REGULATION OF NUCLEOCYTOPLASMIC TRANSPORT

这项工作的长期目标是了解蛋白质的核输入和输出是如何通过磷酸化来调节信号转导途径的。我们正在研究Pho4的调控，Pho4是酵母PHO5基因转录诱导响应磷酸盐限制所需的DNA结合蛋白。PHO5转录调控的遗传和生化研究使我们对细胞外无机磷酸盐响应的信号转导途径有了详细的了解。我们之前的研究表明，磷酸盐响应信号转导通路调节Pho4的磷酸化状态，进而控制Pho4的亚细胞定位来调节PHO5的转录。我们最近的工作表明，Pho4通过非经典的输入途径，利用输入受体Pse1/Kap121导入细胞核。Pho4的磷酸化调节其与Pse1的相互作用，表明Pho4的磷酸化调节其进入细胞核。此外，Pho4的输出受磷酸化调控。我们已经开发了一种体外进口试验，我们可以用来研究酵母的不同进口途径。我们的目标是：(1)确定核输入受体Pse1与转录因子Pho4相互作用的条件，确定Pho4的磷酸化如何调节其与Pse1的相互作用，并确定Pse1的其他货物；(2)鉴定Pho4的核输出受体，并确定Pho4磷酸化如何调节其从细胞核输出；(3)为了进一步表征酵母体外导入系统，使用该系统直接测试Pho4导入是否需要Pse1，并使用纯化的可溶性组分开发酵母体外导入系统。这是一个很好的系统来研究如何调节核转运来控制蛋白质的活性，因为Pho4的磷酸化和定位的生理相关性已经很好地确立了。由于这种受调控的核定位系统与其他系统有相似之处，我们预计，对磷酸化如何调节Pho4定位的理解将作为理解其他系统的范例，并将为核胞质转运机制提供基本见解。

The long-term goal of this work is to understand how the nuclear import and export of proteins is regulated by phosphorylation in response to signal transduction pathways. We are studying the regulation of Pho4, a DNA binding protein required for transcriptional induction of the yeast PHO5 gene in response to phosphate limitation. Genetic and biochemical studies of the regulation of PHO5 transcription have led to a detailed understanding of the signal transduction pathway responsive to extracellular inorganic phosphate. Our prior studies have shown that the phosphate-responsive signal transduction pathway regulates the phosphorylation state of Pho4, which in turn controls its subcellular localization to regulate PHO5 transcription. Our recent work demonstrates that Pho4 is imported into the nucleus via a non-classical import pathway utilizing the import receptor Pse1/Kap121. Phosphorylation of Pho4 regulates its interaction with Pse1, suggesting that phosphorylation of Pho4 regulates its import into the nucleus. Additionally, export of Pho4 is regulated by phosphorylation. We have developed an in vitro import assay which we can use to study different import pathways in yeast. Our goals are: (1) To define the requirements for interaction between the nuclear import receptor Pse1 and the transcription factor Pho4, determine how phosphorylation of Pho4 regulates its interaction with Pse1, and identify other cargoes for Pse1; (2) To identify the nuclear export receptor for Pho4 and to determine how phosphorylation of Pho4 regulates its export from the nucleus; and (3) To characterize further a yeast in vitro import system, use this system to test directly if Pse1 is required for the import of Pho4, and develop a yeast in vitro import system using purified soluble components. This is an excellent system to study how nuclear transport is regulated to control the activity of a protein, as the physiological relevance of the phosphorylation and localization of Pho4 is well established. Because this system of regulated nuclear localization has parallels to others, we anticipate that an understanding of how Pho4 localization is regulated by phosphorylation will serve as a paradigm for understanding other systems and will provide fundamental insight into the mechanisms of nucleocytoplasmic transport.