Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C δ1

Background: Although phospholipase C (PLC)δ1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCδ1 contains an NLS sequence. Results: A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCδ1 in Madin–Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429–442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCδ1 412–498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm. Conclusion: The nuclear import of PLCδ1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.