Physarum amoebae express a distinct fragmin-like actin-binding protein that controls in vitro phosphorylation of actin by the actin-fragmin kinase

Amoebae and plasmodia constitute the two vegetative growth phases of the My xomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmod ia is tightly controlled by fragmin P, a plasmodium specific actin-binding protein that enables actin phosphorylation by the actin-fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spi te of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin-fragmin kinase to cell-free extrac ts of amoebae, suggesting that amoebae contain a protein with properties si milar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes pho sphorylated actin, we demonstrate that Thr203 in actin can be phosphorylate d in this complex. A full-length amoebal fragmin cDNA was cloned and the de duced amino acid sequence shows 65% identity with plasmodial fragmin. Howev er, the fragmins are encoded by different genes. Northern blots using RNA f rom a developing Physarum strain demonstrate that this fragmin isoform (fra gmin A) is not expressed in plasmodia. In situ localization showed that fra gmin A is present mainly underneath the plasma membrane. Our results indica te that Physarum amoebae express a fragmin P-like isoform which shares the property of binding actin and converting the latter into a substrate for th e actin-fragmin kinase.