Isolated THATCH domain of End4 is unable to bind F-actin independently in the fission yeast Schizosaccharomyces pombe

Clathrin mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe critically depends on the connection between the lipid membrane and F-actin. The fission yeast endocytic protein End4 (homologous to Sla2 in budding yeast and HIP1R in human) contains a N-terminal domain that binds to PIP2 on the membrane, and a C-terminal THATCH domain that is postulated to be a binding partner of F-actin in vivo. Purified THATCH domain of the budding yeast Sla2, however, shows low affinity to F-actin in vitro. We tested if isolated THATCH domain still has low affinity to F-actin in vivo, using TEV protease (TEVp)-mediated protein cleaving to separate the THATCH domain from the rest of End4. Our results indicate that the isolated THATCH domain of End4 is unable to bind F-actin independently in vivo, consistent with the low affinity of the THATCH domain to F-actin measured from in vitro binding assays.