LIPOLYTIC MEMBRANE RELEASE OF 2 PHOSPHATIDYLINOSITOL-ANCHORED CAMP RECEPTOR PROTEINS IN YEAST ALTERS THEIR LIGAND-BINDING PARAMETERS

Two new cAMP-binding proteins have been discovered recently in Sacchar omyces cerevisiae. They are genetically distinct from the regulatory s ubunit of cytoplasmic cAMP-dependent protein kinase A and are distingu ished from the latter, in addition, by their anchorage through phospha tidylinositol-containing lipid and glycolipid structures to mitochondr ial and plasma membranes, respectively (Muller and Bandlow, 1989 Bioch emistry 28, 9957-9967, 1991, Biochemistry 30, 10181-10190). A nutritio nal upshift induces the cleavage of the anchor by a phospholipase C (M uller and Bandlow, 1993, J. Cell Biol. 122, 225-236). To test the idea that anchorage by (glycosyl)phosphatidyl-inositol influences cAMP-bin ding and has a regulatory function, we analyzed ligand binding to the two purified cAMP receptors (46,000 and 54,000 Da) in comparison to th e regulatory subunit of the cytoplasmic protein kinase A (52,000 Da). We find that lipolytic cleavage of the two membrane anchors by phospha tidylinositol-specific phospholipases C and D results in significantly higher association and lower dissociation rates of cAMP, thus leading to a dramatic increase in ligand affinity of the two cAMP receptors. Use of cAMP analogues identifies two different cAMP-binding centers in each membrane-embedded protein, one of which is noticeably affected b y the cleavage of the anchor. In both phosphatidylinositol-anchored cA MP receptor proteins a single Trp residue in one of the binding center s is photoaffinity-labeled by 8-N-3-cAMP, whereas two amino acids, Trp and Tyr, are modified after lipolytic removal of the anchor. The diff erences in the labeling patterns are interpreted as to result from a c onformational rearrangement induced by the cleavage of the anchor. Tog ether with the increased affinity to the ligand these changes document alterations of the properties and folding structure of lipid-anchored proteins following cleavage of the PI-containing anchor by specific p hospholipases and provide the first molecular evidence for a regulator y role of the anchorage by a lipid structure. The cytoplasmic regulato ry subunit of yeast protein kinase A is not photolabeled to a signific ant extent under any condition. (C) 1994 Academic Press, Inc.