Müller G, Bandlow W
Hoechst AG, Pharmaceutical Research Division, Metabolism, Frankfurt, Federal Republic of Germany.
Eur J Biochem. 1991 Dec 5;202(2):299-308. doi: 10.1111/j.1432-1033.1991.tb16376.x.
We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP-binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins. Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.
我们发现，除了已被充分表征的细胞质环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）的调节（R）亚基外，酿酒酵母还含有两种cAMP结合蛋白。我们提供的证据表明，它们构成了一种新型的cAMP受体，通过共价连接的脂质结构锚定在膜上。它们在基因上与细胞质R亚基无关。在sral突变体中可以检测到相应的蛋白质，在该突变体中PKA的R亚基基因已被破坏，并且针对细胞质R亚基产生的单克隆抗体与这两种膜结合的cAMP结合蛋白不发生交叉反应。此外，它们在位置以及光亲和标记蛋白的肽图方面也与细胞质蛋白不同。尽管它们在分子量和亚细胞定位上彼此不同，但cAMP结合域的肽图彼此相似，并且两种蛋白质都通过脂质结构锚定在膜上，一种锚定在质膜的外表面，另一种锚定在内线粒体膜的外表面。两种锚定都可以被乙醇胺、肌醇和脂肪酸进行代谢标记。此外，来自质膜的cAMP受体的锚定结构可以被葡糖胺和甘露糖进行放射性标记。用来自锥虫的糖基磷脂酰肌醇特异性磷脂酶C切割锚定后，从质膜溶解的cAMP结合蛋白与特异性识别可溶性锥虫表面蛋白交叉反应决定簇的抗体发生反应，表明锚定具有相似性。降解研究也表明质膜锚定具有糖基磷脂酰肌醇的性质，而线粒体对应物则不太复杂，因为它缺乏碳水化合物。此外，质膜cAMP受体还被一个N - 糖苷键连接的碳水化合物侧链修饰，这主要是其较高分子量的原因。
