AP-1 in Toxoplasma gondii Mediates Biogenesis of the Rhoptry Secretory Organelle from a Post-Golgi Compartment

Abstract

We have previously demonstrated that Toxoplasma gondii has a tyrosine-based sorting system, which mediates protein targeting to the lysosome-like rhoptry secretory organelle. We now show that rhoptry protein targeting is also dependent on a dileucine motif and occurs from a post-Golgi endocytic organelle to mature rhoptries in an adaptin-dependent fashion. The T. gondii AP-1 adaptin complex is implicated in this transport because the 1 chain of T. gondii AP-1 (a) was localized to multivesicular endosomes and the limiting and luminal membranes of the rhoptries; (b) bound to endocytic tyrosine motifs in rhoptry proteins, but not in proteins from dense granule secretory organelles; (c) when mutated in predicted tyrosine-binding motifs, led to accumulation of the rhoptry protein ROP2 in a post-Golgi multivesicular compartment; and (d) when depleted via antisense mRNA, resulted in accumulation of multivesicular endosomes and immature rhoptries. These are the first results to implicate AP-1 in transport from a post-Golgi compartment to a mature secretory organelle and substantially expand the role for AP-1 in anterograde protein transport.Obligate intracellular protozoa of the phylum Apicomplexa are highly sophisticated secretory cells. Although the host cell type and the nature of the intracellular vacuole differ when Plasmodium, Toxoplasma, Cryptosporidium, Eimeria, Theileria, and Babesia are compared, these organisms share common secretory organelles: micronemes, rhoptries, and dense granules. Differential secretion from these organelles directs invasion of host cells, formation of an intracellular parasitophorous vacuole, and subsequent modification of the vacuole for replication (reviewed in Ref. 1).Most unique morphologically and biochemically are the rhoptries. These are flask-shaped secretory organelles that are assembled and positioned at the apical pole of the invasive forms of Apicomplexa (reviewed in Refs. 2-4). At the onset of host cell invasion, prepackaged proteins and membranous materials from rhoptries are secreted through an apical opening. Rhoptry constituents are likely involved in a plethora of essential functions. These include directing host cell attachment and invasion (3), expansion and maintenance of the parasitophorous vacuole membrane surrounding the intracellular parasite (5), and attachment of host mitochondria and the endoplasmic reticulum to the parasitophorous vacuole membrane (6). Furthermore, rhoptry proteins are targets of the normal immune response; antibodies to rhoptry proteins block invasion; and immunization with selected rhoptry proteins provides partial protection against parasite challenge (2). Hence, the unique rhoptry organelle can serve as a potential target for blocking parasite invasion and growth. The biogenesis and phylogenetic relationship of rhoptries to secretory organelles in other cells are not well understood. Rhoptries and pre-rhoptries are described as the only acidified organelles in the parasite (7) and are packaged with specialized hydrolases (8) and choles...

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 inToxoplasma gondii

“…Moreover, the expression of an abundant, soluble and secreted NTPase protein was not affected in these parasites. Taken together, these data confirm the specificity of the antisense approach in abrogating gene expression and is consistent with our previous observations (Nakaar et al, 1999;Nakaar et al, 2000;Ngô et al, 2003).…”

“…Dominant negative interference with the tyrosine-binding pocket of µ1 adaptin abolished its binding to transmembrane rhoptry proteins ROP2 and ROP4, and disrupted the steady state formation of mature rhoptries (Ngô, et al, 2003). Ablation of µ1 adaptin expression by antisense RNA altered rhoptry biogenesis and was detrimental to parasite survival.…”

“…Antisense RNA-mediated depletion of either ROP2 transmembrane cargo (this study) or µ1 adaptin (Ngô et al, 2003) disrupts normal rhoptry formation and consequently causes defects in cell division and growth. In contrast to parasites expressing ROP2 antisense RNA, the TGN and endosomal vacuoles are drastically distorted in AP-1-depleted parasites, but the rhoptries maintain their club shape.…”

Transmembrane Domain Modulates Sorting of Membrane Proteins in Toxoplasma gondii

“…Clearly, mutation of the GRA4 cytoplasmic tail within the context of the various constructs used herein is a logical future direction of study. Such experiments are likely to shed light on the molecular mechanism in which TMD-mediated sorting of GRA4 (demonstrated in this study) is influenced by coexisting conventional sorting signals proposed in previous studies (32,43).…”

“…We have demonstrated previously that selected YXXØ motifs are recognized by the 1 chain of the T. gondii AP-1 adaptor complex (42,43) and that mutation of these motifs alters targeting of the rhoptry proteins ROP2 and ROP4 (42). Although secretion of GRA4 is impaired by mutation of the YXXØ motif (32), the GRA4 cytoplasmic tail is not recognized by the T. gondii 1 chain (43) suggesting that another adaptor may be involved. Clearly, mutation of the GRA4 cytoplasmic tail within the context of the various constructs used herein is a logical future direction of study.…”