## 2 Results and Discussion
### Proteomic Identification of PbI2 Interactions in Schizonts and Gametocytes  
Next, the interactions of the well-known _Plasmodium_ Inhibitor-2 of PP1c [12] were investigated, as it appears as one of the proteins with the most significant interaction in schizonts and gametocytes presented in the above data. To this end, the _P. berghei_ strain expressing mCherry-I2, described above, was used for IP experiments with anti-RFP antibody. Five biological replicates from the mCherry-PbI2 strain and parental WT were included (Figure 1). Using LC-MS/MS analysis and applying statistical filters, 49 and 35 proteins in schizonts and gametocytes were identified, respectively (Supplementary Materials Table S1, sheets 5 and 6) among which eight proteins were detected in both stages. Of these eight proteins, the bait protein mCherry-PbI2 as well as PbPP1c were found to be highly enriched, confirming the data obtained with the bait mCherry-PbPP1c. The combined PbI2-interacting proteins were first analyzed for GO enrichment annotations. This analysis did not show significant enrichment of any specific pathway in terms of  
Figure 5: STRING network visualization of PbPP1c-interacting proteins belonging to “protein transport” GO Slim in gametocyte stages. Using the Cytoscape STRING application, a network was retrieved for the 24 proteins from the protein transport GO Slim of the PbPP1c interactome in gametocyte stages. With a protein–protein interaction enrichment _p_-value \(<\) 1 \(\times\) 10\({}^{-16}\), the resulting network contained 69 functional associations among 20 proteins; four proteins (i.e., PBANKA_0912200, signal recognition particle subunit SRP72; PBANKA_0931200, heat shock protein 101; PBANKA_1217800, signal peptidase complex subunit 2; PBANKA_1359100, nuclear import protein MOG1) with no associations to other proteins in the network were removed. Proteins from KEGG pathways map03060 (protein export) and map04141 (protein processing in endoplasmic reticulum) are colored in blue and red, respectively. Proteins from both of these KEGG pathways are colored in purple. Assignation to coat protein complexes, clathrin-associated adaptor protein complex 1 and to Sec machinery was conducted manually based on protein description in the PlasmoDB and literature. Black stars indicate proteins with RVxF motifs based on the Wakula consensus sequence [19]. Proteins of each network are listed in adjacent tables showing PlasmoDB geneID, short name, and protein description. STRING analysis was performed using the 11.0b version of the database.  
biological processes and molecular processes (data not shown). However, the analysis revealed significant enrichment of cellular components in schizonts (GO term \"cytoplasmic microtubule\") and gametocytes (GO term \"cytosol\"), including two and six proteins respectively (data not shown). Next, in order to set-up possible functional associations, all PbI2-interacting proteins detected in each parasite stage were further searched using the STRING database for interaction mapping. These analyses did not lead to any protein-protein interaction enrichment (_p_-value = 0.237 for schizonts and 0.604 for gametocytes). However, significant functional enrichment in axonemal dynein light chains kinesin motor, tubulin, and actin clusters were observed in the PbI2 interactome in schizonts (data not shown). These clusters involved five proteins: PBANKA_0202700 (kinesin 8B), PBANKA_0925400 (dynein heavy-chain, putative), PBANKA_1018300 (dynein intermediate-chain, putative), PBANKA_0708100 (dynein light-chain 1), and PBANKA_0417700 (alpha tubulin 1). From these data, it appears that PbI2's functions diverge from those deduced from the human I2 interactome that exhibits a main enriched pathway for the regulation of glycogen biosynthesis ([[http://thebiogrid.org/ID5504](http://thebiogrid.org/ID5504)]([http://thebiogrid.org/ID5504](http://thebiogrid.org/ID5504)), accessed on 15 September 2021).