Freeman N L, Chen Z, Horenstein J, Weber A, Field J
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1995 Mar 10;270(10):5680-5. doi: 10.1074/jbc.270.10.5680.
The Saccharomyces cerevisiae adenylyl cyclase complex contains at least two subunits, a 200-kDa catalytic subunit and a 70-kDa cyclase-associated protein, CAP (also called Srv2p). Genetic studies suggested two roles for CAP, one as a positive regulator of cAMP levels in yeast and a second role as a cytoskeletal regulator. We present evidence showing that CAP sequesters monomeric actin (Kd in the range of 0.5-5 microM), decreasing actin incorporation into actin filaments. Anti-CAP monoclonal antibodies co-immunoprecipitate a protein with a molecular size of about 46 kDa. When CAP was purified from yeast using an anti-CAP monoclonal antibody column, the 46-kDa protein co-purified with a stoichiometry of about 1:1 with CAP. Western blots identified the 46-kDa protein as yeast actin. CAP also bound to muscle actin in vitro in immunoprecipitation assays and falling ball viscometry assays. Experiments with pyrene-labeled actin demonstrated that CAP sequesters actin monomers. The actin monomer binding activity is localized to the carboxyl-terminal half of CAP. Together, these data suggest that yeast CAP regulates the yeast cytoskeleton by sequestering actin monomers.
酿酒酵母腺苷酸环化酶复合物至少包含两个亚基，一个200 kDa的催化亚基和一个70 kDa的环化酶相关蛋白CAP（也称为Srv2p）。遗传学研究表明CAP有两个作用，一是作为酵母中cAMP水平的正向调节因子，二是作为细胞骨架调节因子。我们提供的证据表明，CAP隔离单体肌动蛋白（Kd在0.5 - 5 microM范围内），减少肌动蛋白掺入肌动蛋白丝。抗CAP单克隆抗体共免疫沉淀出一种分子量约为46 kDa的蛋白质。当使用抗CAP单克隆抗体柱从酵母中纯化CAP时，46 kDa的蛋白质与CAP以约1:1的化学计量比共纯化。蛋白质免疫印迹法将46 kDa的蛋白质鉴定为酵母肌动蛋白。在免疫沉淀试验和落球粘度测定试验中，CAP在体外也与肌肉肌动蛋白结合。用芘标记的肌动蛋白进行的实验表明，CAP隔离肌动蛋白单体。肌动蛋白单体结合活性定位于CAP的羧基末端一半区域。这些数据共同表明，酵母CAP通过隔离肌动蛋白单体来调节酵母细胞骨架。
