Identification of Exported Proteins That Bind to the Erythrocyte Cytoskeleton.

proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein-protein interactions between erythrocyte cytoskeletal proteins and predicted exported proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with -deficient parasites.