Higher-Order Septin Assembly Is Driven by GTP-Promoted Conformational Changes: Evidence From Unbiased Mutational Analysis in Saccharomyces cerevisiae

Abstract

Septin proteins bind GTP and heterooligomerize into filaments with conserved functions across a wide range of eukaryotes. Most septins hydrolyze GTP, altering the oligomerization interfaces; yet mutations designed to abolish nucleotide binding or hydrolysis by yeast septins perturb function only at high temperatures. Here, we apply an unbiased mutational approach to this problem. Mutations causing defects at high temperature mapped exclusively to the oligomerization interface encompassing the GTP-binding pocket, or to the pocket itself. Strikingly, cold-sensitive defects arise when certain of these same mutations are coexpressed with a wild-type allele, suggestive of a novel mode of dominance involving incompatibility between mutant and wild-type molecules at the septin–septin interfaces that mediate filament polymerization. A different cold-sensitive mutant harbors a substitution in an unstudied but highly conserved region of the septin Cdc12. A homologous domain in the small GTPase Ran allosterically regulates GTP-binding domain conformations, pointing to a possible new functional domain in some septins. Finally, we identify a mutation in septin Cdc3 that restores the high-temperature assembly competence of a mutant allele of septin Cdc10, likely by adopting a conformation more compatible with nucleotide-free Cdc10. Taken together, our findings demonstrate that GTP binding and hydrolysis promote, but are not required for, one-time events—presumably oligomerization-associated conformational changes—during assembly of the building blocks of septin filaments. Restrictive temperatures impose conformational constraints on mutant septin proteins, preventing new assembly and in certain cases destabilizing existing assemblies. These insights from yeast relate directly to disease-causing mutations in human septins.

Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

“…In parallel, we compared the kinetics of accumulation of a G-interface mutant, Cdc3(G365R)-GFP, which was expressed from an otherwise identical plasmid in cells also expressing an untagged cdc3(G365R) allele at the endogenous CDC3 locus. Whereas septin ring assembly and cytokinesis fail at high temperatures 13 (and see below), at this temperature cdc3 (G365R) cells divide normally and assemble septin rings at the bud neck that are indistinguishable from WT ( Fig. 2A).…”

“…We chose this mutant because high temperatures only inhibit new ring assembly in cdc3(G365R) cells, as opposed to other mutants (e.g., cdc10(D182N)) in which high temperatures both inhibit new assembly and destabilize existing assemblies. 13 Yeast cells are able to utilize nitrogen from other sources (e.g., proline) in the absence of added nitrogen, allowing us to restrict nitrogen availability. We used a cdc3(G365R) strain that is auxotrophic for leucine (Leu), uracil (Ura), and methionine (Met) to survey 8 different nutrient limitations in otherwise complete synthetic media (5 mg/ml ammonium, 100 mg/ml Leu, 100 mg/ml Ura, and 50 mg/ml Met): nitrogen (0 or 1 mg/ ml ammonium), Leu (10 or 80 mg/ml), Ura (5 or 20 mg/ml), or Met (10 or 20 mg/ml).…”

“…The TS defects in cdc10(D182N) cells are almost completely suppressed by cdc3(D210G), which we have interpreted as conformational compatibility between the G interfaces of the 2 mutant septins. 13 Since neither mutation is likely to itself affect G1 duration, the 2 mutant polypeptides must, despite chaperone engagement, be faster to (or more likely than) either WT allele to adopt the dimerization-compatible conformation, allowing dimers to form within the G1 "window" even at high temperatures. Thus, as is also illustrated with the case of the Cdc3(G261V) mutant described above, septin "folding kinetics" depend on what conformation is most appropriate in a given cellular circumstance.…”

“…from most other TS septin mutants in that it is semidominant 13,34 and evades QC-mediated exclusion from the bud neck. 15 However, deletion of SWE1 in cdc3(G365R) cells did not improve proliferation at 28 C (Fig.…”

“…11,12 We previously reported that mutant yeast septin proteins (Cdc3, Cdc10, Cdc11, or Cdc12) carrying substitutions affecting the G oligomerization interface display temperature-sensitive (TS) defects in their ability to incorporate into higher-order structures, reflecting the temperature-dependent acquisition of non-native conformations. 13 Chaperone proteins bind transiently to nonnatively-folded proteins to prevent aggregation and inappropriate intermolecular associations, effecting a "kinetic partitioning" mechanism by which "on-pathway" folding states are favored due to the kinetics of their acquisition, not their thermodynamic stability. 14 We further found that, when a wild-type (WT) allele of the same septin is available and the supply of other septin subunits is limiting, these same TS mutant septin proteins are subject to chaperone-dependent exclusion from higher-order assemblies, even at temperatures permissive for function.…”