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Abstract Features of the natural life cycle of the budding yeast Saccharomyces cerevisiae were crucial to its domestication as a laboratory experimental model, especially the ability to maintain stable haploid clones and cross them at will to combine alleles via meiosis. Stable haploidy results from mutations in HO, which encodes an endonuclease required for haploid-specific mating-type switching. Previous studies found an unexpected diversity of HO alleles among natural isolates within a small geographic area. We developed a hands-on field and laboratory activity for middle-school students in Denver, CO, USA, to isolate wild yeast from oak bark, identify species via DNA sequencing, and sequence HO from S. cerevisiae isolates. We find limited HO diversity in North American oak isolates, pointing to efficient, continuous dispersal across the continent. In contrast, we isolated the “dairy yeast,” Kluyveromyces lactis, from a tree <10 m away and found that it represents a new population distinct from an oak population in an adjacent state. The outreach activity partnered middle-school, high-school, and university students in making scientific discoveries and can be adapted to other locations and natural yeast habitats. Indeed, a pilot sampling activity in southeast Texas yielded S. cerevisiae oak isolates with a new allele of HO and, from a nearby prickly pear cactus, a heat-tolerant isolate of Saccharomyces paradoxus. 

The septin family of eukaryotic proteins comprises distinct classes of sequence-related monomers that associate in a defined order into linear hetero-oligomers, which are capable of polymerizing into cytoskeletal filaments. Like actin and ⍺ and β tubulin, most septin monomers require binding of a nucleotide at a monomer-monomer interface (the septin “G” interface) for assembly into higher-order structures. Like ⍺ and β tubulin, where GTP is bound by both subunits but only the GTP at the ⍺–β interface is subject to hydrolysis, the capacity of certain septin monomers to hydrolyze their bound GTP has been lost during evolution. Thus, within septin hetero-oligomers and filaments, certain monomers remain permanently GTP-bound. Unlike tubulins, loss of septin GTPase activity–creating septin “pseudoGTPases”—occurred multiple times in independent evolutionary trajectories, accompanied in some cases by non-conservative substitutions in highly conserved residues in the nucleotide-binding pocket. Here, we used recent septin crystal structures, AlphaFold-generated models, phylogenetics andin siliconucleotide docking to investigate how in some organisms the septin G interface evolved to accommodate changes in nucleotide occupancy. Our analysis suggests that yeast septin monomers expressed only during meiosis and sporulation, when GTP is scarce, are evolving rapidly and might not bind GTP or GDP. Moreover, the G dimerization partners of these sporulation-specific septins appear to carry compensatory changes in residues that form contacts at the G interface to help retain stability despite the absence of bound GDP or GTP in the facing subunit. During septin evolution in nematodes, apparent loss of GTPase activity was also accompanied by changes in predicted G interface contacts. Overall, our observations support the conclusion that the primary function of nucleotide binding and hydrolysis by septins is to ensure formation of G interfaces that impose the proper subunit-subunit order within the hetero-oligomer. 

Abstract Septin proteins contribute to many eukaryotic processes involving cellular membranes. In the budding yeast Saccharomyces cerevisiae , septin hetero‐oligomers interact with the plasma membrane (PM) almost exclusively at the future site of cytokinesis. While multiple mechanisms of membrane recruitment have been identified, including direct interactions with specific phospholipids and curvature‐sensitive interactions via amphipathic helices, these do not fully explain why yeast septins do not localize all over the inner leaflet of the PM. While engineering an inducible split‐yellow fluorescent protein (YFP) system to measure the kinetics of yeast septin complex assembly, we found that ectopic co‐overexpression of two tagged septins, Cdc3 and Cdc10, resulted in nearly uniform PM localization, as well as perturbation of endogenous septin function. Septin localization and function in gametogenesis were also perturbed. PM localization required the C‐terminal YFP fragment fused to the C terminus of Cdc3, the septin‐associated kinases Cla4 and Gin4, and phosphotidylinositol‐4,5‐bis‐phosphate (PI[4,5]P 2 ), but not the putative PI(4,5)P 2 ‐binding residues in Cdc3. Endogenous Cdc10 was recruited to the PM, likely contributing to the functional interference. PM‐localized septins did not exchange with the cytosolic pool, indicative of stable polymers. These findings provide new clues as to what normally restricts septin localization to specific membranes. 

Gametogenesis in diploid cells of the budding yeast Saccharomyces cerevisiae produces four haploid meiotic products called spores. Spores are dormant until nutrients trigger germination, when they bud asexually or mate to return to the diploid state. Each sporulating diploid produces a mix of spores of two haploid mating types, a and α. In asexually dividing haploids, the mating types result from distinct, mutually exclusive gene expression programs responsible for production of mating pheromones and the receptors to sense them, all of which are silent in diploids. It was assumed that spores only transcribe haploid- and mating-type-specific genes upon germination. We find that dormant spores of each mating type harbor transcripts representing all these genes, with the exception of Mata1, which we found to be enriched in a spores. Mata1 transcripts, from a rare yeast gene with two introns, were mostly unspliced. If the retained introns reflect tethering to the MATa locus, this could provide a mechanism for biased inheritance. Translation of pheromones and receptors were repressed at least until germination. We find antisense transcripts to many mating genes that may be responsible. These findings add to the growing number of examples of post-transcriptional regulation of gene expression during gametogenesis.