Evolutionary history of the septin cytoskeletal proteins in opisthokonts

Septins are cytoskeletal proteins important in morphogenesis, cell division and establishing and maintaining cell polarity. Over the course of more than a billion years as Animals and Fungi originated and diversified, their septin genes duplicated and diverged, giving rise to paralogs that encode modular proteins. The septin monomers assemble into heteropolymeric higher order structures that affect cell form by creating physical barriers to diffusion or serving as scaffolds organizing groups of diverse proteins. Here we take advantage of newly sequenced genomes to track the history of septin gene expansions and losses within the phylogeny of Animals and Fungi, including their close protist relatives. By sampling broadly across Opisthokonts, we identified the likely presence of early-diverging animal lineages within Groups 4 and 2A and discovered a novel group of fungal septins not found in Ascomycetes or Basidiomycetes. We hypothesized that previously identified sequence conservation is linked to interface interactions. Using protein homology folding, we mapped interacting residues across Opisthokonts and found that all previously identified motifs were involved in interface interactions, and contained almost all interacting residues. As septin subunit interactions are likely driven by residue identity, we categorized the interacting residues and found specific interface residues associated with each septin Group. We suggest that these residues may explain patterns of septin subunit binding affinity. Notably, we found that Group 3 septins show little conservation within the polybasic region that forms the first alpha helix, found in the NC interface of other septin Groups. This may explain the capping role of Group 3 septins in the yeast septin octamer. With increased sampling, this work identified increased diversity of Opisthokont septins. These proteins show patterns of sequence conservation that are largely driven by conserved interface interactions, in addition to binding of GTP. This work highlights the likely duplications that predate the Opisthokont ancestor, and the structural constraints that shaped the evolution of these multi-purpose septins. Additionally, I attempted to validate and optimize an Agrobacterium-mediated transformation protocol for the chytrid fungus Blastocladiella. While I was unable to conclusively repeat previously published experiments, my work highlighted the difficulties in transformation of these distinct fungi.Science, Faculty ofBotany, Department ofGraduat