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.
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