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反应信号转导途径来调节的。 我们正在研究Pho 4的调节，Pho 4是一种DNA结合蛋白，用于酵母PHO 5基因在磷酸盐限制下的转录诱导。 对PHO 5转录调节的遗传和生化研究使人们对响应细胞外无机磷酸盐的信号传导途径有了详细的了解。 我们先前的研究表明，磷酸盐响应信号转导途径调节磷酸化状态的磷酸化，这反过来又控制其亚细胞定位调节PHO 5转录。我们最近的工作表明，Pho 4是通过一个非经典的进口途径，利用进口受体Pse 1/Kap 121输入到细胞核。 Pho 4的磷酸化调节其与Pse 1的相互作用，表明Pho 4的磷酸化调节其进入细胞核。 此外，Pho 4的输出受磷酸化调节。 我们已经开发了一种体外输入测定法，我们可以用来研究不同的输入途径在酵母。 我们的目标是：（1）确定核输入受体Pse 1与转录因子Pho 4相互作用的条件，确定Pho 4的磷酸化如何调节其与Pse 1的相互作用，并确定Pse 1的其他货物：（2）确定Pho 4的核输出受体，并确定Pho 4的磷酸化如何调节其从核的输出;（3）为了进一步表征酵母体外导入系统，利用该系统直接检测Pho 4的导入是否需要Pse 1，并利用纯化的可溶性组分建立酵母体外导入系统。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，因为磷酸化和磷酸化磷酸化磷酸 因为这个系统的调节核定位与其他人有相似之处，我们预计，了解磷酸化磷酸

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.