High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate

Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca²⁺-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca²⁺/myristoyl switch that allows their reversible membrane association in response to Ca²⁺ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K⁺ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] with high affinity (K_d 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P₂. Co-expression of hippocalcin-(1–14)–ECFP (enhanced cyan fluorescent protein) or NCS-1–ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase δ1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P₂ than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P₂.