Abstract
Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the related actin depolymerizing factor (ADF) and cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin-binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the ADF/cofilin (AC) family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer-sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.
Highlights
Oocysts that are shed by cats and that can contaminate water [2]
Jasplakinolide-treated parasites move with 3-fold increased speed [6], the prevention of filament turnover results in the inability of parasites to pursue directional movement and invade host cells [6]
actin depolymerizing factor (ADF) Sequesters Monomers in T. gondii high G-actin concentrations in apicomplexan parasites, it is surprising that no dedicated G-actin sequestering proteins are present, such as -thymosin, which is found in higher eukaryotes [20]
Summary
TgACT, T. gondii actin; ADF, actin depolymerizing factor; AC, ADF and cofilin; TgADF, T. gondii actin depolymerizing factor; TIRF, total internal reflection fluorescence; CAP, cyclase-associated protein; SpCOF, S. pombe cofilin; ADF-t, TgADF containing the S. pombe cofilin C terminus. Conserved residues are found at the N terminus, which includes the putative phosphorylation site serine 2 or 3 in eukaryotes or serine 6 in plants [25], in the long ␣3 helix, and in the turn connecting strand 6 and ␣4 These sites cluster together in the three-dimensional structure [35,36,37] and constitute a general actin-binding surface. Deletion of a charged residue in the C-tail of the Caenorhabditis elegans AC homologue, Unc60B, results in a loss of severing activity and increased depolymerization activity [32, 39] These studies highlight how a reduction in the affinity for the filament can uncouple the various activities of AC proteins, and identify specific molecular features that may influence function in other AC proteins. Because other apicomplexan ADFs share the same molecular features, it is likely that the properties observed here are conserved in the phylum
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