Mapping of Nuclear Localization Signal in Secreted Liver-Specific Protein 2 of .

The secretory proteome of exhibits differential spatial and functional activity within host cells. secretes proteins that translocate into the human host cell nucleus. Liver-specific protein 2 of (LISP2) shows nuclear accumulation in human hepatocytes during the late liver stage of malaria parasite development. However, the nuclear translocation mechanism for LISP2 remains largely uncharacterized. Here, we identified a classical bipartite nuclear localization signal (NLS) located in the C-terminal region of LISP2. Phylogenetic analysis revealed that this NLS is unique to and its close relative , suggesting an evolutionary adaptation linked to their shared primate hosts. Functional assays confirmed the NLS's nuclear import activity, as fusion constructs of the LISP2 NLS with aldolase (aldolase-NLS-EGFP) localized exclusively to the nucleus of HepG2 cells. Mutation analysis of key lysine and arginine residues in the bipartite NLS demonstrated that the basic amino acid clusters are essential for nuclear localization. Importin-α/β interaction was found to be crucial for LISP2 nuclear transport, as coexpression of the NLS constructs with the importin-α/β inhibitor mCherry-Bimax2 significantly blocked nuclear translocation. Specific interactions between the lysine and arginine residues of LISP2's NLS and the conserved tryptophan and asparagine residues of human importin-α1 facilitate the cytosol-to-nuclear translocation of LISP2. Additionally, LISP2 lacks any nuclear export signal. These results provide new insights into the mechanisms of nuclear transport in , potentially contributing to the understanding of its pathogenicity and host-cell interactions during liver-stage infection.