A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Abstract

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.

Myosin genes in Tetrahymena

“…In the apicomplexa, a group of parasitic protists that includes Babesia, Gregarina, Plasmodium, and Toxoplasma, 17 myosins have been identified; all but one cluster in class XIV, and all have been identified only in apicomplexa [Chaparro-Olaya et al, 2005;Heintzelman and Schwartzman, 1997;Heintzelman, 2004;Hettmann et al, 2000;Lew et al, 2002]. In the ciliophora, Whether the myosins in Tetrahymena are limited to this organism will have to await further identification of myosins in other protists.…”

Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion

“…Plasmids pT8mycTgROM1, pT8mycTgROM2 and pT8mycTgROM4 and pT8mycTgROM5 were all constructed by cloning the corresponding cDNA between the NsiI and the PacI sites in T8MycGFP-HX (Hettmann et al, 2000). Total RNA was isolated from freshly released parasites using TRIzol (Invitrogen).…”

The Induction and Kinetics of Antigen-Specific CD8 T Cells Are Defined by the Stage Specificity and Compartmentalization of the Antigen in Murine Toxoplasmosis

“…All manipulations were conducted at room temperature as described previously (23). In brief, after fixation with 4% paraformaldehye/0.05% glutaraldehyde in PBS, neutralization with 0.1 M glycine in PBS, permeabilization with 0.2% Triton X-100 in PBS, and blocking with 2% BSA/0.2% Triton X-100 in PBS, glass coverslips attached with the tachyzoite-infected HFF cells were incubated with rabbit polyclonal Abs directed against E. coli ␤-gal (BioTrent, Cologne, Germany) and/or a mouse mAb against T. gondii BAG1, a bradyzoite-specific Ag, (24) (kindly provided by Dr. W. Bohne, Universität Göttingen, Goettingen, Germany), followed by FITCor Alexa Fluor 594 reactive dye-conjugated goat anti-mouse or goat antirabbit Ab, respectively (Molecular Probes, Leiden, The Netherlands).…”