| Formins are cellular machines found in all eukaryotes that assemble unbranched actin filaments essential for fundamental processes such as cell division, shape, and movement. All formins contain two actin assembly domains (FH1 and FH2) that utilize profilin-actin to nucleate and processively elongate actin filaments. Formin isoforms differ dramatically in rates of actin assembly, raising the possibility that the FH1FH2 domains as well as flanking regulatory domains specialize formin isoforms for their cellular roles. Fission yeast contains three formin isoforms that assemble distinct actin structures in the cell, the contractile ring (Cdc12), polar actin cables (For3), and mating fusion tip (Fus1). Using both genetic and biochemical approaches, we demonstrated that the FH1FH2 domains of the three fission yeast formins have unique actin assembly properties that are tailored for their cellular function. In fission yeast where the FH1FH2 region of the cdc12 gene was replaced with FH1FH2 regions from Fus1 and For3, we found that contractile ring assembly was severely disrupted. In bulk actin assembly and single molecule TIRF assays, we discovered that the three fission yeast formins have remarkably distinct biochemical activities. Cdc12 and Fus1 are 100-fold more efficient nucleators than For3. Cdc12 and For3 are 10-fold more processive and 2-fold faster elongators than Fus1, however Fus1 uniquely bundles actin filaments. We also found that both the FH1 and FH2 domains contribute to overall formin elongation rates of profilin-actin. Using both sequence alignment and biochemical techniques, we discovered that fission yeast formins contain diaphanous-related N- and C-terminal organizations with divergent regulatory motifs. We found that fission yeast formin N- and C-termini do not exhibit autoinhibition in vitro and proteins such as the polarity factor Bud6 and the side-binding protein tropomyosin impact formin-mediated actin assembly by different mechanisms. |
