CALCIUM SIGNALING AND TRANSPORT IN S CEREVISIAE

钙信号是正常免疫细胞功能和心脏和大脑发育的关键组成部分。萌芽酵母利用一种钙信号机制，这种机制与T淋巴细胞等人类细胞对抗原的反应密切相关。在这两个系统中，钙信号都可以被钙调神经磷酸酶感受到。钙调神经磷酸酶是一种被钙离子/钙调蛋白激活并被免疫抑制剂环孢素A和FK506抑制的蛋白磷酸酶。反过来，激活的钙调神经磷酸酶使特定的转录因子去磷酸化，然后集中在细胞核中，诱导特定目标基因的表达。最近发现了一个新的蛋白质家族，它从酵母到人类都是保守的，可以结合和调节钙调神经磷酸酶的功能。这项建议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的影响。最近的研究还表明，酵母在淋巴细胞中维持着一种与容量性钙进入(CCE)相关的调节机制，有助于产生钙信号。通过失活Pmr1p钙泵耗尽酵母分泌细胞器中的钙离子，可以极大地增强与Cch1p有关的高亲和力钙通道的活性，Cch1p是动物体内电压门控钙通道的同源物。这一建议的其他目标包括鉴定和表征参与细胞内细胞器释放钙离子的因素，感知这些隔室管腔内的钙离子浓度，调节依赖于Cch1p的钙离子通道，以及激活质膜上另一个未知的钙离子内流通道。钙调神经磷酸酶依赖的反馈调节这些钙离子通道和钙离子转运体，耗散钙信号也将被评估。这些目标将通过结合遗传、分子、生理和生化方法来实现。这个项目的长期目标是阐明在所有细胞中运行的钙信号机制的组成部分，并为它们的相互作用和功能开发一个详细的工作模型。

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.