A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae.
Purified plasma membranes from the yeast Saccharomyces cerevisiae bind about 1.2 pmol of cAMP/mg of protein with high affinity (Kd = 6 nM). By using photoaffinity labeling with 8-N3-[32P]cAMP, we have identified in plasma membrane vesicles a cAMP-binding protein (Mr = 54,000) that is present also in bcy1 disruption mutants, lacking the cytoplasmic R subunit of protein kinase A (PKA). This argues that it is genetically unrelated to PKA. Neither high salt, nor alkaline carbonate, nor cAMP extract the protein from the membrane, suggesting that it is not peripherally bound. The observation that (glycosyl)phosphatidylinositol-specific phospholipases (or nitrous acid) release the amphiphilic protein from the membrane, thereby converting it to a hydrophilic form, indicates anchorage by a glycolipidic membrane anchor. Treatment with N-glycanase reduces the Mr to 44,000-46,000 indicative of a modification by N-linked carbohydrate side chain(s). In addition to the action of a phospholipase, the efficient release from the membrane requires the removal of the carbohydrate side chain(s) or the presence of high salt or methyl alpha-mannopyranoside, suggesting complex interactions with the membrane involving not only the glycolipidic anchor but also the glycan side chain(s). Topological studies show that the protein is exposed to the periplasmic space, raising intriguing questions for the function of this protein.
Müller
G
G
Institut für Biochemie I, Universität Heidelberg, Federal Republic ofGermany.
Bandlow
W
W
D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein; D003593:Cytoplasm / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry*; D006031:Glycosylation; D008055:Lipids / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics; D011953:Receptors, Cyclic AMP / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000201:enzymology
D002241:Carbohydrates; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D005656:Fungal Proteins; D008055:Lipids; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D011494:Protein Kinases