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Müller, Günter; Schubert, Karin; Fiedler, Franz und Bandlow, Wolfhard (1992): The CAMP-binding Ectoprotein from Saccharomyces cereuisiae Is Membrane-anchored by Glycosyl-Phosphatidylinositol. In: Journal of Biological Chemistry, Bd. 267: S. 25337-25346 [PDF, 6MB]

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Abstract

Saccharomyces cerevisiae contains an amphiphilic CAMP-binding glycoprotein at the outer face of the plasma membrane (M, = 54,000). It is converted to a hydrophilic form byt reatment withg lycosyl-phosphatidylinositol- specific phospholipases C and D (GPIPLC/ D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxylterminal amino acido f the Pronase-digested proteitno which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphoruiss resistant to alkalinep hosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitroaucisd , and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of Nglycosylation by tunicamycin or removal of proteinbound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangemoefn t constitutents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast CAMP-binding protein by a GPI anchoris strengthened additionallyb y the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinoaf nttr ypanosomal variant surface glycoproteins.

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