Role of heterogeneous N-terminal acylation of recoverin in rhodopsin phosphorylation.
Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.
Sanada
K
K
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan.
Kokame
K
K
Yoshizawa
T
T
Takao
T
T
Shimonishi
Y
Y
Fukada
Y
Y
D000215:Acylation; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D005136:Eye Proteins; D051597:Hippocalcin; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D051603:Recoverin; D012243:Rhodopsin / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D051603:Recoverin; D051597:Hippocalcin; D012243:Rhodopsin; D002118:Calcium