MYRISTOYLATION OF HIPPOCALCIN IS LINKED TO ITS CALCIUM-DEPENDENT MEMBRANE ASSOCIATION PROPERTIES

Hippocalcin, a recently identified Ca2+-binding protein of the recover in family exclusively expressed in the hippocampus, has a primary stru cture containing three putative Ca2+-binding sites (EF-hands) and a po ssible NH2-terminal myristoylation site. Ca-45 blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escheric hia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/m ol of protein. To determine whether hippocalcin is myristoylated, hipp ocalcin mRNA was translated in vitro in the presence of [H-3]myristic acid. H-3 label was resistant to hydroxylamine treatment, and replacem ent of NH2-terminal glycine with alanine prevented H-3 label incorpora tion, indicating that in vitro translated hippocalcin covalently bound [H-3]myristic acid at the NH2-terminal glycine. In vitro translated h ippocalcin is quantitatively myristoylated, as evidenced by an electro phoretic mobility shift of [S-35]methionine-labeled protein on two-dim ensional gels. Native hippocalcin comigrated precisely with the in vit ro translated hippocalcin on two-dimensional gels, suggesting that nat ive hippocalcin is myristoylated. Native and in vitro translated hippo calcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca2+-dependent membrane association, indicating that myristo ylation participates in its Ca2+-dependent membrane association proper ties. In vitro translated hippocalcin bound to phospholipid vesicles s omewhat, however, phospholipid association was insufficient for its me mbrane association properties, suggesting that the NH2-terminal myrist oyl moiety on hippocalcin interacts with lipid bilayers and facilitate s interaction with other membrane proteins.