AN ACTIN MONOMER BINDING-ACTIVITY LOCALIZES TO THE CARBOXYL-TERMINAL HALF OF THE SACCHAROMYCES-CEREVISIAE CYCLASE-ASSOCIATED PROTEIN
AN ACTIN MONOMER BINDING-ACTIVITY LOCALIZES TO THE CARBOXYL-TERMINAL HALF OF THE SACCHAROMYCES-CEREVISIAE CYCLASE-ASSOCIATED PROTEIN
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DOI：
10.1074/jbc.270.10.5680
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发表时间：
1995-03-10
影响因子：
4.8
通讯作者：
FIELD, J
中科院分区：
文献类型：
--
作者：
FREEMAN, NL;CHEN, ZX;FIELD, J
酿酒酵母腺苷酸环化酶复合物含有至少两个亚基，200-kDa催化亚基和70-kDa环化酶相关蛋白CAP（也称为Srv 2 p）。遗传研究表明CAP有两个作用，一个是作为酵母中cAMP水平的正调节剂，另一个是作为细胞骨架调节剂。我们目前的证据表明，CAP螯合单体肌动蛋白（K-D在0.5-5 μ M的范围内），减少肌动蛋白纳入肌动蛋白丝。抗CAP单克隆抗体共免疫沉淀分子大小约为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蛋白质与CAP以约1：1的化学计量共纯化。蛋白质印迹鉴定46 kDa蛋白为酵母肌动蛋白。CAP也结合到肌肉肌动蛋白在体外的免疫沉淀试验和落球粘度测定。芘标记的肌动蛋白的实验表明，CAP螯合肌动蛋白单体。肌动蛋白单体结合活性定位于CAP的羧基末端的一半。总之，这些数据表明，酵母CAP调节酵母细胞骨架螯合肌动蛋白单体。
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 (K-d in the range of 0.5-5 mu M), 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.