Huân M. Ngô

Golgi biogenesis in Toxoplasma gondii

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.

Targeting to rhoptry organelles of Toxoplasma gondii involves evolutionarily conserved mechanisms.

Intracellular parasites of the phylum Apicomplexa contain specialized rhoptry secretory organelles that have a crucial function in host-cell invasion and establishment of the parasitophorous vacuole. Here we show that localization of the Toxoplasma gondii rhoptry protein ROP2 is dependent on a YEQL sequence in the cytoplasmic tail that binds to µ-chain subunits of T. gondii and mammalian adaptors, and conforms to the YXXφ mammalian sorting motif. Chimaeric reporters, containing the transmembrane domains and cytoplasmic tails of the low-density lipoprotein receptor and of Lamp-1, are sorted to the Golgi or the trans-Golgi network (TGN), and partially to apical microneme organelles of the parasite, respectively. Targeting of these reporters is mediated by YXXφ- and NPXY-type signals. This is the first demonstration of tyrosine-dependent sorting in protozoan parasites, indicating that T. gondii proteins may be targeted to, and involved in biogenesis of, morphologically unique organelles through the use of evolutionarily conserved signals and machinery.

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

We have previously demonstrated thatToxoplasma 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. TheT. gondii AP-1 adaptin complex is implicated in this transport because the μ1 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.

Differential sorting and post-secretory targeting of proteins in parasitic invasion

Toxoplasma gondii uses a highly coordinated arsenal of three structurally and biochemically distinct secretory granules to invade and develop in a wide range of host cells. Proteins of these secretory granules are sorted to strategic subcellular locations using distinctive sorting signals and are then triggered differentially for exocytosis. These secreted proteins are subsequently targeted and inserted into membrane domains.

Are rhoptries in Apicomplexan parasites secretory granules or secretory lysosomal granules

The club‐shaped rhoptries in Apicomplexan parasites are one of the most unusual secretory organelles among the eukaryotes, containing unusual lipid and protein cargo that is specialized for intracellular parasitism. Rhoptries have traditionally been viewed strictly as regulated secretory granules. We discuss in this article recent data on the cargo, function and biogenesis of rhoptries in two parasitic model systems, Toxoplasma and Plasmodium. Current findings suggest that rhoptries receive products from both biosynthetic and endocytic pathways and, therefore, they are most analogous to secretory lysosomal granules found in mammalian cells.

Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii.

Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.

Toxoplasma gondii Rab5 enhances cholesterol acquisition from host cells

The role of endocytosis in nutrient uptake by Toxoplasma gondii is unknown. To explore this issue, we characterized an endosomal compartment by identifying a T. gondii Rab5 homologue, a molecular marker for early endosomes in eukaryotic cells. The deduced amino acid sequence of the T. gondii Rab5 gene encodes a protein of 240 amino acids, which we termed TgRab51. TgRab51 was epitope‐tagged at the N‐terminus, expressed in the parasite, and localized by immunofluorescence and immunoelectron microscopy to tubulovesicular structures anterior to the parasite nucleus and adjacent to, but distinct from the Golgi. By immunofluorescence analysis, TgRab51wt‐HA staining partially overlapped with Golgi/TGN markers, but not with the T. gondii secretory organelles. A dominant positive mutant, TgRab51Q103L‐HA, enhanced uptake of exogenous cholesterol analogues in intracellular parasites, augmented formation of lipid droplets and accelerated parasite growth. Brefeldin A disrupted the TgRab51 compartment, and altered the distribution of fluorescent exogenous cholesterol in cells expressing TgRab51Q103L‐HA. These results suggest that TgRab51 facilitates sterol uptake, possibly through a Golgi‐dependent pathway.

Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii.

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

Toxoplasma gondii ADP-ribosylation Factor 1 Mediates Enhanced Release of Constitutively Secreted Dense Granule Proteins

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5′-3-O-(thio)triphosphate (GTPγS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424–2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPγS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter,Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.