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Our research focuses on identifying molecular mechanisms underlying the assembly of macromolecular complexes, with a focus on multisubunit complexes formed by septin proteins. All cellular processes require the function of multisubunit complexes, and while much attention has been given to solving the final structures of such assemblies, comparatively little is known about how individual subunits adopt oligomerization-competent conformations and find their partner subunits in the crowded, dynamic cellular milieu.

In our research we mostly use the premiere eukaryotic experimental system for genetic analysis, the budding yeast Saccharomyces cerevisiae. Studies of septin protein function in yeast gametogenesis led us to begin to explore some fundamental unanswered questions about yeast gametes.
Below is a summary of recent research from our group in these two areas.

“What does GTP do for septins?” This question was posed in 2002 in Current Biology by Tim Mitchison and Chris Field, reflecting the awareness that septins clearly evolved from an ancestral GTPase and most septins bind GTP, but some do not hydrolyze it and nucleotide exchange is very slow. How GTP binding and/or hydrolysis relates to septin structure or function was unknown. Over the last 13 years, my lab answered this question. Using simple genetic approaches, we demonstrated that GTP binding is dispensable for septin function and merely guides de novo folding towards an oligomerization-competent state (). We next developed a new method for determining the temporal order of protein-protein interactions in living cells, and with it revealed the step-wise pathway for septin hetero-octamer assembly in yeast (). In doing so, we found that slow GTP hydrolysis by one septin enforces order of assembly and mediates the choice of incorporation of the two alternative “competing” subunits at one subunit position in septin octamers (). During yeast evolution loss of GTP hydrolysis by another septin eliminated an alternate assembly pathway that humans and other fungi use to make hexamers in addition to octamers (). Slow GTP hydrolysis controls septin assembly pathways by creating a transient GTP-bound septin that has a distinct affinity for partner septins compared to the GDP-bound form (Weems and McMurray, 2017; ). Crucially, this feature allows septin assembly pathways to respond to the GTP:GDP ratio in the cytoplasm (), providing a way for cells to tailor the composition of their septin complexes to current cellular demands. Human cells almost certainly use this mechanism to choose what kinds of complexes they assemble from the 13 distinct human septin genes. Finally, we revealed additional examples throughout phylogeny of how loss of GTP hydrolysis or GTP/GDP binding altogether drove changes in septin-septin assembly interfaces that likely reflect adaptation to metabolic changes resulting in varying nucleotide levels ().

Requirements for de novo septin complex assembly. Monomers of actin and tubulin require folding assistance from cytosolic chaperones. We first discovered that cytosolic chaperones mediate a kind of “quality control” over septin assembly, imposing a kinetic disadvantage on slowly-folding mutant septins that favors incorporation of wild-type septins into complexes (). These findings demonstrated a new role for general cytosolic chaperones in the expression of subtly misfolded mutant alleles, representing a new paradigm in cytosolic cellular proteostasis (). We next used direct in vivo approaches to identify septin-chaperone interactions () and a combination of in vivo crosslinking and in vitro reconstitution to show that specific cytosolic chaperones engage nascent septin polypeptides to promote efficient septin complex assembly (). We also discovered that simultaneous co-overexpression of two yeast septins (one fused to a small tag) is sufficient to drive an unprecedented phenotype: filaments composed of only those two septins localized all over the inner leaflet of the plasma membrane (). These findings represent key advances in our understanding of the molecular requirements for septin-septin interactions, complex assembly, and membrane association.

Chemical rescue of mutant protein function in living cells. During our septin studies we serendipitously discovered that the small molecule guanidine hydrochloride is able to restore the folding and/or function of certain missense mutants in vivo (; ). In at least some cases (including a mutant allele of actin that is linked to cardiac disease, and a mutant allele of ornithine transcarbamylase (OTC) linked to OTC deficiency) this rescue involves replacement by the guanidinium ion of an arginine side chain. Such replacement had been shown for some proteins in vitro but had never been demonstrated in living cells. We went on to explore chemical rescue by other naturally occurring small molecules, and found hundreds of examples of mutant rescue in living cells by molecules like DMSO and trimethylamine-N-oxide (TMAO) (). TMAO, found naturally in marine organisms that experience high levels of urea or hydrostatic pressure, rescued ~20% of the mutants we tested (). These findings point to influences of intracellular small molecules on protein evolution, as well as possible therapeutic interventions for genetic diseases.

Establishment of sexual identity and cell polarity during budding yeast gametogenesis. Diploid yeast cells produce haploid gametes by coupling meiosis to sporulation, a specialized form of cytokinesis. We studied septin assembly and function in sporulation (; ) and found that septins mark a cortical site in spores that directs polarized outgrowth away from contacts with sister spores (). We also explored a surprisingly open question: Since the four gametes produced by a single diploid cell are of two different sexual identities, and mating/fertilization requires mutually exclusive gene expression programs, how is it that these expression programs begin early in gametogenesis, before the gametes are fully formed and isolated from each other? We found evidence of a variety of post-transcriptional mechanisms that may inhibit translation of sex-specific mRNAs made “too early”, including antisense transcription and extended 5’ UTRs with upstream open reading frames (uORFs) ().

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