Mintzer K A, Field J
Department of Biological Sciences, Columbia University, New York, NY 10027.
Cell Signal. 1994 Aug;6(6):681-94. doi: 10.1016/0898-6568(94)90050-7.
The adenylyl cyclase complex from Saccharomyces cerevisiae contains at least two subunits, a catalytic subunit of M(r) 200,000, encoded by CYR1 and a cyclase associated subunit, of apparent M(r) 70,000, encoded by CAP. The complex is a major effector of RAS proteins in S. cerevisiae. The interactions between CAP, adenylyl cyclase and RAS were explored in a strain of yeast that lacked CAP and contained an epitope tagged adenylyl cyclase. Adenylyl cyclase activity in this strain was not immunoprecipitated with anti-CAP antibodies, but was immunoprecipitated with anti-epitope antibodies. Two anti-CAP polyclonal antisera and five anti-CAP monoclonal antibodies were used in these studies. Like CAP-bound adenylyl cyclase, the CAP-free adenylyl cyclase was fully activated by yeast RAS2. Transformation of cap strains with plasmids expressing portions of CAP allowed the adenylyl cyclase binding sites on CAP to be mapped by immunoprecipitation experiments. In other experiments, deletion mutations of adenylyl cyclase were used to map the CAP binding site on adenylyl cyclase. The adenylyl cyclase binding site localized to the amino one third of CAP (amino acids 1-168), and the CAP binding site localized to the carboxyl terminus of adenylyl cyclase (amino acids 1768-2026).
来自酿酒酵母的腺苷酸环化酶复合物至少包含两个亚基，一个由CYR1编码的分子量为200,000的催化亚基，以及一个由CAP编码的表观分子量为70,000的环化酶相关亚基。该复合物是酿酒酵母中RAS蛋白的主要效应物。在一个缺乏CAP且含有表位标记的腺苷酸环化酶的酵母菌株中，研究了CAP、腺苷酸环化酶和RAS之间的相互作用。该菌株中的腺苷酸环化酶活性不能用抗CAP抗体进行免疫沉淀，但能用抗表位抗体进行免疫沉淀。在这些研究中使用了两种抗CAP多克隆抗血清和五种抗CAP单克隆抗体。与结合了CAP的腺苷酸环化酶一样，无CAP的腺苷酸环化酶也能被酵母RAS2完全激活。用表达CAP部分片段的质粒转化cap菌株，通过免疫沉淀实验可以定位CAP上的腺苷酸环化酶结合位点。在其他实验中，利用腺苷酸环化酶的缺失突变来定位腺苷酸环化酶上的CAP结合位点。腺苷酸环化酶结合位点定位于CAP的氨基端三分之一（氨基酸1 - 168），而CAP结合位点定位于腺苷酸环化酶的羧基末端（氨基酸1768 - 2026）。
