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(1976) NMR in Biological Research: Peptides and Proteins, North-Holland Publishing Co., Amsterdam. 117-120. zyxwv A CAMP-Binding Ectoprotein in the Yeast Saccharomyces cerevisiae? Gunter Muller**f*i.and Wolfhard Bandlow11 - Institut fur Biochemie I der Universitat Heidelberg, Im Neuenheimer Feld 328, 0-6900 Heidelberg 1, Federal Republic of Germany, and Institut fur Genetik und Mikrobiologie der Universitat Miinchen, Maria- Ward-Strasse l a , 0-8000 Miinchen 19, Federal Republic zyxwv of Germany Received February 12, 1991; Revised Manuscript Received May 14, 1991 ABSTRACT: 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 (M, = 54 000) that is present also in bcyl 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 (glyco- sy1)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 M, to 44000-46000 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 a-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. In the yeast Saccharomyces cerevisiae, CAMP‘is known to influence a number of cellular processes and to link them to the nutritional situation of the cell. Among them are storage carbohydrate metabolism (Pall, 1981; Thomer, 1982), cell size regulation and cell cycle progression (Matsumoto et al., 1985; Baroni et al., 1989), sporulation (Matsumoto et al., 1983a,b), and transcription (Merino et al., 1989). Control of these functions is thought to be achieved through the activation of ‘This work was, in part, supported by a grant to W.B. from the *Present address: Hoechst AG Frankfurt a.M., Pharmaceutical Re- search Division, Metabolism, P.O. Box 80 03 20, D-6230 Frankfurt am Main 80, Federal Republic of Germany. 11 lnstitut fiir Genetik und Mikrobiologie der Universitat Miinchen. Deutsche Forschungsgemeinschaft. Institut fur Biochemie I der Universitat Heidelberg. the well-characterized cytoplasmic CAMP-dependent protein kinases (Beebe & Corbin, 1986) in response to the modulation I Abbreviations: CAMP, adenosine 3’,5’-cyclic monophosphate; C (R) subunit, catalytic (regulatory) subunit; DTT, dithiothreitol; EDTA, ethylendiaminetetraacetic acid; EGTA, ethylene glycol bis(&aminoethyl ether)-N,N,N’,N’-tetraacetic acid; GPI, glycosylphosphatidylinositol; HEPES, N-( 2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid; IBMX, 3-isobutyl-1-methylxanthine; MES, 2-(N-morpholino)-ethanesulfonic acid; a-MMP, methyl a-mannopyranoside; MOPS, 3-(N-morpholino)- propanesulfonic acid; N-glycanase, peptide N-glycohydrolase F; PEG, poly(ethy1ene glycol); (G)PI-PLC (D), (g1ycosyl)phosphatidylinositol- specific phospholipase C (D); PKA, CAMP-dependent protein kinase; PMSF, phenylmethanesulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCA, trichloroacetic acid; Tris, tris(hydroxymethy1)aminomethane; TX-114, Triton X-114, poly- (ethylene glycol) mono(octy1 phenyl ether). 0006-2960/91/0430-10181$02.50/0 0 1991 American Chemical Society