Toxoplasma gondii Actin Depolymerizing Factor Acts Primarily to Sequester G-actin

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 steadystate 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.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1

“…Despite these differences in structure and apparent low binding affinity for filaments as compared with other AC proteins, PfADF1 is capable of mediating filament disassembly via severing. This result is in line with observations from its homolog in T. gondii, TgADF1 (28). Importantly, PfADF1 is capable of disassembling PfAct1 filaments, which are markedly divergent from those of other eukaryotes (3).…”

“…However, we clearly demonstrate that PfADF1 is capable of F-actin severing. This observation is strongly supported by recent biochemical data from the malaria-related parasite T. gondii, in which the direct ortholog of PfADF1, TgADF, also mediated actin filament disassembly and severing (28). The ability to mediate disassembly independent of the implicated filament-binding motifs (19,21) is inconsistent with expectations.…”

“…In the recent characterization of TgADF1 (also assumed to lack the anchored fold), the addition of the C-terminal α4 helix of SpCOF reduced the ability of this AC protein to mediate filament disassembly with a reported, although not shown, increase in Factin binding potential (28). We predict that addition of an α4 helix structurally altered this protein, making it more akin to ScABP1 in which the previously exposed Lys 68 is drawn into a polar interaction with the C-terminal helix.…”

“…Filament disassembly was assayed by analytical ultracentrifugation at 100,000 × g (TLA100 rotor, Beckman Coulter Optima TL Ultracentrifuge) with rabbit actin (4 μM; Cytoskeleton, Inc.) or PfAct1 (4 μM) essentially as described (28) with varying concentrations of recombinant PfADF1, PfADF2, or control GST added to preformed actin filaments prepared under polymerizing conditions for 1 h in a predominately ADP + P i -rich form 1 h before sedimentation. Quantification of protein in pellet and supernatant fractions was performed by densitometry analysis using a GS-800 calibrated densitometer (Bio-Rad).…”

Un complexe moléculaire unique à l’origine de la motilité et de l’invasion des Apicomplexes

“…Plusieurs protéines liant l'actine et conservées à travers le phylum ont d'ailleurs récemment été décrites, et leur rôle s'est révélé critique pour la motilité et l'invasion du toxoplasme. Les formines TgFRM1 (Toxoplasma formin 1) et TgFRM2 sont impliquées dans la polymérisation des filaments d'actine [16,17], alors que la cofiline TgADF (Toxoplasma acting depolymerizing factor) joue un rôle dans leur dépolymérisation [18,19] et que la profiline TgPRF (Toxoplasma profilin) séquestre les monomères [17,20] (Figure 2B). Normalement, formine et profiline fonctionnent de façon coopérative afin de stimuler la formation du filament d'actine de part et d'autre du domaine FH2 (formin homology 2 domain) de la formine ( Figure 2B).…”