PRIMARY STRUCTURE OF AND STUDIES ON ACANTHAMOEBA-ACTOPHORIN

We determined the amino acid sequence of the actin monomer binding/act in filament severing protein actophorin from Acanthamoeba castellanii by automated Edman degradation of peptide fragments and by sequencing of full-length cDNA. Actophorin consists of 138 amino acids (calculate d molecular weight of 15 543) and shares a high degree of sequence sim ilarity to other low molecular weight actin monomer sequestering prote ins, especially vertebrate cofilin, vertebrate actin depolymerizing fa ctor/destrin, and echinoderm depactin. Actophorin is smaller and does not contain a nuclear localization sequence like the related vertebrat e-proteins. Southern blot analysis indicates that actophorin is a sing le-copy gene; however, Northern blots show two distinct mRNA species o f 1 and 0.9 kb in size. Homogeneous recombinant actophorin purified fr om Escherichia coli is indistinguishable from the native protein in it s physical properties and in biochemical assays of its interaction wit h actin, but is less reactive with three monoclonal antibodies raised against the native protein. The NH2 terminus of native actophorin is b locked, while the initiating methionine residue is removed from recomb inant actophorin. This difference has no measurable effect on activity . By fluorescent antibody staining of Acanthamoeba, actophorin colocal izes with actin filaments in the cortical cytoplasm, especially at the leading edge of the cell. Additionally, actophorin binds phosphatidyl inositol 4',5'-bisphosphate. The recombinant actophorin forms X-ray di ffraction quality crystals of superior quality in poly(ethylene glycol )/2-propanol and, like the native crystal form, belongs to space group P2(1)2(1)2(1).