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重要组成部分。出芽酵母酿酒酵母（Saccharomyces cerevisiae）利用一种钙信号传导机制，这种机制与人类细胞（如T淋巴细胞）对抗原的反应密切相关。在这两种系统中，钙信号都可以通过钙调磷酸酶感知，钙调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素a和FK506抑制。反过来，活化的钙调磷酸酶使特定的转录因子去磷酸化，然后这些转录因子集中在细胞核中并诱导特定靶基因的表达。最近发现了一个从酵母到人类保守的结合和调节钙调磷酸酶功能的新蛋白家族。本研究的一个主要目标是确定酵母蛋白Rcn1p如何对钙调磷酸酶的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一种与容性钙进入（CCE）相关的调节机制，有助于产生钙信号。通过Pmr1p Ca2+泵的失活，酵母分泌细胞器中的Ca2+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。本提案的其他目标包括鉴定和表征参与从胞内细胞器释放Ca2+的因素，感知这些室腔内Ca2+浓度，调节cch1p依赖性Ca2+通道，并激活质膜中另一个未知的Ca2+内流通道。钙调磷酸酶依赖性反馈调节这些Ca2+通道和Ca2+转运体消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。该项目的长期目标是阐明在所有细胞中运作的钙信号机制的组成部分，并为它们的相互作用和功能建立详细的工作模型。

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.