ABSTRACT
Information can be transferred between the nucleus and the cytoplasm by translocating macromolecules across the nuclear envelope. Communication of extracellular or intracellular changes to the nucleus frequently leads to a transcriptional response that allows cells to survive in a continuously changing environment. Eukaryotic cells have evolved ways to regulate this movement of macromolecules between the cytoplasm and the nucleus such that the transfer of information occurs only under conditions in which a transcriptional response is required. This review focuses on the ways in which cells regulate movement of proteins across the nuclear envelope and the significance of this regulation for controlling diverse biological processes.
Subject(s)
Cell Nucleus/metabolism , Animals , Biological Transport , Humans , Nuclear Localization Signals/physiologyABSTRACT
The movement of many transcription factors, kinases and replication factors between the nucleus and cytoplasm is important in regulating their activity. In some cases, phosphorylation of a protein regulates its entry into the nucleus; in others, it causes the protein to be exported to the cytoplasm. The mechanism by which phosphorylation promotes protein export from the nucleus is poorly understood. Here we investigate how the export of the yeast transcription factor Pho4 is regulated in response to changes in phosphate availability. We show that phosphorylation of Pho4 by a nuclear complex of a cyclin with a cyclin-dependent kinase, Pho80-Pho85, triggers its export from the nucleus. We also find that the shuttling receptor used by Pho4 for nuclear export is the importin-beta-family member Msn5, which is required for nuclear export of Pho4 in vivo and binds only to phosphorylated Pho4 in the presence of the GTP-bound form of yeast Ran in vitro. Our results reveal a simple mechanism by which phosphorylation can control the nuclear export of a protein.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins , Fungal Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Biological Transport , Cloning, Molecular , Cytoplasm/metabolism , Escherichia coli , GTP Phosphohydrolases/metabolism , Karyopherins , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , ran GTP-Binding ProteinABSTRACT
The transcription factor Pho4 is phosphorylated and localized predominantly to the cytoplasm when budding yeast are grown in phosphate-rich medium and is unphosphorylated and localized to the nucleus upon phosphate starvation. We have investigated the requirements for nuclear import of Pho4 and find that Pho4 enters the nucleus via a nonclassical import pathway that utilizes the importin beta family member Pse1/Kap121. Pse1 binds directly to Pho4 and is required for its import in vivo. We have defined the nuclear localization signal on Pho4 and demonstrate that it is required for Pse1 binding in vitro and is sufficient for PSE1-dependent import in vivo. Phosphorylation of Pho4 inhibits its interaction with Pse1, providing a mechanism by which phosphorylation may regulate import of Pho4 in vivo.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins , Fungal Proteins/metabolism , Membrane Transport Proteins , Receptors, Cytoplasmic and Nuclear/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Binding Sites , Cloning, Molecular , Cytoplasm/metabolism , Escherichia coli , Kinetics , Phosphates/metabolism , Phosphorylation , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Transcription Factors/metabolismABSTRACT
PHO4, a transcription factor required for induction of the PHO5 gene in response to phosphate starvation, is phosphorylated by the PHO80-PHO85 cyclin-CDK (cyclin-dependent kinase) complex when yeast are grown in phosphate-rich medium. PHO4 was shown to be concentrated in the nucleus when yeast were starved for phosphate and was predominantly cytoplasmic when yeast were grown in phosphate-rich medium. The sites of phosphorylation on PHO4 were identified, and phosphorylation was shown to be required for full repression of PHO5 transcription when yeast were grown in high phosphate. Thus, phosphorylation of PHO4 by PHO80-PHO85 turns off PHO5 transcription by regulating the nuclear localization of PHO4.
Subject(s)
Cell Nucleus/metabolism , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA-Binding Proteins , Fungal Proteins/metabolism , Phosphate Transport Proteins , Repressor Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Culture Media , Cytoplasm/metabolism , Dipeptides/metabolism , Gene Expression Regulation, Fungal , Membrane Transport Proteins/genetics , Molecular Sequence Data , Mutation , Phosphates/metabolism , Phosphorylation , Saccharomyces cerevisiae/geneticsABSTRACT
Induction of the yeast gene PHO5 is mediated by the transcription factors PHO2 and PHO4. PHO5 transcription is not detectable in high phosphate; it is thought that the negative regulators PHO80 and PHO85 inactivate PHO2 and PHO4. Here it is reported that PHO80 has homology to yeast cyclins and interacts with PHO85, a p34cdc2/CDC28-related protein kinase. The PHO80-PHO85 complex phosphorylates PHO4; this phosphorylation is correlated with negative regulation of PHO5. These results demonstrate the existence of a cyclin-cdk complex that is used for a regulatory process other than cell-cycle control and identify a physiologically relevant substrate for this complex.
