| Formins stimulate actin assembly through a novel mechanism by remaining processively associated with the elongating barbed end and driving the rapid addition of profilin-actin. Profilin is an essential protein that stimulates rapid formin-mediated actin elongation by simultaneously binding both to the proline-rich formin FH1 domain and actin monomers. We found that formins variously utilize different profilin isoforms. The molecular basis for this isoform specificity does not stem solely from differences in the FH1 domain, suggesting that profilin's interaction with actin and/or the formin FH2 domain might also be critical. We hypothesize that formin utilization of profilin-actin explains why profilin is required by fission yeast. To test this hypothesis, we mutated a budding yeast profilin (ScPFY) to complement the fission yeast profilin-ts cdc3-124. We identified a ScPFY mutant that complements the fission yeast profilin-ts strain and is utilized better by Cdc12 than wild-type ScPFY in vitro. The mutations lie in the actin-binding region of profilin, which also interacts with the formin FH2 region, suggesting a mechanism of specificity where the formin interacts with the profilin at the barbed end of the actin filament and this interaction helps release the profilin and allow subsequent cycles of filament elongation. We also hypothesize that in addition to activation at the right time and place, the specific actin assembly properties tailor formin isoforms for a particular cellular role. We are investigating the physiological importance of actin assembly properties in fission yeast, which expresses three formins with unique cellular roles: Cdc12 (cytokinesis), For3 (polarization) and Fus1 (mating). We constructed fission yeast strains that exclusively express cytokinesis formin Cdc12 chimeras, in which its actin assembly domains were replaced with the actin assembly domains from biochemically diverse formins. Quantitative time-lapse imaging reveals significant delays in contractile ring assembly and maturation. |
