Three Distinct F-Actin Binding Sites in the Dictyostelium discoideum 34 000 Dalton Actin Bundling Protein

Abstract

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.

Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton inDictyostelium

“…However, the 34 kDa protein is tightly associated with cortical actin, whereas α-actinin reveals both cortical staining and a significant amount of diffuse cytoplasmic localization (Brier et al, 1983;Fechheimer, 1987). Further, biochemical studies in vitro show directly that the 34 kDa protein has a higher affinity for actin filaments than does amoeba α-actinin (Fechheimer, 1987;Lim et al, 1999;Wachsstock et al, 1993). Thus, the results can be reconciled and do demonstrate a requirement for calcium-regulation of the actin-crosslinking in vivo.…”

The C Terminus of Annexin II Mediates Binding to F-actin

“…For example, the F-actin-binding site of vinculin is 123 amino acid residues in length (35). In addition, the three identified F-actin-binding sites within the D. discoideum 34-kDa F-actin-bundling protein vary in length from 16 to 123 amino acid residues in length, although the 16-amino acid residue domain has only weak binding to F-actin (34).…”

Requirements for Hirano Body Formation

“…Full-length and truncated forms of the 34-kDa protein have been previously shown to inhibit F-actin depolymerization in a concentration-dependent manner (23,37). ) ϫ 100.…”

“…The 34-kDa actin-binding protein is one of many actin cross-linking proteins found in Dictyostelium (3,19) and has been biochemically characterized as a calcium-sensitive actinbundling protein (20)(21)(22). Molecular dissection of recombinant forms of the 34-kDa protein has revealed three actin-binding sites (residues 1 to 123, 193 to 254, and 279 to 295), as well as an N-terminal inhibitory domain (residues 1 to 76) that is proposed to regulate 34-kDa protein binding to F-actin (23,24). A purified C-terminal fragment (CT) (residues 124 to 295) was characterized in vitro and found to possess a calcium-insensitive enhanced F-actin affinity (23,24).…”

“…Molecular dissection of recombinant forms of the 34-kDa protein has revealed three actin-binding sites (residues 1 to 123, 193 to 254, and 279 to 295), as well as an N-terminal inhibitory domain (residues 1 to 76) that is proposed to regulate 34-kDa protein binding to F-actin (23,24). A purified C-terminal fragment (CT) (residues 124 to 295) was characterized in vitro and found to possess a calcium-insensitive enhanced F-actin affinity (23,24). Therefore, it has been proposed that CTinduced model Hirano body formation may be the result of increased F-actin binding and loss of calcium regulation (16).…”