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.
Ren Y, Berro J