Acetylation of fission yeast tropomyosin does not promote differential association with cognate formins

It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro, we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although our in vitro findings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

Automated summary

It was proposed that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations based on the presence or absence of amino-terminal acetylation, which determines its binding to formin-mediated F-actin networks. However, there was no supporting biochemistry. In vitro experiments using recombinant S. pombe proteins showed that acetyl-Tpm tightly binds to actin, while unacetylated Tpm binds weakly. Contrary to the differential recruitment model, Tpm did not show preferential binding to filaments assembled by the FH1-FH2 domains of two S. pombe formins.