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

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 Golgi-localizing protein (ULP1), which is structurally similar to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness that is the result of defects in microneme secretion, invasion, replication, and egress. Using ULP1 as bait for TurboID proximity labeling and immunoprecipitation, we identify 11 more Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii-conserved oligomeric Golgi (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 11 proteins on parasite fitness. Together, this work reveals a diverse set of 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 fitness and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labeling to identify 11 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.

Keywords: ER exit sites; Golgi apparatus; Toxoplasma gondii; apicomplexan parasites; protein trafficking; secretory pathway; vesicular trafficking.

Fig 1

ULP1 is a Golgi-localizing protein that is important for parasite fitness. (A) Gene model of TGGT1_289120 (ULP1) showing its p115/Uso1-like domain and predicted CC domain. (B) Immunofluorescence assay (IFA) showing that ULP1 colocalizes with the Golgi apparatus marker GRASP55-YFP. Magenta, anti-HA detecting ULP1^3xHA; green, GRASP55-YFP. (C) IFA showing that ULP1^AID localizes normally and is depleted after 24 hours of indoleacetic acid (IAA) treatment. Magenta, anti-HA detecting ULP1^AID; green, anti-IMC6. (D) Plaque assay for ULP1^AID parasites ±IAA shows that depletion of ULP1 results in a severe reduction in overall lytic ability. (E) Quantification of plaque size for plaque assays shown in panel D. Significance was determined using a two-tailed t test (****P < 0.0001). (F) PCR verification for genomic DNA of wild-type (WT) and Δulp1 parasites. Diagram indicates the binding location of primers used to amplify the ULP1 coding sequencing (blue arrows) and the site of recombination for the knockout (red arrows). (G) IFA of Δulp1 parasites confirms loss of ULP1^3xHA signal. Magenta, anti-HA; green, anti-IMC6. Scale bars for IFAs = 2 µm. (H) Plaque assays of WT and Δulp1 parasites. Scale bars for plaque assays = 0.5 mm. (I) Quantification of plaque size for plaque assays shown in panel H. Statistical significance was determined using a two-tailed t test (****P < 0.0001). (J) Quantification of the number of parasites per vacuole for ULP1^AID parasites treated with IAA or vehicle control after 30 hours of growth. Significance was determined using multiple two-tailed t tests (*P < 0.05; ns = not significant). (K) Quantification of the number of parasites per vacuole for ULP1^AID parasites treated with IAA or vehicle control after 36 hours of growth. Significance was determined using multiple two-tailed t tests (*P < 0.05; ns = not significant). (L) Quantification of invasion for ULP1^AID parasites treated with IAA or vehicle control. Significance was determined using a two-tailed t test (**P < 0.01). (M) Quantification of egress induced by calcium ionophore for ULP1^AID parasites treated with IAA or vehicle control. Significance was determined using a two-tailed t test (***P < 0.001).

Fig 2

Depletion of ULP1 causes a defect in microneme secretion despite normal organellar morphology and trafficking to secretory organelles. IFAs comparing the overall morphology of various T. gondii organelles in control vs. ULP1-depleted parasites. (A) Golgi apparatus morphology is unaffected by depletion of ULP1. Magenta, anti-V5 detecting TgTrs85^3xV5; green, anti-IMC6. (B) Post-Golgi vesicle morphology is unaffected by depletion of ULP1. Magenta, anti-DrpB; green, anti-IMC6. (C) PLVAC morphology is unaffected by depletion of ULP1. Magenta, anti-NHE3; green, anti-IMC6. (D) ER morphology is unaffected by depletion of ULP1. Magenta, anti-SERCA; green, anti-IMC6. (E) ELC morphology is unaffected by depletion of ULP1. Magenta, anti-V5 detecting Vps9^3xV5; green, anti-IMC6. (F) Dense granule morphology is unaffected by depletion of ULP1. Magenta, anti-GRA12; green, anti-IMC6. (G) Rhoptry morphology is unaffected by depletion of ULP1. Magenta, anti-ROP7; green, anti-IMC6. (H) Western blot showing that proROP13 is processed into its mature form normally in ULP1-depleted parasites. Parasites were treated with IAA or vehicle control for 30 hours. IMC6 is used as a loading control. (I) Microneme morphology is unaffected by depletion of ULP1. Magenta, anti-MIC2; green, anti-IMC6. (J) Western blot and quantification of ULP1^AID parasites treated with the calcium ionophore A23187 to induce microneme secretion in the presence and absence of IAA. MIC2 was used to assess microneme secretion, and the constitutively secreted dense granule protein GRA39 was used as a control. Scale bars = 2 µm.

