Effects of Bni5 Binding on Septin Filament Organization

Abstract

Septins are a protein family found in all eukaryotes (except higher plants) that have roles in membrane remodeling, formation of diffusion barriers, and as a scaffold to recruit other proteins. In budding yeast, proper execution of cytokinesis and cell division requires formation of a collar of circumferential filaments at the bud neck. These filaments are assembled from apolar septin hetero-octamers. Currently, little is known about the mechanisms that control the arrangement and dynamics of septin structures. In this study we utilized both Förster resonance energy transfer and electron microscopy to analyze the biophysical properties of the septin-binding protein Bni5 and how its association with septin filaments affects their organization. We found that interaction of Bni5 with the terminal subunit (Cdc11) at the junctions between adjacent hetero-octamers in paired filaments is highly cooperative. Both the C-terminal end of Bni5 and the C-terminal extension of Cdc11 make important contributions to their interaction. Moreover, this binding may stabilize dimerization of Bni5, which, in turn, forms cross-filament braces that significantly narrow, and impose much more uniform spacing on, the gap between paired filaments.

Septin Assembly and Remodeling at the Cell Division Site During the Cell Cycle

“…Right , depiction of Cdc11-capped rods forming paired filaments in either low salt (<75 mM) ( Bertin et al, 2008 ) or on the surface of PIP2-containing lipid monolayers ( Bertin et al, 2010 ; Bridges et al, 2014 ); dashed box indicates zoomed in inset shown to the right. (C) Left , depiction of two Bni5 proteins (red molecules) interacting with Cdc11-CTEs found in paired septin filaments to lower the average interfilament distance to 10 nm ( Finnigan et al, 2015 ; Booth et al, 2016 ). Right , depiction of Bni5 bundling septin filaments into large clusters in vitro in a dose-dependent manner ( Patasi et al, 2015 ).…”

“…One such SAP, Bni5, localizes to the bud neck in a septin-dependent manner ( Lee et al, 2002 ). Bni5 appears to interact with the CTEs of the terminal subunits Cdc11 and Shs1 in vitro and in vivo ( Figure 1C ) ( Finnigan et al, 2015 ; Booth et al, 2016 ). Recombinant Bni5 can reduce the distance between individual filaments of a paired filament and can also bundle septin filaments, thereby contributing to a higher-order organization ( Figures 1B,C ) ( Patasi et al, 2015 ; Booth et al, 2016 ).…”

“…Bni5 appears to interact with the CTEs of the terminal subunits Cdc11 and Shs1 in vitro and in vivo ( Figure 1C ) ( Finnigan et al, 2015 ; Booth et al, 2016 ). Recombinant Bni5 can reduce the distance between individual filaments of a paired filament and can also bundle septin filaments, thereby contributing to a higher-order organization ( Figures 1B,C ) ( Patasi et al, 2015 ; Booth et al, 2016 ). It is possible that a combination of phospholipid composition, PTMs, and SAPs is able to bypass the need for positive curvature during the nascent septin ring formation, as there is no discernable bud neck at this point and the membrane topology at the presumptive bud site does not match the curvature preference of the septins.…”

“…Besides the PTMs, different SAPs are also involved in the regulation of septin organization during the cell cycle. For example, Bni5 is associated with the septin hourglass before cytokinesis ( Lee et al, 2002 ; Fang et al, 2010 ) and can bundle septin filaments in vitro ( Patasi et al, 2015 ; Booth et al, 2016 ). The Rho guanine nucleotide-exchange factor (RhoGEF) Bud3 and the anillin-like protein Bud4 can stabilize the double ring structure during cytokinesis ( Wloka et al, 2011 ; Eluere et al, 2012 ; Mcquilken et al, 2017 ).…”

Architecture, remodeling, and functions of the septin cytoskeleton

“…Bni5 is essential for the localization of Myo1, the sole heavy chain of myosin-II in budding yeast, to the bud neck prior to cytokinesis by serving as a linker between the septins and the tail region of Myo1 (Fang et al 2010; Finnigan et al 2015). Bni5 dimerizes and regulates the spacing and organization of paired septin filaments by directly interacting with the terminal subunit Cdc11 (Patasi et al 2015; Booth et al 2016). Thus, Bni5 is thought to play a key role in stabilizing the septin hourglass.…”