Plasmodium actin is incompletely folded by heterologous protein‐folding machinery and likely requires the native Plasmodium chaperonin complex to enter a mature functional state

Abstract

Actin filament turnover underpins several processes in the life cycle of the malaria parasite, Plasmodium falciparum. Polymerization and depolymerization are especially important for gliding motility, a substrate-dependent form of cell movement that underpins the protozoan parasite’s ability to disseminate and invade host cells. To date, given difficulties in extraction of native actins directly from parasites, much of our biochemical understanding of malarial actin has instead relied on recombinant protein extracted and purified from heterologous protein expression systems. Here, using in vitro transcription-translation methodologies and quantitative protein-binding assays, we explored the folding state of heterologously expressed P. falciparum actin 1 (PfACTI) with the aim of assessing the reliability of current recombinant-protein-based data. We demonstrate that PfACTI, when expressed in non-native systems, is capable of binding to and release from bacterial, yeast, and mammalian chaperonin complexes but appears to be incompletely folded. Characterization of the native Plasmodium folding machinery in silico, the chaperonin containing t-complex protein-1 complex, highlights key divergences between the different chaperonin systems that likely underpins this incomplete folded state. These results highlight the importance of characterizing actin’s folded state and raise concerns about the interpretation of actin polymerization kinetics based solely on protein derived from heterologous expression systems.—Olshina, M. A., Baumann, H., Willison, K. R., Baum, J. Plasmodium actin is incompletely folded by heterologous protein-folding machinery and likely requires the native Plasmodium chaperonin complex to enter a mature functional state.

Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

“…Since apicomplexan actin cannot be functionally heterologously expressed (Olshina et al, 2016), we used skeletal chicken actin to estimate the binding characteristics of Cb to F-actin. Recombinant Cb with a C-terminal 6-His tag (rCb) was expressed and purified from bacteria (Figure 5A,B).…”

“…Since then, polymerization kinetics of heterologously expressed apicomplexan actin showed that it polymerized in an unusual isodesmic process, in contrast to conventional eukaryotic actin (Skillman et al, 2013). These findings have been recently questioned, as heterologously expressed apicomplexan actin was shown to be misfolded (Olshina et al, 2016). In parallel, a number of recent genetic studies have demonstrated important functions of parasite actin during intracellular development, including maintenance of the apicoplast (Andenmatten et al, 2013; Egarter et al, 2014), daughter cell replication (Haase et al, 2015) and motility of secretory organelles (Heaslip et al, 2016).…”

“…We speculate that this network consists of either short F-actin bundles that are cross-linked via unknown actin-binding proteins (based on the formation of short actin filaments in vitro) (Skillman et al, 2011) or that the presence of actin binding proteins such as formin, profilin, and coronin may coordinate their activities in vivo to produce longer actin filaments than those formed in vitro (Skillman et al, 2011; Olshina et al, 2016;Salamun et al, 2014). While this study focuses on the characterization of the membranous network within the PV, it is worth nothing that highly dynamic actin filaments have been also detected within the cytosol of the parasite, and we show that these dynamics are almost completely abolished upon depletion of ADF in good agreement with previous findings (Haase et al, 2015; Mehta and Sibley, 2011) (Figure 6; Videos 5–7).…”

“…Puzzlingly, formins and other actin nucleating proteins have been shown to have essential roles in Toxoplasma and Plasmodium, begging the question of their function if they are not required to initiate actin polymerisation or accelerate filament elongation (Baum et al, 2008; Jacot et al, 2013). A recent study suggested that the polymerization process of apicomplexan actin needs to be reinvestigated, as heterologously expressed apicomplexan actin, the basis for many of the previous studies, is incorrectly folded (Olshina et al, 2016). Furthermore, it was demonstrated that conditional deletion of act1 in T. gondii results in complete abrogation of known actin functions, long before G-actin levels are fully depleted, suggesting that in vivo the formation of F-actin depends on a critical monomer concentration (Whitelaw et al, 2017).…”

Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion

“…Previous polymerisation studies used G-actin produced in heterologous protein expression systems. However, a recent study demonstrated that apicomplexan actin is not properly folded when heterologously expressed due to differences in the chaperonin T-complex [50]. Furthermore, a recent study suggests that Toxoplasma actin is capable of forming F-actin structures in vivo that depend on a critical monomer concentration and demonstrates that F-actin is involved in multiple essential processes during intracellular parasite growth such as daughter cell assembly, vacuole organisation and parasite egress [28].…”