Molecular Evolution, Localization, and Functional Analysis of Toxoplasma Gondii Coated Vesicle (Copi) Beta Subunit

Toxoplasma gondii is an obligate intracellular pathogen that infects many individuals worldwide. Protein secretion from three unique secretory organelles; the micronemes, rhoptries and dense granules, is pivotal for T. gondii infection and survival within host cells. T. gondii is polarized with a single Golgi stack apical to the nucleus. Few studies have been done to characterize the retrograde transport processes involved in Golgi to ER protein trafficking in T. gondii. The work in this dissertation discusses the characterization of the T. gondii ̢COP homolog, a protein involved retrograde transport in mammalian and yeast cells as a subunit of coatomer coated vesicles (COPI). Experiments were designed to identify both conserved and novel characteristics of Tg̢COP and include a phylogenetic analysis, localization studies, and a functional assessment. Studies in mammalian and yeast cells have shown that ̢COP localizes to the Golgi, is recruited to the Golgi via the GTPase ARF1, interacts with proteins containing the retrieval KKXX or KDEL motif, and disperses to the cytosol after treatment with Brefeldin A (BFA). In the following studies we show that Tg̢COP localizes to the Golgi region in non-dividing parasites, is involved in retrograde transport of HDEL-tagged proteins (the T. gondii retrieval motif), and initial evidence suggests that Tg̢COP is recruited to the Golgi in a GTP-dependent manner as expected.

We observe several novel features in terms of sequence, location during parasite division, and response to calcium chelation. Several novel inserts were identified within TgÌ¢COP, including one insert that is shared among apicomplexans. In addition to the phylogenetics, localization changes were monitored. During parasite endodyogeny, TgÌ¢COP staining closely follows the contours of elongating apicoplasts and encircles the Golgi during Golgi duplication and segregation into new daughter cells. Initial functional studies of TÌ¢COP were conducted using pharmacological agents, including BFA, GTPãS, and the calcium chelator, MAPTA-AM. Unlike mammalian cells, there is a population of TgÌ¢COP refractory to the effects of BFA that do not completely disperse to the cytosol after treatment. Lastly, disruption in TgÌ¢COP localization occurs upon MAPTA-AM treatment and appears to be more responsive to the effects of calcium chelation than host cells.