## 2 Results and Discussion
### Identification of PbPP1c-Interacting Proteins in Schizonts and Gametocytes  
The digested samples were directly analyzed by LC-MS/MS using five biological replicates to identify PbPP1c-associated proteins. After statistical analysis, the data revealed a total of 489 proteins including the mCherry-PbPP1c bait protein. Among them, 157 proteins were present in both schizont and gametocyte extracts and considered as common interacting proteins (CIPs) (Supplementary Materials Table S1, sheets 2-4 in pink; Figure 2). In addition, 68 proteins were detected as specific interacting proteins in schizonts (SIPS) (Supplementary Materials Table S1, sheet 2 in green), and 263 proteins were found as specific interacting proteins in gametocytes (SIPGs) (Supplementary Materials Table S1, sheet 3 in blue). Among the CIP, it is important to point out that LRR1, I2, I3, GEXP15, and RCC-PIP proteins as well as NEMF and PBANKA_0621300 proteins, which we previously reported as direct interactors of _Plasmodium_ PP1c, were found among the top interacting proteins (Figure 2, annotated in panels (A) and (B) and listed in panel (C)) [11, 12, 14, 16, 17, 18]. This observation supports the robustness of the interactome data as well as a validation of the IP/MS experiments. Next, we analyzed all potential PbPP1c protein complexes (using the Protein Motif Pattern tool of PlasmoDB) for the presence of the highly conserved RVxF docking motif [19], known to be present in >80% of PP1c direct regulators [7]. Of the 488 proteins, 296 interacting proteins exhibited at least one consensus RVxF binding motif (Supplementary Materials Table S1). These motifs are enriched when compared to the overall presence of the RVxF motif in _P. berghei_ proteins (fold 1.17; hypergeometric _p_-value = 3.17 \(\times\) 10\({}^{5}\), [[https://systems.crump.ucla.edu/hypergeometric/index.php](https://systems.crump.ucla.edu/hypergeometric/index.php)]([https://systems.crump.ucla.edu/hypergeometric/index.php](https://systems.crump.ucla.edu/hypergeometric/index.php)), accessed on 16 August 2021). This suggests that the presence of the RVxF motif could contribute to the molecular basis of interaction with PP1c. These motifs need to be further investigated and confirmed as structural and functional motifs. Figure 1: Workflow and study overview. Schizont and gametocyte stages were prepared from PbWT GFP, PbPP1c-mCherry, and PbI2–mCherry parasites. Immunoprecipitation was performed with anti-RFP antibody on 5 different biological replicates of schizons and gametocytes extracts prepared from each parasite strain (PbWT, PbPP1c-mCherry, and PbI2-mCherry). The presence of mCherry-tagged PbPP1c and PbI2 was checked in the immunoprecipitated fractions (control wild-type vs. tagged proteins) before MS. Proteomic and statistical analyses (detailed in Section 3) allowed for the identification of the interactome of PbPP1c and PbI2 in schizont and gametocyte stages that were further analyzed for GO enrichment, STRING networks, and comparative analyses. Following the PbPP1c immunoprecipitation, it should be noted that 21% of the CIPs (34/157) and 45% of the SIPS (31/68) were found to be ribosomal subunits or ribosome-associated proteins (Supplementary Materials Table S1), suggesting that PbPP1c brings down a large part of the ribosome complex. It is very unlikely that these interactions could be considered as non-specific. In support for this, the MS of IP extracts of WT parasites and of the mCherry-PbI2 parasites generated in the same strain in which the mCherry-PbPP1c line was obtained did not allow for the detection of these proteins (see below). In addition, it is important to bear in mind that previous studies support the selectivity of PP1c binding to ribosome complexes in mammalian cells [20, 21, 22]. Further, by manual curation, we found that 19 ribosome-associated proteins detected in the interactome exhibited at least one RVxF binding motif, supporting their potential direct interactions with PbPP1c (Supplementary Materials Table S1).