Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein

Abstract

Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (ARO) and myosin F (MyoF). Here, we show that two ARO partners, AROinteracting protein (AIP) and adenylate cyclase β (ACβ) localize to a rhoptry subcompartment. In absence of AIP, ACβ disappears from the rhoptries. By assessing the contribution of each ARO armadillo (ARM) repeat, we provide evidence that ARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that ARO resembles the myosin-binding domain of the Caenorhabditis elegans myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative MyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Finally, Plasmodium falciparum ARO functionally complements ARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa.

A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence

“…It is possible that TgCA_RP interacts in a similar way with an unknown binding partner in the nascent rhoptry membranes, promoting adhesion and fusion of the nascent rhoptry vesicles to form the mature organelle. The region containing the annotated CA domain of TgCA_RP was previously shown to be exposed to the cytosol, which would be consistent with this hypothesis (30). In this hypothetical scenario, the absence of TgCA_RP would cause a delay in the assembly of nascent rhoptry vesicles, which would become disorganized.…”

“…2C, red). Localization to rhoptries of TgCA_RP was previously done by expressing an extra copy of the gene with a c-Myc tag (30). To provide fine details of TgCA_RP localization, we performed cryo-immunoelectron microscopy of TgCA_RP-HA extracellular tachyzoites.…”

Dynamic proteinS-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence

“…Three proteins were identified and confirmed as TgARO binding partners: myosin F, adenylate cyclase β, and a conserved hypothetical protein, armadillo interacting protein (Figure 2C) (Mueller et al , 2013, Mueller et al , 2016). Tethering of the rhoptries via TgARO is an actomyosin-dependent process since treatment of cells with either an actin polymerization inhibitor or a myosin ATPase inhibitor resulted in dispersion of rhoptries, with no effect on the micronemes, mimicking the TgAROcKO phenotype (Mueller et al , 2013).…”

“…and vesicles or the nuclear membrane (Frenal et al , 2013). However, many PATs in P. falciparum , P. berghei , and T. gondii are localized to unique apicomplexan organelles, including the inner membrane complex (IMC) of vesicles underlies the plasma membrane (PfDHHC1, PfDHHC9, PfDHHC3, TgDHHC2, TgDHHC14), which is important for gliding motility, the rhoptries , which are involved in secretion of factors necessary for invasion (PfDHHC7, PbDHHC7, TgDHHC7), and the crystalloid bodies in Plasmodium ookinete an oocyst stages (PbDHHC10) (Beck et al , 2013, Frenal et al , 2013, Wetzel et al , 2015, Hopp et al , 2016, Mueller et al , 2016, Santos et al , 2016, Tay et al , 2016). …”

“…The micronemes, dense granules, and apicoplast were correctly positioned in the TgAROcKO knockdown parasites, and microneme secretion was normal. TgARO contains six armadillo repeats, and all of them are required for tethering the rhoptries, but only ARM6 at the C-terminus is required for clustering the rhoptries (Mueller et al , 2016). …”