ROLE OF HETEROGENEOUS N-TERMINAL ACYLATION OF RECOVERIN IN RHODOPSIN PHOSPHORYLATION

Recoverin, a new member of the EF-hand superfamily, plays a critical r ole in the light/dark adaptation of retinal rods by regulating rhodops in phosphorylation in a Ca2+-dependent manner. Recoverin is composed o f four isoforms, each of which is modified at its N terminus by myrist ate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C 14:1). Although the N-fatty acylation is implicated in protein-membran e and protein-protein interactions, the functional difference among th e recoverin isoforms and the significance of the heterogeneous acylati on have not been defined. Here we separated the heterogeneous recoveri n into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixtur e of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the indivi dual 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 mu M). However, the magnitude of the inhibition at higher Ca2+ concen tration 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 th e recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.