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.

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

Septin proteins hetero-oligomerize to form filaments. Mutant septins that subtly misfold a key oligomerization interface retain some function when expressed alone but are excluded from filaments when the wild-type allele is present. Cytosolic chaperones mediate this “quality control” via prolonged interactions with the mutant polypeptides.

No abstract

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.

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Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach to identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Chaperone inhibition liberates the mutant to enter the nucleus where it has a slight dominant-negative effect. These findings provide new insights into the effects of missense mutations.

Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most frequently observed molecular alterations in many human malignancies, including head and neck squamous cell carcinoma (HNSCC). A growing body of evidence demonstrates the prime importance of the PI3K pathway at each stage of tumorigenesis, that is, tumor initiation, progression, recurrence, and metastasis. Expectedly, targeting the PI3K pathway yields some promising results in both preclinical studies and clinical trials for certain cancer patients. However, there are still many questions that need to be answered, given the complexity of this pathway and the existence of its multiple feedback loops and interactions with other signaling pathways. In this paper, we will summarize recent advances in the understanding of the PI3K pathway role in human malignancies, with an emphasis on HNSCC, and discuss the clinical applications and future direction of this field.