). It was discovered that under normal conditions, Cdc10 forms a homodimer before interacting with two Cdc11-Cdc12-Cdc3 trimers in high GTP conditions and Shs1-Cdc12-Cdc3 trimers in low GTP conditions (Weems and McMurray 2017). Cdc10 and Cdc12 were the only yeast septins previously shown to display GTP hydrolysis activity (Versele and Thorner 2004).…”

“…Analysis of mutations leading to a loss of the guanine nucleotide binding in Cdc10 and Cdc12 by BiFC provided novel insights into septin complex formation. Without a bound nucleotide, Cdc10 loses the high propensity for homodimerization prior to interaction with Cdc3 (Weems and McMurray 2017). In this case, mutant Cdc10 interacts with Cdc3-Cdc12-Cdc11 (or Cdc3-Cdc12-Shs1) trimer to form a tetramer, and then two tetramers interact to form an octamer.…”

Meeting report – shining light on septins

“…Building on his recent work (Weems and McMurray, 2017), Michael McMurray (University of Colorado, Denver, USA) explored how some fungi assemble hetero-hexamers lacking the standard central homodimer. McMurray provided evidence that, in budding yeast cells, certain conditions favour a 'central homodimer bypass' assembly pathway involving an evolutionarily ancient molecular mode of septin homodimerization otherwise available only to GTPase-active septins.…”