title: High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate.
abstract: 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 delta1 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.
authors.affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, UK.
authors.forename: Dermott W
authors.initials: DW
authors.lastname: O'Callaghan
authors.forename: Lee P
authors.initials: LP
authors.lastname: Haynes
authors.forename: Robert D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002470:Cell Survival; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D051597:Hippocalcin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D002135:Calcium-Binding Proteins; D008081:Liposomes; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D019269:Phosphatidylinositol 4,5-Diphosphate; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D051597:Hippocalcin