Fig 3

Validation of ULP1 TurboID and IP. (A) The TurboID biotin ligase and a 3xHA tag were fused to the C-terminus of ULP1 to generate the ULP1^TurboID line. (B) IFA showing that ULP1^TurboID localizes as expected and results in biotinylation of proximal proteins in a biotin-dependent manner (yellow arrow). The apicoplast, which contains endogenously biotinylated proteins, is denoted by white arrow. Magenta, anti-HA detecting ULP1^TurboID; green, streptavidin. Scale bar = 2 µm. (C) Western blot showing enrichment of ULP1^3xHA after immunoprecipitation with anti-HA resin despite significant protein breakdown. WCL, whole cell lysate prior to IP; IP, final sample eluted from anti-HA resin.

Fig 4

ULP1 TurboID and IP experiments reveal 11 Golgi-associated proteins. IFAs and corresponding gene models for 11 genes identified as candidate Golgi proteins in the ULP1 TurboID and IP experiments. Each gene was endogenously tagged with mAID-3xHA or mIAA7-3xHA AIDs. White arrows indicate that the protein primarily colocalizes with GRASP55-YFP. Yellow arrows indicate that the protein primarily localizes upstream of GRASP55-YFP. Cyan arrows indicate that the protein primarily localizes downstream of GRASP55-YFP. (A) TGGT1_311400 (Sec31) localizes upstream of GRASP55 and contains a WD40 repeat domain. (B) TGGT1_294730 (Sec16-L) localizes upstream of GRASP55 and contains an ACE1 domain. (C) TGGT1_264090 (TRAPPC11) colocalizes with GRASP55 and contains a TRAPPC11 domain. (D) TGGT1_290310 (COG1) colocalizes with GRASP55 and contains no identifiable functional domains. (E) TGGT1_232190 (GBF1) colocalizes with GRASP55 and contains a GBF1 domain. (F) TGGT1_207370 (GLP2) colocalizes with GRASP55 and contains three predicted CC domains within residues 273–301, 313–373, and 382–423. (G) TGGT1_230400 (WPDP) colocalizes with GRASP55 and contains a large WASP-interacting protein-related domain. (H) TGGT1_216370 (GLP3) localizes downstream of GRASP55 and contains two predicted CC domains within residues 337–370 and 494–527. (I) TGGT1_ 301410 (Tepsin) localizes downstream of GRASP55 and contains ENTH and Tepsin domains. (J) TGGT1_258080 (GLP1) localizes downstream of GRASP55 and contains five predicted CC domains within residues 1117–1332, 1362–1407, 1425–1464, 1603–1646, and 1659–1701. (K) TGGT1_240220 (GLP4) localizes downstream of GRASP55 and contains three predicted CC domains within residues 634–747, 797–830, and 850–881. Magenta, anti-HA detecting degron-tagged proteins; green, GRASP55 YFP. Scale bars = 2 µm.

Fig 5

Conditional knockdown of Sec31, GBF1, COG1, Tepsin, and TRAPPC11. (A) Plaque assays were performed ±IAA to assess how depletion of Sec31, GBF1, COG1, Tepsin, or TRAPPC11 affects overall lytic ability over the course of 7 days. Number of plaques was quantified for each condition. Statistical significance was determined using multiple two-tailed t tests (****P < 0.0001 and ***P < .001). (B, C) Depletion of Sec31 or GBF1 results in growth arrest. Depletion of GBF1 additionally leads to morphological defects in some parasites (white arrow). (D, E) Depletion of COG1 or Tepsin results in morphological defects (white arrows). (F) Depletion of TRAPPC11 for 24 hours has no obvious effect on most parasites (panel i) but causes some parasites to exhibit abnormal rosette formation (panel ii, yellow asterisk) or morphological defects (panel iii, white arrow). (G) Depletion of TRAPPC11 for 43 hours results in severe defects in growth and IMC morphology (white arrow), as well as abnormal vacuolar arrangement (yellow asterisk). Magenta, anti-HA detecting degron-tagged proteins; green, anti-IMC6. Scale bars = 2 µm.

Fig 6

Conditional knockdown of Sec16-L, WPDP, GLP1, GLP2, GLP3, and GLP4. (A) Plaque assays were performed ±IAA to assess how depletion of Sec16-L, WPDP, GLP1, GLP2, GLP3, and GLP4 affects overall lytic ability over the course of 7 days. Mean plaque area was quantified for each condition. Statistical significance was determined using multiple two-tailed t tests (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). (B–G) Depletion of Sec16-L, WPDP, GLP1, GLP2, GLP3, or GLP4 does not result in any obvious defects in morphology. Magenta, anti-HA detecting degron-tagged proteins; green, anti-IMC6. Scale bars = 2 µm.