Mechanism of interaction of Dictyostelium severin with actin filaments.

Abstract

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+-dependent manner, was purified 500-fold to > 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982). Severin has a Stokes radius of 29 .ANG. and consists of a single polypeptide chain. It contains 1 methionyl and 5 cysteinyl residues. The action of severin on actin filaments was studied by EM, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluorescence-labeled severin shows that this protein changes in conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 [half-life] .simeq. 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 .simeq. 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin:severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.