Fig. 3: K13 mutations associate with differential expression of proteins involved in proteasome-mediated turnover and protein folding, and altered levels of TCA cycle, purine, glutamate, and pyruvate metabolites at the ring stage.

a Heat map of the log₂ fold change of significantly different protein levels in Cam3.II^R539T or Cam3.II^C580Y mutants relative to the isogenic Cam3.II^WT parasites for 155 DE proteins identified from isobaric labeling-based quantitative proteomics. Within each biological replicate, two- sided t-tests were performed for peptide levels belonging to each protein between K13 mutant and WT isogenic lines. Word cloud representation of functional analysis of significant biological processes and cellular components (Gene Ontology database) for the 87 up-regulated proteins in K13 mutant rings, along with their corresponding P values. b Relative fold change of K13 protein levels in Cam3.II^R539T and Cam3.II^C580Y mutants vs. Cam3.II^WT, at the ring and trophozoite stages. K13 protein was ~2-fold lower in the Cam3.II^R539T rings and trophozoites, as compared to the isogenic Cam3.II^WT line. Data points are shown with means of the averaged normalized peptide abundance across two independent experiments for each strain and stage, except for C580Y trophozoites. c Scatterplot of the enrichment –log₁₀ P score (using a two-sided t-test adjusted for multiple comparisons using the Holm–Bonferroni method) of the metabolite pathway and % pathway impact calculated from metabolite set enrichment analysis comparing Cam3.II^C580Y and Cam3.II^WT rings at basal level. Differential expression of four pathways (P < 0.05; gold color) is shown. Circle sizes reflect the pathway impact value, which represents the cumulative percentage of matched metabolite nodes (i.e. the importance measure) with respect to the total pathway based on pathway topological analyses. Box plots show reduced normalized log₂ levels of the two TCA cycle metabolites, malate, and 2-ketoglutarate, and the glutamate metabolite, N-acetyl glutamate, but increased level of the purine metabolite, inosine 5′-diphosphate in Cam3.II^C580Y as compared to Cam3.II^WT rings. Data are represented as box and whisker plots with medians, interquartile ranges, and 95% confidence intervals (N = 3 independent experiments). d Interactome network of the 21 putative K13-interacting partners detected by co-immunoprecipitation (co-IP) with the K13-specific monoclonal antibodies E3 and D9 (ref. ⁴¹) and mass spectrometry experiments using K13 WT, C580Y, or R539T samples from either the Cam3.II or CamWT strain background¹⁶. Proteins were clustered using the Markov Cluster Algorithm in STRING. Line thickness depicts the confidence/strength of the relationship between proteins (nodes). Solid lines represent intra-cluster interactions while dashed lines represent the interaction between clusters. Red nodes correspond to mitochondrial proteins, which are over-represented in this list of co-IP proteins. Gene abbreviations are listed in Supplementary Data 5.