A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions

Abstract

Summary Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host’s cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii . This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

Conservation of theToxoplasmaconoid complex proteome reveals a cryptic conoid inPlasmodiumthat differentiates between blood- and vector-stage zoites

“…Of the 95 putative apical protein assignments by hyperLOPIT, 13 had been validated as being located to the very apex of the cell during our hyperLOPIT study [36], 23 with this same specific apical location by us or others previously, and 21 proteins were known to be specific to the apical cap or other IMC elements (Table S1 and refs therein). This left a further 38 new protein candidates for which there was no independent validation of their apical location.…”

“…* References for localization data: TS, this study. Table S2: Oligonucleotide primers used for T. gondii and P. berghei gene tagging Footnotes: a PCR plasmid templates P5 and P6 as described in Barylyuk et al (2020) [36] b Endogenous gene fusion method for epitope-tagging or birA*-tagging as described in Barylyuk et al (2020) or Wall et al (2016), respectively. Table S3: Sources and numbers of detected putative orthologues of apical proteins (Table 1, S1 Table) across Alveolata.…”

“…Another 13 proteins were assigned to these clusters but below this high-confidence cut-off. The two high-confidence clusters were verified as comprising proteins specific to the structures associated with the conoid, apical polar rings, and 'apical cap' of the IMC [36]. In addition to the 3832 proteins that we reported in this highresolution spatial proteome [36], a further 1013 proteins were quantified in either only two or one of the hyperLOPIT datasets due to the stochasticity of mass spectrometry sampling.…”

“…The two high-confidence clusters were verified as comprising proteins specific to the structures associated with the conoid, apical polar rings, and 'apical cap' of the IMC [36]. In addition to the 3832 proteins that we reported in this highresolution spatial proteome [36], a further 1013 proteins were quantified in either only two or one of the hyperLOPIT datasets due to the stochasticity of mass spectrometry sampling. While assignment of these proteins consequently had less data support, a further 32 proteins were assigned to the apical clusters (not reported in the Barylyuk et al study) from analysis of either the pairs of LOPIT experiments, or the single experiments.…”

“…To expand our knowledge of the proteins that contribute to conoid structure and function we applied multiple spatial proteomics discovery approaches. The primary method used was hyperplexed Localisation of Organelle Proteins by Isotope Tagging (hyperLOPIT) that we have recently applied to extracellular T. gondii tachyzoites [36,37]. This approach entailed generating hyperLOPIT datasets from three independent samples.…”