Lipolytic membrane release of two phosphatidylinositol-anchored cAMP receptor proteins in yeast alters their ligand-binding parameters.

G Müller, W Bandlow

文献索引：Arch. Biochem. Biophys. 308(2) , 504-14, (1994)

全文：HTML全文

摘要

Two new cAMP-binding proteins have been discovered recently in Saccharomyces cerevisiae. They are genetically distinct from the regulatory subunit of cytoplasmic cAMP-dependent protein kinase A and are distinguished from the latter, in addition, by their anchorage through phosphatidylinositol-containing lipid and glycolipid structures to mitochondrial and plasma membranes, respectively (Müller and Bandlow, 1989 Biochemistry 28, 9957-9967, 1991, Biochemistry 30, 10181-10190). A nutritional upshift induces the cleavage of the anchor by a phospholipase C (Müller and Bandlow, 1993, J. Cell Biol. 122, 225-236). To test the idea that anchorage by (glycosyl)phosphatidyl-inositol influences cAMP-binding and has a regulatory function, we analyzed ligand binding to the two purified cAMP receptors (46,000 and 54,000 Da) in comparison to the regulatory subunit of the cytoplasmic protein kinase A (52,000 Da). We find that lipolytic cleavage of the two membrane anchors by phosphatidylinositol-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 by the cleavage of the anchor. In both phosphatidylinositol-anchored cAMP receptor proteins a single Trp residue in one of the binding centers is photoaffinity-labeled by 8-N3-cAMP, whereas two amino acids, Trp and Tyr, are modified after lipolytic removal of the anchor. The differences in the labeling patterns are interpreted as to result from a conformational rearrangement induced by the cleavage of the anchor. Together 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 phospholipases and provide the first molecular evidence for a regulatory role of the anchorage by a lipid structure. The cytoplasmic regulatory subunit of yeast protein kinase A is not photolabeled to a significant extent under any condition.