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 signalling, 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 1000 sites, were identified on proteins with diverse localisations 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 localise 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.

Post‐translational modifications as key regulators of apicomplexan biology: insights from proteome‐wide studies

“…O‐GlcNAcylation was detected in T. gondii using antibodies against the PTM and numerous high molecular weight (above 130 kDa) O‐GlcNAc modified proteins were found (Perez‐Cervera et al ., ). O‐GlcNAcylation appears to be abundant on a diverse set of intracellular proteins in T. gondii (Silmon de Monerri et al ., and Silmon de Monerri and Kim, unpublished data).…”

“… Proteins modified by PTMs are enriched in cell cycle regulated genes in T. gondii . Cell cycle enrichment analysis of published (Braun et al ., ; Treeck et al ., ; Jeffers and Sullivan, ; Li et al ., ; Foe et al ., ; Silmon de Monerri et al ., ; Yakubu et al ., ) and unpublished (O‐GlcNAc, Silmon de Monerri and Kim, in preparation) PTM datasets. Proteins identified in proteomic studies of PTM were compared against cell cycle gene sets by a hypergeometric test of enrichment.…”

“… PTM display preferences for different metabolic pathways in T. gondii . Proteins identified as PTM targets by proteomic studies in T. gondii were compared to sets of genes with different functions (classified by Gene Ontology [GO] terms) using a hypergeometric test of enrichment using methodology described by Silmon de Monerri (Silmon de Monerri et al ., ). The five most significantly enriched GO terms are shown in a clustered heatmap.…”

“…Similar patterns are found with regard to arginine monomethylation, where this PTM has been found outside of the AP2 DNA‐binding domain in ten ApiAP2 proteins (Yakubu et al ., ). Ubiquitination sites have been found in five ApiAP2 in T. gondii (Silmon de Monerri et al ., ), with only AP2X‐7 (TGME49_214840) containing ubiquitin (at K1368) within its third ApiAP2 domain. In the case of AP2X‐4, AP2X‐9, AP2X‐3 and AP2XII‐8 the ubiquitin sites lie outside of the ApiAP2 domain.…”

“…(). Both published (Braun et al ., ; Treeck et al ., ; Jeffers and Sullivan, ; Li et al ., ; Foe et al ., ; Silmon de Monerri et al ., ; Yakubu et al ., ) and unpublished (O‐GlcNAc dataset, Silmon de Monerri and Kim, in preparation) PTM datasets were analyzed. Color key is in ‐log2 ( p ‐value).…”