Identification of functional regions on the tail of Acanthamoeba myosin-II using recombinant fusion proteins. II. Assembly properties of tails with NH2- and COOH-terminal deletions

Abstract

We used purified fusion proteins containing parts of the Acanthamoeba myosin-II tail to localize those regions of the tail responsible for each of the three steps in the successive dimerization mechanism (Sinard, J. H., W. F. Stafford, and T. D. Pollard. 1989. J. Cell Biol. 107:1537-1547) for Acanthamoeba myosin-II minifiliment assembly. Fusion proteins containing the terminal ∼90% of the myosin-II tail assemble normally, but deletions within the last 100 amino acids of the tail sequence alter or prevent assembly. The first step in minifilament assembly, formation of antiparallel dimers, requires the COOH-terminal ∼30 amino acids that are thought to form a nonhelical domain at the end of the coiled-coil. The second step, formation of antiparallel tetramers, requires the last ∼40 residues in the coiled-coil. The final step, the association of two antiparallel tetramers to form the completed octameric minifilament, requires residues ∼40-70 from the end of the coiled-coil. A region of the tail near the junction with the heads is important for tight packing of the tails in the minifilaments. Divalent cations induce the lateral aggregation of minifilaments formed from native myosin-II or fusion proteins containing a nonmyosin "head," but under the same conditions fusion proteins composed essentially only of myosin tail sequences with very little nonmyosin sequences form paracrystals. The region of the tail necessary for this paracrystal formation lies NH2-terminal to amino acid residue 1,468 in the native myosin-II sequence.