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

Abstract

Septins are a family of GTP-binding proteins considered to be cytoskeletal elements because they self-assemble into filaments and other higher-order structures in vivo. In budding yeast, septins establish a diffusion barrier at the bud neck between a mother and daughter cell, promote membrane curvature there, and serve as a scaffold to recruit other proteins to the site of cytokinesis. However, the mechanism by which any septin engages a partner protein has been unclear. The two most related and recently evolved subunits appear to be Cdc11 and Shs1, and the basic building blocks for assembling septin structures are hetero-octameric rods (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1). Loss of Cdc11 is not normally tolerated, whereas cells lacking Shs1 do not appear grossly abnormal. We established several different sensitized genetic backgrounds wherein Shs1 is indispensable, which allowed us to carry out the first comprehensive and detailed genetic analysis of Shs1 in vivo. Our analysis revealed several novel insights, including: (i) the sole portion of Shs1 essential for its function is a predicted coiled-coil-forming segment in its C-terminal extension (CTE); (ii) the CTE of Cdc11 shares this function; (iii) this role for the CTEs of Cdc11 and Shs1 is quite distinct from that of the CTEs of Cdc3 and Cdc12; and (iv) heterotypic Cdc11 and Shs1 junctions likely occur in vivo. KEYWORDS yeast; cytoskeleton; complexes; filaments; mutants S EPTINS are GTP-binding proteins conserved across Eukarya (except higher plants) (Pan et al. 2007;Nishihama et al. 2011). This protein family was first identified because the corresponding loci were among the temperature-sensitive (ts) cell division cycle (cdc) mutations isolated in Saccharomyces cerevisiae (Hartwell 1978). At the restrictive temperature, cdc3, cdc10, cdc11, and cdc12 mutants continued to bud, replicate, and segregate their chromosomes, yet failed to execute cell division, resulting in the formation of chains of multinucleate cells (Hartwell 1971). Shifting a ts allele of any of these four homologous gene products to restrictive temperature prevented formation of what appeared to be rings of highly ordered membrane-associated filaments at the bud neck (Byers and Goetsch 1976). Antibody decoration (Haarer and Pringle 1987;Ford and Pringle 1991;Kim et al. 1991) and, later, use of fusions to green fluorescent protein (GFP) (Cid et al. 1998) demonstrated that these four proteins colocalized with and were likely constituents of these filaments. Because loss of the function of these proteins prevents cytokinesis and cell septation, and they seemed to be integral components of the filamentous structures erected at the bud neck, they were dubbed septins (Sanders and Field 1994;Pringle 2008). Indeed, subsequent purification of these proteins from yeast (Frazier et al. 1998), and their production as recombinant proteins in bacteria Versele and Thorner 2004;Farkasovsky et al. 2005), demonstrated that Cdc3, Cdc10, Cdc11...

Detection of protein–protein interactions at the septin collar inSaccharomyces cerevisiaeusing a tripartite split-GFP system

“…Consistent with other evidence that the C-terminal end of Bni5 is required for its function (Finnigan et al ., 2015a), Bni5-β11 interacted exclusively with N-terminally β10-tagged Cdc11 and Shs1 and no other septin. In agreement with structure predictions that Bni5 is an elongated, highly α-helical protein, β10-Bni5 had a longer “reach,” yielding a readily detectable fluorescence signal with every C-terminally β11-tagged septin.…”

“…These basic residues are believed to mediate interaction with phosphatidylinositol-4,5- bis -phosphate (PtdIns4,5P 2 ) on the inner leaflet of the PM (Bertin et al ., 2010) and are required for the full in vivo function of these septins (Finnigan et al ., 2015b). Thus access to a tag placed at the N-terminus of either of these two septins may be limited, resulting in a weaker signal in this assay format.…”

Endosomal assembly and transport of heteromeric septin complexes promote septin cytoskeleton formation

“…Interactions between, for example, Cdc12-Cdc3, Cdc10-Cdc10 and Cdc11-Cdc11 at the so-called 'NC' interface are essential (Beise and Trimble, 2011;Sirajuddin et al, 2007). The terminal subunit Cdc11 can be replaced by an alternative septin subunit Shs1, resulting in the formation of alternative heterooctamers (Booth et al, 2015;Finnigan et al, 2015;Garcia et al, 2011).…”

Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae

“…The advent of GFP and other genetically encoding fluorescent protein tags to follow protein dynamics in live cells in real time revealed further that septin organization undergoes dramatic changes during cell cycle progression. Septins first accumulate as a patch at the incipient site of bud emergence that rapidly resolves into a filamentous ring (Kozubowski et al, 2005; Okada et al, 2013), which then expands, concomitant with bud growth, into an hourglass-shaped tube or collar composed of at least 30–40 gyres of circumferential filaments at the bud neck (Byers and Goetsch, 1976; McMurray et al, 2011; Finnigan et al, 2015b; Patasi et al, 2015). At the onset of cytokinesis, the collar splits into two fllamentous bands of roughly equal size with a prominent gap in between (Dobbelaere et al, 2003; Dobbelaere and Barral, 2004) wherein factors needed for actomyosin contractile ring assembly and new plasma membrane (PM) and cell wall (CW) synthesis accumulate (Bi et al, 1998; Nishihama et al, 2009).…”

“…In vitro the former can associate end-to-end into paired filaments in low-salt solution and into sheets of paired filaments on the surface of a lipid monolayer containing phosphatidylinositol-4,5- bis phosphate (Bertin et al, 2010). Mutagenesis studies have shown that the residues needed for these characteristic in vitro behaviors are also essential for viability in vivo (Bertin et al, 2008; McMurray et al, 2011; Finnigan et al, 2015b). The Shs1-containing hetero-octamers associate laterally in a staggered fashion, rather than end-on-end, forming curved bundles, rings, and bird's nest-like structures in vitro (Garcia et al, 2011).…”