Bni5p, a Septin-Interacting Protein, Is Required for Normal Septin Function and Cytokinesis in Saccharomyces cerevisiae

Abstract

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7⌬ strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7⌬ mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.Cytokinesis in animal cells involves an actomyosin-based contractile ring, which forms late in the cell cycle and constricts the plasma membrane, resulting in the division of one cell into two cells. In the budding yeast Saccharomyces cerevisiae, the cleavage plane is specified early in the cell cycle, and cytokinesis involves the concerted action of actomyosin ring contraction and septum formation (5, 37, 55). Despite some differences in the morphological features and timing of certain cytokinetic events between yeast and animal cells, many of the components involved are conserved (18). In addition to the conserved involvement of the actomyosin contractile ring, it is now widely appreciated that the septins also play a critical role in cytokinesis in both yeast and animal cells.Septins are a family of proteins that were identified first in yeast and subsequently in various other fungi and animals (for review, see references 20, 32, and 39). Septin family members possess at least 26% amino acid sequence identity. All of the known septins possess an N-terminal P-loop and other sequences conserved in the GTPase superfamily of nucleotidebinding proteins (8). In addition, at or near their C termini, most septins possess a predicted coiled-coil domain (for review, see reference 39) that may be important in proteinprotein interactions.In yeast, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p (or Shs1p) septins all localize to the presumptive bud site before bud emergence and remain at the mother-bud neck until after cytokinesis (11,21,28,33,45; M. S. Longtine, unpublished observations). Recent studies...

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

“…Further study of two cdc42 effector-loop mutants and of the roles of Bem3p, Rga1p, and Rga2p, which are three GTPase-activating proteins (GAPs) for Cdc42p (Zheng et al, 1993;Stevenson et al, 1995;Johnson, 1999;Smith et al, 2002), has suggested that Cdc42p GTPase cycling is involved in septin-ring formation (Gladfelter et al, 2002). In particular, it was suggested that Cdc42p, like the assembly GTPase EF-Tu in protein synthesis, participates in septin-ring formation by shuttling septin complexes to the assembling ring structure (Gladfelter et al, 2002), a view that contrasts with the more common view of activated (i.e., GTP-bound) Cdc42p activating effectors in signaling pathways in a Ras-like manner (Johnson, 1999).In addition to Cdc42p, its GEF, and its GAPs, several other proteins including Bni5p, Nap1p, and the protein kinases Cla4p, Gin4p, and Elm1p are also known to be involved in formation of the normal septin ring (Cvrcková et al, 1995;Longtine et al, 1998aLongtine et al, , 2000Sreenivasan and Kellogg, 1999;Bouquin et al, 2000;Weiss et al, 2000;Lee et al, 2002). Cla4p may function in the same pathway as Cdc42p, because it is a Cdc42p effector (Cvrcková et al, 1995;Weiss et al, 2000), although it is not clear whether the interaction between Cla4p and Cdc42p is required for its role in septin organization.…”

“…In addition to Cdc42p, its GEF, and its GAPs, several other proteins including Bni5p, Nap1p, and the protein kinases Cla4p, Gin4p, and Elm1p are also known to be involved in formation of the normal septin ring (Cvrcková et al, 1995;Longtine et al, 1998aLongtine et al, , 2000Sreenivasan and Kellogg, 1999;Bouquin et al, 2000;Weiss et al, 2000;Lee et al, 2002). Cla4p may function in the same pathway as Cdc42p, because it is a Cdc42p effector (Cvrcková et al, 1995;Weiss et al, 2000), although it is not clear whether the interaction between Cla4p and Cdc42p is required for its role in septin organization.…”

“…Despite some differences in the details of their results, Dobbelaere et al (2003) have recently reached the same conclusion from independent FRAP experiments. Second, in a variety of mutants (certain cdc42 mutants, Cdc42p GAP mutants, and cla4, gin4, bni5, nap1, and elm1 mutants), the septins appear to be recruited to an appropriate site in the cell cortex but fail to form a normal structure there; the caps, patches, and bars that do form differ both in appearance and in their continuing lability (as judged by FRAP) from normal septin rings (Figures 3, 5, 6, 8, and 9;Cvrcková et al, 1995;Longtine et al, 1998aLongtine et al, , 2000Sreenivasan and Kellogg, 1999;Weiss et al, 2000;Bouquin et al, 2000; Gladfelter et al, 2001Gladfelter et al, , 2002Lee et al, 2002;Smith et al, 2002;Dobbelaere et al, 2003). These observations suggest that Cdc42p itself, the Cdc42p GAPs, Cla4p, Gin4p, Bni5p, Nap1p, and Elm1p are all involved in a distinct assembly step (or steps; see below) that follows the initial Cdc42p-dependent recruitment of the septins to the presumptive bud site.…”

Shs1 Plays Separable Roles in Septin Organization and Cytokinesis in Saccharomyces cerevisiae

“…Those are proteins functioning in septin recruitment and ring assembly (see above), cytokinesis (see below), bud-site selection (Chant et al 1995;Sanders and Herskowitz 1996;Kang et al 2004), cell cycle control (Frenz et al 2000;Yoshida and Toh-e 2001), and mitotic signaling network (Carroll et al 1998;Longtine et al 1998Longtine et al , 2000Barral et al 1999;Lee et al 2002;. Septin serves as a scaffold for these proteins to localize and maintain at the bud neck.…”

“…Approximately 15 min before bud emergence, septins are recruited to the presumptive bud site (Kim et al 1991), which depends on activated Rho-type GTPase Cdc42 and its effectors Gic1 and Gic2 (Iwase et al 2006). After recruitment at the incipient bud site, septins are reorganized and assembled as a higher-ordered ring structure at the mother-bud neck, which requires Cdc42, its GTPase-activating factors (Bem3, Rga1, and Rga2), Bni5, Nap1, Bni1, Cla4, Gin4, and Elm1 (Cvrckova et al 1995;Richman et al 1999;Bouquin et al 2000;Longtine et al 2000;Weiss et al 2000;Gladfelter et al 2001aGladfelter et al , 2002Gladfelter et al , 2004Lee et al 2002;Caviston et al 2003;Goehring et al 2003;Kadota et al 2004;Versele and Thorner 2004).…”