Mapping of Nuclear\nLocalization Signal in Secreted\nLiver-Specific Protein 2 of Plasmodium falciparum

The secretory proteome of <i>Plasmodium</i> exhibits\ndifferential spatial and functional activity within host cells. <i>Plasmodium</i> secretes proteins that translocate into the human\nhost cell nucleus. Liver-specific protein 2 of Plasmodium\nfalciparum (<i>Pf-</i>LISP2) shows nuclear\naccumulation in human hepatocytes during the late liver stage of malaria\nparasite development. However, the nuclear translocation mechanism\nfor <i>Pf-</i>LISP2 remains largely uncharacterized. Here,\nwe identified a classical bipartite nuclear localization signal (NLS)\nlocated in the C-terminal region of <i>Pf-</i>LISP2. Phylogenetic\nanalysis revealed that this NLS is unique to Plasmodium\nfalciparum and its close relative Plasmodium\nreichenowi, suggesting an evolutionary adaptation\nlinked to their shared primate hosts. Functional assays confirmed\nthe NLS’s nuclear import activity, as fusion constructs of\nthe <i>Pf-</i>LISP2 NLS with <i>Pf-</i>aldolase\n(<i>Pf-</i>aldolase-NLS-EGFP) localized exclusively to the\nnucleus of HepG2 cells. Mutation analysis of key lysine and arginine\nresidues in the bipartite NLS demonstrated that the basic amino acid\nclusters are essential for nuclear localization. Importin-α/β\ninteraction was found to be crucial for <i>Pf-</i>LISP2\nnuclear transport, as coexpression of the NLS constructs with the\nimportin-α/β inhibitor mCherry-Bimax2 significantly blocked\nnuclear translocation. Specific interactions between the lysine and\narginine residues of <i>Pf-</i>LISP2’s NLS and the\nconserved tryptophan and asparagine residues of human importin-α1\nfacilitate the cytosol-to-nuclear translocation of <i>Pf-</i>LISP2. Additionally, LISP2 lacks any nuclear export signal. These\nresults provide new insights into the mechanisms of nuclear transport\nin Plasmodium falciparum, potentially\ncontributing to the understanding of its pathogenicity and host–cell\ninteractions during liver-stage infection.