The Carboxy-Terminal Tails of Septins Cdc11 and Shs1 Recruit Myosin-II Binding Factor Bni5 to the Bud Neck in Saccharomyces cerevisiae

Abstract

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...

Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 inSaccharomyces cerevisiae

“…Revealing, however, the dramatic change to the charge of the Shs1 CTE was not tolerated in cells lacking Cdc10 ( Figure 1D). This observation alerted us to the possibility that one role of the Shs1 CTE may be shared, in part, with a function also carried out by Cdc10 (see accompanying article, Finnigan et al 2015). On the other hand, our in vivo studies also highlight the difficulty in interpreting the effects of phosphomimetic mutations observed in vitro.…”

“…However, genetic or physical links between Shs1 (and/ or Cdc11) and other cellular proteins involved in morphogenesis at the bud neck have also been described, including IQGAP Iqg1 (Iwase et al 2007), formin Bnr1 (Buttery et al 2012), F-BAR protein Hof1 (Meitinger et al 2013), polarisome component Spa2 (Mino et al 1998), septin-interacting protein Nis1 (Iwase and Toh-E 2001), and the Myo1-binding factor Bni5 (Lee et al 2002). As described in detail in the accompanying article (Finnigan et al 2015), in our hands, the critical function shared by the CC elements in the CTEs of Shs1 and Cdc11 is physical association with Bni5.…”

Effects of Bni5 Binding on Septin Filament Organization

“…At the NC interface responsible for the homotypic Cdc11-Cdc11 interaction required for the polymerization of hetero-octamers, an N-terminal alpha-helix (α0; residues 6–19) plays a pivotal role [14, 16, 20]. Furthermore, Cdc11 possesses a pronounced C-terminal extension (CTE; residues 301–415), which contains a C-terminal segment (residues 377–415) with strongly predicted coiled-coil forming propensity and is required for full Cdc11 function [13, 30]. …”

“…Bni5 localizes to the bud neck after bud emergence during the period when the superstructure in the septin collar is being stabilized [42]. Absence of Bni5 results in growth defects and exacerbates the phenotypes ( e.g ., elongated cell shape and cell cycle arrest) conferred by septin mutations [30, 42]. Reportedly, phosphorylation of Bni5 contributes to its ability to interact with Cdc11 (and Cdc12) and for its dislocation from the bud neck in late mitosis [43], as well as for proper bud morphogenesis [44].…”

“…Reportedly, phosphorylation of Bni5 contributes to its ability to interact with Cdc11 (and Cdc12) and for its dislocation from the bud neck in late mitosis [43], as well as for proper bud morphogenesis [44]. However, subsequent site-directed mutagenesis studies demonstrated that none of the purported sites, alone or in combination, had any effect in two in vivo assays for Bni5 function [30]. Most importantly, Bni5 binding to Cdc11 within the septin collar is necessary for efficient recruitment, in turn, of Myo1, the type II myosin required for assembly of the actomyosin contractile ring that drives plasma membrane ingression for cytokinesis [30, 45–47].…”