Septins are a conserved family of GTP-binding proteins that form heterooctameric complexes that assemble into higher-order structures. In yeast, septin superstructure at the bud neck serves as a barrier to separate a daughter cell from its mother and as a scaffold to recruit the proteins that execute cytokinesis. However, how septins recruit specific factors has not been well characterized. In the accompanying article in this issue, , we demonstrated that the C-terminal extensions (CTEs) of the alternative terminal subunits of septin heterooctamers, Cdc11 and Shs1, share a role required for optimal septin function in vivo. Here we describe our use of unbiased genetic approaches (both selection of dosage suppressors and analysis of synthetic interactions) that pinpointed Bni5 as a protein that interacts with the CTEs of Cdc11 and Shs1. Furthermore, we used three independent methods-construction of chimeric proteins, noncovalent tethering mediated by a GFP-targeted nanobody, and imaging by fluorescence microscopy-to confirm that a physiologically important function of the CTEs of Cdc11 and Shs1 is optimizing recruitment of Bni5 and thereby ensuring efficient localization at the bud neck of Myo1, the type II myosin of the actomyosin contractile ring. KEYWORDS yeast; cytokinesis; complexes; filaments; mutants S EPTIN-based structures in metazoans are frequently found at the boundaries between cellular compartments (Saarikangas and Barral 2011;Trimble and Grinstein 2015). The tissue specificity with which septin genes are expressed, and/or their mRNAs undergo alternative splicing, is consistent with specific septins and septin isoforms playing distinct roles in particular developmental and physiological processes (Roeseler et al. 2009;Hall and Russell 2012;Dolat et al. 2014). A prominent septin-containing structure is located at the bottom of the neck of each small membranous protrusion (dendritic spine) that projects from the dendrites that extend from the cell body of a neuron (Tada et al. 2007;Ewers et al. 2014), indicating some role in neurogenesis. A discrete septincontaining structure, dubbed the annulus, separates the head and midpiece from the flagellum in mature mammalian sperm and is required for sperm motility (Sugino et al. 2008;Toure et al. 2011). Similarly, septin function has been implicated in the amoeboid motility of T cells by promoting membrane retraction (Tooley et al. 2009;Gilden et al. 2012). Septincontaining structures are found at the base of the primary (nonmotile) cilium and at discrete regions along the axoneme, suggesting roles in ciliogenesis and in the signaling functions of this sensory organelle (Hu et al. 2010;Ghossoub et al. 2013;Sharma et al. 2013). Septin "cages" assemble around intracellular bacterial pathogens or the vacuolar inclusions that contain them, but whether caging serves as a first line of defense against their spread (Mostowy et al. 2010), or is exploited by the microbes to promote their spread (Volceanov et al. 2014), has not been resolved. In all the instanc...

A tripartite split-GFP system faithfully reports the order of the subunits in septin hetero-octamers (and thus can serve as a “molecular ruler”), conversely yields little or no false signal even with very highly expressed cytosolic proteins, and detects authentic interactions of other cellular proteins that are bona fide septin-binding proteins.

A long-standing conundrum is resolved about the underlying sequence determinants and molecular mechanism responsible for the recruitment of the protein kinase Hsl1 (an indispensable component of the so-called “morphogenesis checkpoint”) exclusively to the septin collar at the bud neck.

Septins are GTP-binding proteins conserved across metazoans. They can polymerize into extended filaments and, hence, are considered a component of the cytoskeleton. The number of individual septins varies across the tree of life—yeast (Saccharomyces cerevisiae) has seven distinct subunits, a nematode (Caenorhabditis elegans) has two, and humans have 13. However, the overall geometric unit (an apolar hetero-octameric protomer and filaments assembled there from) has been conserved. To understand septin evolutionary variation, we focused on a related pair of yeast subunits (Cdc11 and Shs1) that appear to have arisen from gene duplication within the fungal clade. Either Cdc11 or Shs1 occupies the terminal position within a hetero-octamer, yet Cdc11 is essential for septin function and cell viability, whereas Shs1 is not. To discern the molecular basis of this divergence, we utilized ancestral gene reconstruction to predict, synthesize, and experimentally examine the most recent common ancestor (“Anc.11-S”) of Cdc11 and Shs1. Anc.11-S was able to occupy the terminal position within an octamer, just like the modern subunits. Although Anc.11-S supplied many of the known functions of Cdc11, it was unable to replace the distinct function(s) of Shs1. To further evaluate the history of Shs1, additional intermediates along a proposed trajectory from Anc.11-S to yeast Shs1 were generated and tested. We demonstrate that multiple events contributed to the current properties of Shs1: (1) loss of Shs1–Shs1 self-association early after duplication, (2) co-evolution of heterotypic Cdc11–Shs1 interaction between neighboring hetero-octamers, and (3) eventual repurposing and acquisition of novel function(s) for its C-terminal extension domain. Thus, a pair of duplicated proteins, despite constraints imposed by assembly into a highly conserved multi-subunit structure, could evolve new functionality via a complex evolutionary pathway.