The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

Abstract

Protein ubiquitination plays key roles in protein turnover, cellular signaling, and intracellular transport. The genome of Toxoplasma gondii encodes ubiquitination machinery, but the roles of this posttranslational modification (PTM) are unknown. To examine the prevalence and function of ubiquitination in T. gondii, we mapped the ubiquitin proteome of tachyzoites. Over 500 ubiquitin-modified proteins, with almost 1,000 sites, were identified on proteins with diverse localizations and functions. Enrichment analysis demonstrated that 35% of ubiquitinated proteins are cell-cycle regulated. Unexpectedly, most classic cell-cycle regulators conserved in T. gondii were not detected in the ubiquitinome. Furthermore, many ubiquitinated proteins localize to the cytoskeleton and inner membrane complex, a structure beneath the plasma membrane facilitating division and host invasion. Comparing the ubiquitinome with other PTM proteomes reveals waves of PTM enrichment during the cell cycle. Thus, T. gondii PTMs are implicated as critical regulators of cell division and cell-cycle transitions.

Figure 1. Ubiquitin is ubiquitous in T. gondii and changes during the cell cycle

A. Western blot of 50 μg of intracellular tachyzoite lysate, probed with anti-ubiquitin antibody. B. Immunofluorescence staining of methanol/acetone-fixed intracellular T. gondii tachyzoites with anti-ubiquitin antibody (red). Nuclei are stained with DAPI (blue). Ubiquitin staining foci resembling IMC (arrows), centrosomes (see single nuclear apical dot in bottom parasite with arrow in top panel and double dots marked with triangle arrowheads in parasites beginning to divide in middle panel), and daughter buds (V-shaped arrowheads, bottom panel) are indicated. An illustration of parasite morphology shown in each microscopy image is displayed on the right; IMC (green), nucleus (blue), plasma membrane (grey). See also Figure S1.

Figure 2. Identification of ubiquitinated proteins in T. gondii

A. Diglycyllysine remnant affinity purification. Protein lysates containing ubiquitinated proteins (green dots) and non-ubiquitinated proteins were digested with trypsin, creating a remnant diglycine moiety (GG) on the ubiquitinated lysine residue. A monoclonal antibody specific to diglycine was used to purify peptides, preceding LC-MS/MS. B. Pie charts showing ubiquitinated proteins classified by localisation, excluding proteins whose localisation is unknown. C. Pie charts showing ubiquitinated proteins classified by function, excluding proteins whose function is unknown. D. Significance of enrichment of ubiquitinated proteins in different cellular compartments, as determined by hypergeometric testing against a background of all T. gondii predicted proteins; −Log₂(p-value) is displayed. E. Gene ontology (GO) terms that are significantly enriched in ubiquitinated proteins; −Log₂(p-value) is displayed. See also Figure S2 and supplemental Tables S1 and S2.

Figure 3. Amino acid environment surrounding ubiquitinated lysine residues

A. Heat map showing abundance of residues surrounding ubiquitinated lysine residues (7 residues upstream and downstream) relative to the entire T. gondii predicted proteome (green = enriched, red = depleted, scale as indicated). B. Consensus graphic of ubiquitination sites. Relative sizes of residue letters indicate their relative abundance across all peptides. The X-axis denotes residues surrounding the ubiquitinated Lysine residue. Residues are coloured according to side chain chemistry (green = polar uncharged, purple = polar amide, blue = basic, red = acidic, black = hydrophobic).

Figure 4. Detection of ubiquitinated IMC components

A. Western blots of immunoprecipitated ISP1, ILP1, ISC2, IMC18, IMC21, or IMC24, from lysates of HA-tagged intracellular parasites, were probed with anti-HA (left panel) or anti-ubiquitin antibodies (right). Similar amounts of each HA-tagged protein were enriched, and ubiquitin immunoblots showed varying degrees of higher molecular weight species consistent with polyubiquitination. For ILP1, a major band consistent with monoubiquitination was visible. Bands corresponding to antibody heavy and light chains are denoted by one or two asterisks, respectively. B. Immunofluorescence staining of paraformaldehyde-fixed ILP1-HA-tagged parasites (upper panel) and IMC18-HA-tagged parasites (lower panel) with ubiquitin antibodies (red) and anti-HA (green). Nuclei were stained with DAPI (blue). Partial colocalisation of ILP1-HA signal (yellow) with ubiquitin at daughter buds is labelled (arrowheads). See also Figure S4.

