The Function of Yeast Adenylyl Cyclase-Associated Proteins in Regulation of Cell Growth and Cytoskeletal Structure

在芽殖酵母中，腺苷酸环化酶受Ras蛋白调节。我们发现腺苷酸环化酶与两种环化酶相关蛋白70-kDa CAP和50-kDa p50形成复合物，并分析了它们与环化酶的相互作用方式及其细胞功能. CAP是一种双功能蛋白;其N-末端区域是与环化酶结合以及环化酶对Ras的适当反应所必需的，而其C-末端区域可能参与细胞骨架调节。通过对CAP的突变分析，我们将其腺苷酸环化酶结合位点定位于N端36个氨基酸残基。在缺乏CAP的酵母中，36个残基足以引起适当的cAMP反应。此外，我们将环化酶的CAP结合位点定位到其C末端附近的118个残基。这两个结合位点都含有LXXLXXX的串联重复，表明它们相互作用的卷曲螺旋机制。当检查这些序列中亮氨酸残基的氨基酸取代的效果时，突变 ...更多信息 消除了CAP-环化酶的相互作用，证明了卷曲螺旋机制的重要性.我们发现Ras的翻译后修饰（尤其是法尼基化）是激活腺苷酸环化酶所必需的，而它对环化酶与Ras的结合亲和力没有影响。当环化酶缺乏其相关CAP时，Ras修饰对环化酶活化的刺激作用丧失。在该系统中，CAP的过表达恢复了有效的CAP结合以及修饰对环化酶的刺激作用。缺乏CAP结合位点的环化酶突变体对CAP过表达不能恢复的刺激效应不敏感。这些结果表明，与CAP的关联介导Ras翻译后修饰对环化酶激活的刺激.通过芘基肌动蛋白的方法，我们表明，CAP的C-末端区域具有G-肌动蛋白结合和螯合活性。发现p50不是环化酶复合物的固有组分，因此没有进一步分析。少

In budding yeast, adenylyl cyclase is regulated by Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their modes of interaction with cyclase and their cellular functions.1. CAP is a bifunctional protein ; its N-terminal region is required for association with cyclase and for proper response of cyclase to Ras, while its C-terminal region is presumably involved in cytoskeletal regulation. By mutational analyzes of CAP,we mapped its adenylyl cyclase-binding site to N-terminal 36-amino acid residues. The 36-residues were sufficient for invoking proper cAMP response in yeast lacking CAP.Also, we mapped the CAP-binding site of cyclase to 118 residues near its C terminus. Both of these binding sites contained tandem repetition of LXXLXXX,suggesting the coiled-coil mechanism for their interaction. When the effect of amino acid substitutions of the leucine residues in these sequences was examined, the mutations … More abolished the CAP-cyclase interaction, demonstrating the importance of the coiled-coil mechanism.2. We found that posttranslational modification (especially farnesylation) of Ras is required for activation of adenylyl cyclase, whereas it has no effect on the binding affinity of cyclase for Ras. When cyclase was devoid of its associating CAP,the stimulatory effect of Ras modification on cyclase activation was lost. In this system, overexpression of CAP resumed efficient CAP binding as well as the stimulatory effect of the modification on cyclase. Cyclase mutants lacking the CAP-binding site were insensitive to the stimulatry effect, which was not resumed by CAP overexpression. These results indicate that association with CAP mediates the stimulation of cyclase activation by Ras posttranslational modification.3. By the pyrenyl actin method, we showed that the CAP C-terminal region has G-actin-binding and -sequestering activities. p50 was found not an inherent component of the cyclase complex, and, therefore, was not analyzed any further. Less