Bil2 Is a Novel Inhibitor of the Yeast Formin Bnr1 Required for Proper Actin Cable Organization and Polarized Secretion.

Thomas J Rands,Bruce L Goode

doi:10.3389/fcell.2021.634587

Thomas J Rands, Bruce L Goode

Open Access

https://doi.org/10.3389/fcell.2021.634587

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Abstract

Cell growth in budding yeast depends on rapid and on-going assembly and turnover of polarized actin cables, which direct intracellular transport of post-Golgi vesicles to the bud tip. Saccharomyces cerevisiae actin cables are polymerized by two formins, Bni1 and Bnr1. Bni1 assembles cables in the bud, while Bnr1 is anchored to the bud neck and assembles cables that specifically extend filling the mother cell. Here, we report a formin regulatory role for YGL015c, a previously uncharacterized open reading frame, which we have named Bud6 Interacting Ligand 2 (BIL2). bil2Δ cells display defects in actin cable architecture and partially-impaired secretory vesicle transport. Bil2 inhibits Bnr1-mediated actin filament nucleation in vitro, yet has no effect on the rate of Bnr1-mediated filament elongation. This activity profile for Bil2 resembles that of another yeast formin regulator, the F-BAR protein Hof1, and we find that bil2Δ with hof1Δ are synthetic lethal. Unlike Hof1, which localizes exclusively to the bud neck, GFP-Bil2 localizes to the cytosol, secretory vesicles, and sites of polarized cell growth. Further, we provide evidence that Hof1 and Bil2 inhibitory effects on Bnr1 are overcome by distinct mechanisms. Together, our results suggest that Bil2 and Hof1 perform distinct yet genetically complementary roles in inhibiting the actin nucleation activity of Bnr1 to control actin cable assembly and polarized secretion.

Highlights

Formins are a conserved family of actin assembly-promoting proteins that perform essential roles in numerous actin-based cellular and physiological processes (Kovar, 2006; Chhabra and Higgs, 2007; Chesarone et al, 2010)
Our data show that bil2 cells have a lower coefficient of variation (CoV) compared to wildtype cells, which agrees with our SOAX analysis, and together these results indicate that Bil2 is required for the formation of properly organized actin cable networks
A previous proteomic screen had identified this gene as being required for normal delivery of a marker protein to the cell surface via the secretory pathway, revealing that in bil2 cells the surface protein aberrantly accumulated in the trans-Golgi compartment (Proszynski et al, 2005)

Summary

Introduction

Formins are a conserved family of actin assembly-promoting proteins that perform essential roles in numerous actin-based cellular and physiological processes (Kovar, 2006; Chhabra and Higgs, 2007; Chesarone et al, 2010). Formins nucleate the assembly of new actin filaments and accelerate actin filament elongation while protecting growing barbed ends from capping proteins (Goode and Eck, 2007; Breitsprecher and Goode, 2013). Studies in yeast have led to important advances in our understanding of formin activities, mechanism, and regulation (Breitsprecher and Goode, 2013). Mammalian genomes encode 15 different formins (Higgs and Peterson, 2005), which are used to assemble a wide variety of cellular actin structures, including filopodia, stress fibers, stereocilia, and lamellipodia (Faix and Grosse, 2006). The relative simplicity of the yeast system, combined with its genetic tractability, have made it a powerful model for elucidating formin regulatory mechanisms

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