Abstract
Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.
Highlights
We recently identified a novel membeorf the brain-derived recoverin familyh, ippocalcin(Kobayashiet al., 1992).Interest in hippocalcin is heightenedby the finding that it is expressed exclusively inthe pyramidal neurons of the hippocampus.The predicted amino acid sequence of hippocalcin has three putative Ca2+-bindingdomains of the EF-hand structure (Kretsinger, 1979)and a possibleNH2-~rminal m~stoylatsioitne (Towleret al., 1988).Both structuralfeatures are common to labeled protein on two-dimensional gels
NH2- crude membrane fraction from rat hippocampuswas prepared terminal sequencing showedthat LysHip begins with glycine, in the presence of 1 mM Ca2+,immunoblot analysis showed since N-myristoyl transferase (NMT) activity is absent in the E. coli translation system that hippocalcin remained largely associated with the mem
When [35S] hippocalcin was incubated with the membrane fraction from rHipA and LysHipA was subjected to two-dimensional gels, rat hippocampusin the presence of 1 mM Ca2+or 1 mM
Summary
Bacterial Expression ofFull-length Hippocalcin-The SnaI site of (1970),and two-dimensional gel electrophoresis was performed essenhippocalcin cDNA (nucleotide position 974 inthe 3'-non-coding tially according to the method of O'Farrell (1975). ?+Labeled hippocalcin (10,000 cpm) was incubated at 30 "C mids PET-Hip and PET-HipA were linearized by EcoRV digestion for 60 min with 50 pl of the crude membrane fraction at theapproand transcribed in vitro with T7 RNA polymerase according to the priate Ca2' concentrations. MM EGTA or 1mM CaC12.%-Labeled hippocalcin (10,000cpm) was Synthesis and Myristoyhtwn of Hippocalcin in Vitro-A incubated at 30 "Cfor 60 min with the phospholipid vesicles (100 pg) homogeneous population of hippocalcinmRNAwas tranin a total volume of 200 pl of the appropriate binding buffer. The precipitate was washed twicwe ith the appropriate binding buffer, scribed from PET-Hip in uitro This mRNA was translated in a rabbit reticulocyte lysate, and the reaction products were and the radioactivity of the resultant precipitate was determined analyzed by SDS-PAGE. A single polypeptide band was synthesized with a molecular size of 23 kDa.The identity of the 23 kDaband as hippocalcin
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