CYCLIC-AMP-DEPENDENT PHOSPHOPROTEIN COMPONENTS-I AND COMPONENTS-II INTERACT WITH BETA-GAMMA-SUBUNITS OF TRANSDUCIN IN FROG ROD OUTER SEGMENTS

Components I and II (CI&II) in frog rod outer segments (ROS) are promi nent cAMP-dependent protein kinase (PK-A) substrates, Their phosphoryl ation level is high in the dark, and illumination causes dephosphoryla tion. In order to understand their physiological role in phototransduc tion, biochemical characterization of CI&II phosphorylation was perfor med. Fractionation of phosphorylated ROS proteins showed that CI&II in the soluble fraction were highly phosphorylated by endogenous PK-A, w hereas those in the membrane-associated protein fractions were not: Th e latter proteins could be phosphorylated by purified catalytic subuni t of PK-A (PK-A(cat)) while the former proteins were not, suggesting t hat membrane-bound CI&II are normally much less phosphorylated. Treatm ents that dissociate the alpha subunit (alpha(t)) of transducin (G(t)) from beta gamma subunits (beta gamma(t)) and thus produce excess free subunits of G(t) in the soluble fraction caused inhibition of CI&II p hosphorylation in the soluble fraction and enhancement of CI&II phosph orylation in the peripheral membrane fractions containing less G(t). U nphosphorylated CI&II tightly associated with the washed ROS membranes could be extracted after phosphorylation by PK-A(cat). Phosphorylatio n also caused elution of beta gamma(t) from the membrane under the sam e conditions. Cross-linking by the maleimidobenzoyl-N-hydroxysuccinimi de ester of the peripheral membrane fraction produced a distinct phosp horylated 50 kDa product with concurrent disappearance of the beta sub unit of transducin (beta(t)) and phosphorylated CI&II. This phosphoryl ated cross-linked product was not recognized by a monoclonal anti-alph a(t) antibody but was recognized by antiserum against beta(t), suggest ing that the 50 kDa protein is a complex of beta gamma(t) and CI&II. A mino terminal sequencing of components I and II suggests that they are identical proteins with a unique sequence unrelated to other proteins in protein data bases. Phosphopeptide mapping of phosphorylated CI&II in the soluble fraction yielded two trypsinized phosphopeptides, whil e that in the peripheral membrane fractions sheaved only one phosphope ptide. These data suggest that multiple phosphorylation of CI&II alter s their cellular localization, We conclude that phosphorylation of CI& II controls their localization in frog ROS and an interaction of CI&II with subunits of G(t) regulates their phosphorylation.