MOLECULAR-CLONING, OVER-EXPRESSION, DEVELOPMENTAL REGULATION AND IMMUNOLOCALIZATION OF FRAGMINP, A GELSOLIN-RELATED ACTIN-BINDING PROTEIN FROM PHYSARUM-POLYCEPHALUM PLASMODIA

FragminP is a Ca2+-dependent actin-binding and microfilament regulator y protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA c lone of 1,104 bp encoding 368 amino acids of fragminP, revealing two c onsensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the c entral part of the protein. The first methionine is modified by an ace tyl group, and three amino acids were missing from the protein coded f or by the cDNA clone. Full-length recombinant fragminP was generated b y PCR, purified after over-expression from Escherichia coli and displa yed identical properties to native Physarum fragminP. Northern blot an alysis against RNA, isolated from cultures at various stages of develo pment, indicated that fragminP is absent from amoebae and that express ion is initiated at an early stage during apogamic development, in a s imilar way to that observed for the profilin genes. In situ immunoloca lization of fragminP in Physarum microplasmodia revealed that the prot ein is localized predominantly at the plasma membrane, suggesting a ro le in the regulation of the subcortical actin meshwork. Our data indic ate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polyce phalum uses a different fragmin isoform that performs different functi ons.