The Evolutionary Origins and Ancestral Features of Septins

Abstract

Septins are a family of membrane-associated cytoskeletal GTPases that play crucial roles in various cellular processes, such as cell division, phagocytosis, and organelle fission. Despite their importance, the evolutionary origins and ancestral function of septins remain unclear. In opisthokonts, septins form five distinct groups of orthologs, with subunits from multiple groups assembling into heteropolymers, thus supporting their diverse molecular functions. Recent studies have revealed that septins are also conserved in algae and protists, indicating an ancient origin from the last eukaryotic common ancestor. However, the phylogenetic relationships among septins across eukaryotes remained unclear. Here, we expanded the list of non-opisthokont septins, including previously unrecognized septins from rhodophyte red algae and glaucophyte algae. Constructing a rooted phylogenetic tree of 254 total septins, we observed a bifurcation between the major non-opisthokont and opisthokont septin clades. Within the non-opisthokont septins, we identified three major subclades: Group 6 representing chlorophyte green algae (6A mostly for species with single septins, 6B for species with multiple septins), Group 7 representing algae in chlorophytes, heterokonts, haptophytes, chrysophytes, and rhodophytes, and Group 8 representing ciliates. Glaucophyte and some ciliate septins formed orphan lineages in-between all other septins and the outgroup. Combining ancestral-sequence reconstruction and AlphaFold predictions, we tracked the structural evolution of septins across eukaryotes. In the GTPase domain, we identified a conserved GAP-like arginine finger within the G-interface of at least one septin in most algal and ciliate species. This residue is required for homodimerization of the singleChlamydomonasseptin, and its loss coincided with septin duplication events in various lineages. The loss of the arginine finger is often accompanied by the emergence of the α0 helix, a known NC-interface interaction motif, potentially signifying the diversification of septin-septin interaction mechanisms from homo-dimerization to hetero-oligomerization. Lastly, we found amphipathic helices in all septin groups, suggesting that curvature-sensing is an ancestral trait of septin proteins. Coiled-coil domains were also broadly distributed, while transmembrane domains were found in some septins in Group 6A and 7. In summary, this study advances our understanding of septin distribution and phylogenetic groupings, shedding light on their ancestral features, potential function, and early evolution.

A single septin from a polyextremotolerant yeast recapitulates many canonical functions of septin hetero-oligomers

“…Pinto and colleagues report a charge reversal mutation to the catalytic arginine finger at the G-interface of Chlamydomonas reinhardtii ’s Group 6 septin completely abrogates dimer formation (Pinto et al, 2017). This arginine residue is conserved in some Group 5 septins, including Kp AspE (Delic et al, 2024), so we made the analogous mutation: R275E. This mutation did not completely eliminate dimer formation by Kp AspE, but it did shift the distribution of the population to monomers as the more abundant species (Figure 3).…”

“…The number of septin genes varies between organisms, from as few as one in the green alga Chlamydomonas reinhardtii to as many as a dozen in humans (Shuman & Momany, 2021). Opisthokont septins are presently classified into five Groups, with members of the same group able to occupy orthologous positions in septin heteropolymers (Delic et al, 2024; Kinoshita, 2003; Shuman & Momany, 2021). For example, S. cerevisiae Cdc11 and Shs1 are both Group 3 septins, as one or the other occupies the terminal positions in every septin octamer.…”

“…The vast majority of studies on opisthokont septins focus on members of Groups 1-4, which assemble into canonical hetero-octamers. Far less is known about Group 5 septins, which are found in filamentous fungi (Delic et al, 2024; Shuman & Momany, 2021). Septins have also been identified in the genomes of some non-opisthokonts, including ciliates, diatoms, chlorophyte algae and brown algae (Delic et al, 2024; Shuman & Momany, 2021; Vargas-Muñiz et al, 2016; Yamazaki et al, 2013).…”

“…Far less is known about Group 5 septins, which are found in filamentous fungi (Delic et al, 2024; Shuman & Momany, 2021). Septins have also been identified in the genomes of some non-opisthokonts, including ciliates, diatoms, chlorophyte algae and brown algae (Delic et al, 2024; Shuman & Momany, 2021; Vargas-Muñiz et al, 2016; Yamazaki et al, 2013). In the ciliate Tetrahymena thermophila , all three septins in its genome are Group 8 septins; they localize to and stabilize mitochondria (Wloga et al, 2008).…”

“…Crystal structures of Cr SEPT reveal that it dimerizes across a G-interface and that a catalytic arginine residue is essential for dimerization (Pinto et al, 2017). Intriguingly, this arginine finger, which is present in the majority of non-opisthokont septins, is also present in some Group 5 septins, and they are the only group of opisthokont septins with this feature (Delic et al, 2024). To our knowledge, there is no structural data on Group 5 septins, not even in the model filamentous fungus Aspergillus nidulans , in which the Group 5 septin known as AspE was first described (Hernández-Rodríguez et al, 2014; Momany et al, 2001).…”