A Conserved Mechanism for Bni1- and mDia1-induced Actin Assembly and Dual Regulation of Bni1 by Bud6 and Profilin

Abstract

Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

“…Bud6p cooperates with Bni1p in promoting actin assembly in vivo Sagot et al, 2002a) and in vitro (Moseley et al, 2004) and associates with secretory vesicles (Jin and Amberg, 2000), and in the absence of Bud6p, cables are very sparse (Amberg et al, 1997). Spa2p is required for the proper localization of Bni1p to the bud tip (Fujiwara et al, 1998), and in its absence, cables are specifically absent from the bud, similar to bni1⌬ cells (our unpublished data).…”

“…In vitro, the FH2 domain nucleates actin filaments from monomers Sagot et al, 2002b;Kovar et al, 2003;Li and Higgs, 2003;Harris et al, 2004;Kobielak et al, 2004), but distinct from other nucleators, it remains associated with the growing barbed end to promote elongation, even in the presence of barbed-end capping proteins Kovar et al, 2003;Li and Higgs, 2003;Pring et al, 2003;Harris et al, 2004;Kobielak et al, 2004;Moseley et al, 2004). The FH1 region is a proline-rich stretch that recruits profilin, an actin-monomer binding protein essential for formin function in vivo , to participate in FH2-mediated nucleation in vitro (Sagot et al, 2002b;Kovar et al, 2003;Pring et al, 2003).…”

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

“…The two homologues to ScBnr1p differ much more in size and, interestingly, these size differences are located exclusively within the FH1 domains leading to differences in the number of short polyproline stretches ( Figure 2B). It was shown that the FH1 domain of formins can mediate interactions with profilin, an abundant actin-binding protein, thereby sequestering actin monomers Sagot et al, 2002b;Moseley et al, 2004). The FH2 domain provides the activity for nucleation of actin cables Sagot et al, 2002a;Kovar et al, 2003;Harris et al, 2004;Moseley et al, 2004).…”

“…It was shown that the FH1 domain of formins can mediate interactions with profilin, an abundant actin-binding protein, thereby sequestering actin monomers Sagot et al, 2002b;Moseley et al, 2004). The FH2 domain provides the activity for nucleation of actin cables Sagot et al, 2002a;Kovar et al, 2003;Harris et al, 2004;Moseley et al, 2004). This domain is the most conserved and best characterized domain within formins and was used as a basis for the phylogenetic classification of over a hundred formins (Higgs and Peterson, 2005).…”

Regulation of the Formin for3p by cdc42p and bud6p

“…These proteins are necessary for the formation of numerous actin structures, including stress fibers, filopodia, cytokinetic actin rings, junctional actin structures, or actin cables. The proper regulation of formins is likely to be critical for cellular processes such as cell migration, cytokinesis, cell adhesion, and cell polarity.A well characterized biochemical activity of formins is to nucleate and elongate linear actin filaments Sagot et al, 2002b;Kovar et al, 2003;Li and Higgs, 2003;Moseley et al, 2004). This activity occurs through the formin-homology (FH) 2 domain, which dimerizes to form a doughnut-shaped structure containing multiple actin-binding sites in its core Otomo et al, 2005b).…”