Regulation of yeast formin-mediated actin assembly by Bud6 and Hof1

Actin filaments play crucial roles in a variety of cellular processes, including migration, polarized secretion, and cytokinesis. Formins, a class of highly-conserved actin assembly factors, promote the polymerization of filaments from a pool of actin monomers. The mechanisms used by formins to catalyze actin assembly are coming into focus; but the extent to which formins collaborate with additional factors to drive actin assembly is not yet clear. In this thesis, I use a combination of genetics, live cell imaging, actin assembly assays, and in vitro binding experiments to uncover the biochemical mechanisms and cellular roles for the polarity factor Bud6 and F-BAR protein Hof1 in the regulation of formin-mediated actin assembly. Specifically, I show that the cell polarity factor Bud6 stimulates formin-dependent actin nucleation in vitro, defining it as a `nucleation-promoting factor'. This function of Bud6 is required for robust actin assembly in vivo. In collaboration with Michael Eck's lab, the structure of the formin-binding domain of Bud6 is solved using x-ray crystallography and I identify conserved surfaces that mediate interactions between Bud6 and the formin Bni1. The stoichiometry of a formin:Bud6:G-actin complex is determined to be 2:2:4. Bud6 thus stimulates actin assembly by recruiting up to four actin monomers to the formin. I further demonstrate that Bud6 serves as an NPF for both yeast formins (Bni1 and Bnr1) and identify a co-factor (Bil1) which directly binds Bud6, regulating its NPF activity on Bnr1. These results define a new mode of formin regulation which may be broadly conserved in other systems. I further reveal that the F-BAR protein Hof1 has an unanticipated role in the regulation of formin-mediated actin cable assembly during polarized growth. Purified Hof1 directly inhibits Bnr1's activities and cells lacking HOF1 display disorganized actin cables. These results suggest that Hof1 acts alongside Smy1 and Bud14 to regulate activated Bnr1 and underscores the importance of tightly controlling formin activity to produce robust actin cables that efficiently transport materials to sites of polarized growth.