Characterization and functional analysis ofToxoplasmaGolgi-associated proteins identified by proximity labelling

Abstract

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.

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

“…TgTBC1-18 were tagged using CRISPR/Cas9 with primers P1 to P72 and verified by PCR using primers P73 to P92 (S4 Table ). TgGLP2, TgVps9, Der1-2, TgCep250, and IMC29 were tagged with primers P147 to P166 (S4 Table) [23,42,47,90,91].…”