In immunosuppressed patients the protozoan parasite Toxoplasma gondii can cause severe tissue destruction by rapid invasion, multiplication, and lysis of host cells, notably those of the central nervous system. In order to invade and manipulate the host cell to its needs the parasite secretes the contents of three specialized organelles - micronemes, rhoptries and dense granules. Despite the central role of these organelles in parasite survival, the mechanisms and pathways by which they are formed are poorly understood. We have recently found that, as in mammalian cells, T. gondii has a highly versatile and discriminatory mechanism for targeting transmembrane proteins to diverse intracellular locations based on the recognition of tyrosine-containing amino acid motifs in their cytoplasmic tails. In one case, the evidence suggested that a rhoptry protein, ROP2, requires such a motif for targeting from a putative pre-rhoptry compartment of mature rhoptries, and that adaptor proteins that recognize these motifs are closely involved in rhoptry biogenesis. We propose to further elucidate the protein requirements for rhoptry targeting by preparing and analyzing a series of mutants of ROP2 and other rhoptry proteins and by characterizing their oligomerization. In addition, we propose to use ROP2 mutants as markers to thoroughly characterize the pre-rhoptry compartment by electron microscopy, subcellular fractionation, and in vitro trafficking assays, which would considerably advance our understanding of the rhoptry biogenesis pathway and mechanisms These goals add substantially to the three specific aims of the parent grant, which are to: I.) Characterize the molecular interactions between rhoptry proteins in the PVM and host cell proteins/organelles, Identify the unique features of protein targeting to T. gondii dense granules, and III.) Elucidate the novel features controlling dense granule secretion. By elucidating the mechanisms of rhoptry biogenesis and targeting, a goal which is analogous to specific aim II in the parent grant for dense granules, the proposed studies provide a critical window into the link between rhoptry protein targeting and rhoptry protein function.
