CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE

酿酒酵母中的钙信号传导和运输

• 批准号: 6891165
• 负责人: KYLE W CUNNINGHAM
• 金额: $ 10.9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891165
• 关键词: Saccharomyces cerevisiae; biological signal transduction; calcineurin; calcium channel; calcium flux; calcium transporting ATPase; cell free system; cell membrane; density gradient ultracentrifugation; enzyme activity; fluorescence microscopy; fungal proteins; gene complementation; gene expression; immunoprecipitation; membrane transport proteins; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; southern blotting; transcription factor; western blottings; yeast two hybrid system

Calcium signaling is a critical component of normal immune cell function and development of the heart and brain. The budding yeast Saccharomyces cerevisiae utilizes a calcium signaling mechanism that is closely related to those operating in human cells such as T lymphocytes in their response to antigens. In both of these systems, calcium signals can be sensed by calcineurin, a protein phosphatase that is activated by Ca2+/calmodulin and inhibited by the immunosuppressive drugs Cyclosporin A and FK506. In turn, activated calcineurin dephosphorylates specific transcription factors which then concentrate in the nucleus and induce expression of specific target genes. A new family of proteins conserved from yeast to humans that binds and regulates calcineurin function has been recently identified. One major goal of this proposal is to determine how the yeast protein Rcn1p exerts both positive and negative effects on calcineurin functions. Recent studies also show that yeast maintains a regulatory mechanism related to capacitative calcium entry (CCE) in lymphocytes that helps generate calcium signals. Depletion of Ca2+ from secretory organelles in yeast through inactivation of the Pmr1p Ca2+ pump strongly enhances the activity of a high-affinity Ca2+ channel involving Cch1p, a homolog of voltage-gated Ca2+ channels in animals. Additional goals of this proposal include the identification and characterization of factors involved in releasing Ca2+ from intracellular organelles, sensing Ca2+ concentration in the lumen of these compartments, regulating the Cch1p-dependent Ca2+ channel, and activating another unidentified Ca2+ influx channel in the plasma membrane. Calcineurin- dependent feedback regulation of these Ca2+ channels and of the Ca2+ transporters that dissipate calcium signals also will be evaluated. These goals will be accomplished by combining genetic, molecular, physiological, and biochemical methods. The broad long-term objectives of this project are to elucidate the components of the calcium signaling mechanism operating in all cells and to develop a detailed working model for their interactions and functions.

钙信号传导是正常免疫细胞功能以及心脏和大脑发育的关键组成部分。 芽殖酵母酿酒酵母利用钙信号传导机制，该机制与人类细胞（例如 T 淋巴细胞）对抗原的反应密切相关。 在这两个系统中，钙信号可以被钙调神经磷酸酶感知，钙调神经磷酸酶是一种蛋白磷酸酶，被 Ca2+/钙调蛋白激活，并被免疫抑制药物环孢菌素 A 和 FK506 抑制。 反过来，激活的钙调神经磷酸酶使特定转录因子去磷酸化，然后集中在细胞核中并诱导特定靶基因的表达。 最近发现了从酵母到人类保守的一个新的蛋白质家族，它可以结合并调节钙调神经磷酸酶的功能。 该提案的一个主要目标是确定酵母蛋白 Rcn1p 如何对钙调神经磷酸酶功能产生积极和消极影响。 最近的研究还表明，酵母维持着与淋巴细胞中的电容性钙进入（CCE）相关的调节机制，有助于产生钙信号。 通过 Pmr1p Ca2+ 泵失活，酵母分泌细胞器中 Ca2+ 的消耗强烈增强了涉及 Cch1p 的高亲和力 Ca2+ 通道的活性，Cch1p 是动物中电压门控 Ca2+ 通道的同源物。 该提案的其他目标包括识别和表征与从细胞内细胞器释放 Ca2+、感测这些区室腔内 Ca2+ 浓度、调节 Cch1p 依赖性 Ca2+ 通道以及激活质膜中另一个未识别的 Ca2+ 流入通道相关的因素。 还将评估这些 Ca2+ 通道和消散钙信号的 Ca2+ 转运蛋白的钙依赖磷酸酶依赖性反馈调节。 这些目标将通过结合遗传、分子、生理和生化方法来实现。 该项目的广泛长期目标是阐明在所有细胞中运行的钙信号传导机制的组成部分，并为其相互作用和功能开发详细的工作模型。