Patray, S. | Search bioRxiv

bioRxiv2026

Two anchoring proteins control daughter apical complex assembly in Toxoplasma gondii

During Toxoplasma gondii division, the apical complex--comprising the conoid, apical polar ring (APR), and preconoidal rings--assembles with precise spatiotemporal coordination to form functional daughter buds. Despite their essential roles in invasion, motility, and division, the scaffolding proteins orchestrating this ordered assembly have remained largely unidentified. Here, we identify and characterize RCC1-2 and APR8 as essential factors directing distinct, sequential phases of daughter cell apical complex construction. Both proteins are recruited with precise spatial and temporal dynamics to the daughter buds, where they function as scaffolds rather than static structural components. APR8 transiently occupies the basal region of the APR specifically in early daughter cells. It is dispensable for conoid and PCR initiation, yet its loss causes APR collapse, abolishes SPMT anchoring, and eventually arrests conoid maturation. In contrast, RCC1-2 localizes beneath the APR basal layer and persists throughout daughter cell development, where it contributes to stabilizing the attachment of SPMTs to the APR. Notably, in situ cryo-electron tomography further reveals that the interspersed pillars bridging SPMTs ends to the APR fail to form properly in RCC1-2-depleted parasites. These findings map a hierarchical RCC1-2/APR8-dependent scaffolding process that advances our understanding of parasite replication.

microbiology↗

bioRxiv2024

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of P. falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI-anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for a highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

biochemistry↗