Figure 5. Ubiquitination sites related to transcriptional activation, repression and DNA repair are present in T. gondii

A. Table showing ubiquitination sites identified on T. gondii histones, with previously mapped histone modifications (Nardelli et al., 2013). B. Western blot on acid-extracted histones using anti-ubiquitin, H2BK120 ubiquitination (H2BK120Ub) and T. gondii H2BV. A Ponceau-stained image of the blot is shown in the bottom panel. Specific bands recognised by ubiquitin, H2BK120 ubiquitination antibodies correspond to bands specific to T. gondii H2BV (arrows). C. The antibody against H2BK120Ub was raised against a C-terminal peptide to human H2B. An alignment of H2B C-terminal peptides from human (Hs) and T. gondii (Tg) are shown. Consensus is indicated (* = identical, : = highly similar properties, . = weakly similar properties). D. Immunofluorescence staining of intracellular tachyzoites with antibodies against H2BK120Ub. Nuclei are labelled with DAPI (blue). E. Functions of ubiquitination sites on histones. At sites of DNA damage, ubiquitination of histones H2AX, H3 and H4 recruits DNA repair complexes e.g. XPC to damaged sites to mediate repair (left panel). Ubiquitination of histone H2AK119 inhibits RNA polymerase II elongation phase, causing transcriptional repression (middle panel). Ubiquitination of histone H2BK120 activates transcription via methylation of H3K4 and H3K79 (right panel). Blue dots, ubiquitination; red dots, methylated lysines. See also Figure S5.

Figure 6. Ubiquitinated proteins are enriched for cell cycle regulated proteins

A. Parasite morphology across the cell cycle in T. gondii. Parasites are outlined in grey, nucleus in blue and IMC in green. The schema below indicates parasite cell cycle, progressing from G1 to S, M and C phases and beginning G1 again. B and C. Enrichment analysis of ubiquitinated proteins for genes in stage-specific G1 phase (B) and S/M phase (C). Adjusted p-values (−Log₂-transformed) are plotted. Predefined gene sets corresponding to 12 min time points during the 8 hr cell cycle were used and represent G1 and S/M subtranscriptomes during endodyogeny (Behnke et al., 2010; Croken et al., 2014). D and E. Expression trends of ubiquitination enzymes and ubiquitinated proteins in intracellular parasites. Graph showing number of E3 ligases and deubiquitinating enzymes across the T. gondii cell cycle in G1 (D) and S/M (E) (see Table S3); with transcriptome data from (Behnke et al., 2010), overlaid with −Log₂ transformed p-values from enrichment analysis of ubiquitinated proteins detected in intracellular parasites. F and G. The ubiquitinome of extracellular tachyzoites shows decreased enrichment of cell cycle regulated proteins. Enrichment analysis of ubiquitinated proteins identified in the ubiquitinomes of intracellular and extracellular tachyzoites in G1 gene sets (F) or S/M phase gene sets (G). See also Figure S6, Table S3.

Figure 7. Cell Cycle Enrichment Analysis of PTM Proteomes

A. Table showing overlaps between ubiquitination (total ubiquitinated proteins identified from intracellular and extracellular tachyzoites) and other T. gondii PTM proteomes. B. Heatmap displaying the significance [−Log₂(p-value)] of the overlap of PTM datasets with one another, compared to the entire T. gondii proteome. C. Two waves of PTM modified proteins are evident in G1 (hr 4.5-5.5; hr 6.5-8) and one in S/M (hr 3-4). Proteins identified in PTM proteomic studies were tested for enrichment in predefined gene sets corresponding to the G1 or S/M subtranscriptomes. Adjusted p-values [−(Log₂)-transformed] are plotted.