Identification and characterization of a novel protein in Toxoplasma gondii: The myosin A docking protein

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Apicomplexan parasites move by gliding motility, a substrate-dependant process that does not involve extensions such as pseudopodia or lamellipodia. Instead, the parasites move along the substrate, appearing to glide, twist, and twirl. When this research was begun, it was known that actin and myosin were both essential for gliding motility. Additionally, it had been shown that both actin and myosin are localized beneath the plasma membrane of the parasite. However, one would expect a complex of proteins to be involved in the process of gliding motility; we therefore set out to determine what other factors are involved in this process.; To discover what other proteins may be involved in the process of gliding motility, we isolated membrane-associated proteins and generated antisera to them. Screening of a Toxoplasma cDNA expression library with these antisera resulted in the identification of a novel membrane-associated protein, which we have the named the Toxoplasma gondii myosin A docking protein (TgMADP). TgMADP is found exclusively in a large complex that also includes myosin A, the myosin A light chain, and a currently unidentified 50-kD protein. To determine if one of the members of this complex serves as a membrane anchor, we performed labeling experiments with the photoactive, lipid-soluble membrane probe 5-[125I]iodonaphthyl-1-azide (INA). TgMADP is the only member of the myosin A complex to label with this probe and is, therefore, likely responsible for docking the complex to the plasma membrane.; Further experiments to establish the domains of TgMADP required for membrane association revealed that the N-terminal nine amino acids of TgMADP are necessary and sufficient for membrane targeting and association. This N-terminal domain contains putative myristoylation and palmitoylation sites, and metabolic labeling experiments with these lipids revealed that TgMADP is both myristoylated and palmitoylated at its N-terminus. Additionally, myristoylation and palmitoylation are both necessary and sufficient for membrane targeting. Thus, through the identification of the components in the myosin-containing complex, the studies performed in this dissertation have laid out the foundation for understanding the mechanism underlying gliding motility.