Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties.
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.
Kobayashi
M
M
Department of Physiology, Toho University School of Medicine, Tokyo, Japan.
Takamatsu
K
K
Saitoh
S
S
Noguchi
T
T
D000409:Alanine; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005998:Glycine; D051597:Hippocalcin; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D002132:Calcium Radioisotopes; D002135:Calcium-Binding Proteins; C496691:HPCA protein, human; C496693:Hpca protein, rat; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009227:Myristic Acids; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019814:Myristic Acid; D051597:Hippocalcin; D000409:Alanine; D002118:Calcium; D005998:Glycine