The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5^C domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif’s primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

MESA 红细胞骨架结合 (MEC) 基序是在 14 种输出的恶性疟原虫蛋白中发现的 13 个氨基酸序列。首次在恶性疟原虫中发现成熟寄生虫感染的红细胞表面抗原 (MESA)，MEC 基序足以将蛋白质靶向感染的红细胞细胞骨架。为了鉴定宿主细胞靶标，纯化的​​ MESA MEC 基序与来自未感染红细胞的可溶性提取物一起孵育，沉淀并进行质谱分析。最丰富的共纯化蛋白是红细胞锚蛋白（ANK1）。使用共纯化实验、分裂荧光素酶测定和酵母双杂交测定独立验证了 MEC 基序和 ANK1 之间的直接相互作用。核心 MEC 基序的系统突变分析表明，在 MEC 基序 C 末端保守的天冬氨酸残基与红细胞内外囊泡和 ANK1 结合具有关键作用。使用一组 ANK1 构建体，^C域，它没有已知的功能。当 MEC 基序被引入未感染的红细胞幽灵中时，MEC 基序对红细胞的变形能力没有影响，这表明 MEC 基序的主要功能是将输出的蛋白质靶向细胞骨架。最后，我们证明 PF3D7_0402100 (PFD0095c) 可能分别通过其 MEC 和 PHIST 基序与 ANK1 和带 4.1 结合。总之，我们提供了多条证据表明 MEC 基序与红细胞 ANK1 结合。