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

在萌芽酵母中，腺酰环化酶是RAS蛋白的下游效应器。我们发现腺苷环化酶与两种环化酶相关蛋白：70-kDa CAP和50-kDa p50形成一个复合体，并对它们的结构和功能进行了分析。CAP是一种双功能蛋白，其N-末端与腺苷环化酶结合是必需的，而其C-末端与哺乳动物肌动蛋白结合蛋白ASP-56同源，可能参与细胞骨架的调节。基因破坏和突变研究表明，CAP对环化酶与RAS蛋白的相互作用不是必需的。我们在环化酶的C-末端指定了一个CAP结合位点，并创造了失去与CAP结合的能力的突变体。然而，在激活的RAS2；VAL9&>背景中，它们没有表现出异常的表型和对葡萄糖的夸大的cAMP反应，这是…更多的是RAS2；；lt；Vall9；细胞携带野生型环化酶的特征。相反，在野生型RAS2背景下，类似的周期酶基因替换并不影响cAMP反应。这些结果表明，虽然CAP不参与对野生型RAS的反应，但环化酶对激活的RAS的夸大反应是必需的。因此，我们找到了一种方法来抑制激活的RAS的夸大作用，而不影响野生型RAS的夸大作用。其背后的机制目前正在调查中。P50结合在腺酰环化酶富含亮氨酸的重复结构域上，这是一个RAS结合部位。纯化P50，测定N-末端氨基酸序列。结果表明，p50与核糖体大亚基蛋白L3相同。除了参与毛霉素类耐药外，L3的功能目前尚不清楚。我们正在制备针对L3的抗体，用于分析其功能。我们推测，L3与环化酶的联系可能代表了RAS途径与蛋白质合成机制之间的联系。较少

In budding yeast adenylyl cyclase is a downstream effector of Ras proteins. We found adenylyl cyclase forms a complex with two cyclase-associated proteins ; 70-kDa CAP and 50-kDa p50, and analyzed their structures and functions.1. CAP is a bifunctional protein ; its N-terminal region is required for association with adenylyl cyclase, while its C-terminal region is homologous to mammalian actin-binding protein ASP-56 and presumably involved in cytoskeletal regulation. Gene-disruption and mutational studies showed that CAP is not essential for interaction of cyclase with Ras proteins. We assigned a CAP-binding site of cyclase to its C-terminal domain and created mutants which lost the ability to bind CAP.Yeast cells whose chromosomal cyclase genes were replaced by the CAP-nonbinding mutants posessed cyclase activity fully responsive to Ras in vitro. However, in the activated RAS2^<Vall9> background they did not exhibit abnormal phenotypes and exaggerated cAMP response to glucouse, which … More were characteristic of RAS2^<Vall9> cells carrying wild-type cyclase. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the cAMP response. These results suggested that the association with CAP, although not involved in response to wild-type Ras, is required for the exaggerated response of cyclase to activated Ras. Thus, we found a way to suppress the exaggerated action of activated Ras without affecting that of wild-type Ras. Mechanisms underlying this are currently under investigation.2. p50 binds to leucine-rich repeats domain of adenylyl cyclase, a Ras-binding site. p50 was purified, and N-terminal amino acid sequence determaned. The result indicated p50 was identical with a ribosomal large subunit protein, L3. The function of L3 is presently unknown except for involvement in tricodermin-resistance. We are preparing antibody against L3 for analysis of its function. We speculate that the association of L3 with cyclase may represent a link between Ras pathway and protein synthesis machinery. Less