Abstract
Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction.
Highlights
X-ray crystallography (4 –10) shows that G-actin has four subdomains, and it binds nucleotide, ATP or ADP, and divalent cation in the cleft between subdomains 2 and 4
Molecular dynamics modeling of muscle actin predicted an ionic interaction between Arg-39 of one monomer and a Glu at residue 275 of a neighboring monomer, which is not predicted by the Holmes model
Contrary to the hypothesis originating from the T. gondii study, the D275R mutation had no effect on polymerization kinetics (Fig. 3) indicating that an ionic interaction involving residue 275 is not essential for filament stability
Summary
DNase I (grade D) was purchased from Worthington Biochemicals. The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA), and the DNA primers used for site-directed mutagenesis were obtained from Integrated DNA Technologies (Coralville, IA). Rhodamine-phalloidin, FM 4 – 64, and 4Ј,6-diamidino-2-phenylindole were purchased from Molecular Probes-Invitrogen. 1,N6-Ethenoadenosine 5Ј-triphosphate (⑀-ATP) was purchased from Invitrogen. Yeast cakes for wild-type (WT) yeast actin controls were pur-. 3 The abbreviations used are: ⑀-ATP, 1,N6-ethenoadenosine 5Ј-triphosphate; GFP, green fluorescent protein; WT, wild type; EM, electron microscopy Personal communication. 3 The abbreviations used are: ⑀-ATP, 1,N6-ethenoadenosine 5Ј-triphosphate; GFP, green fluorescent protein; WT, wild type; EM, electron microscopy
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