Alveolin proteins in the Toxoplasma inner membrane complex form a highly interconnected structure that maintains parasite shape and replication.

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins in Toxoplasma, IMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant invasion and replication but surprisingly minor effects on motility. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and 2 other cytoskeletal IMC proteins-IMC3 and ILP1. This provides direct evidence of protein-protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasite's structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here we identify a novel Golgi-localizing protein (ULP1) which contains structural homology to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness and replicative ability. Using ULP1 as bait for TurboID proximity labelling and immunoprecipitation, we identify eleven more novel Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii COG complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these eleven proteins on parasite fitness. Together, this work reveals a diverse set of novel T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites. Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite replication and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labelling to identify eleven additional Golgi-associated proteins which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

Toxoplasma gondii encodes an array of TBC-domain containing proteins including an essential regulator that targets Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host-cell manipulation and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii , precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC)-domain containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC-domain containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. We then use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein that localizes to the ER is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and Golgi apparatus. We show that the conserved dual-finger active site in the TBC-domain of the protein is critical for its GTPase-activating protein (GAP) function and that the P. falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast two hybrid analyses that TgTBC9 directly binds Rab2, indicating that this TBC-Rab pair controls ER to Golgi traffic in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan, provide new insight into intracellular vesicle trafficking in T. gondii , and reveal promising targets for the design of novel therapeutics that can specifically target apicomplexan parasites.