ABSTRACT
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
The bovine papillomavirus type 1 (BPV-1) E2 translational open reading frame encodes three proteins that regulate viral transcription and DNA replication: the E2 transcriptional activator (E2TA), the E2 transcriptional repressor (E2TR) and the E8/E2 transcriptional repressor (E8/E2TR). E2TA is a strong activator of papillomaviral promoters and is required for viral DNA replication. E2TR and E8/E2TR inhibit the activities of E2TA but also possess weak transactivational properties of their own. Two components of the cellular transcription apparatus, TFIID and TFIIB, have previously been shown to associate with other viral and cellular transcriptional activators. We present evidence here that E2TA, the full-length E2 open reading frame gene product, directly binds both of these transcription factors in vitro. Glutathione S-transferase E2TA (GST-E2TA) fusion protein bound in vitro-synthesized TATA-box-binding protein (TBP), a component of TFIID, and in vitro-synthesized TFIIB. Likewise, GST-E2TA bound TFIID and TFIIB present in a nuclear extract from the human cervical cancer-derived cell line, HeLa. The binding of GST-E2TA to TBP and TFIIB required no additional mammalian factors, as shown by direct binding of GST-E2TA to bacterially synthesized recombinant TBP and recombinant TFIIB. The domain of E2TA required for its interaction with both TBP and TFIIB was localized to the C terminus of E2TA, a region also present in E2TR and E8/E2TR. This domain lies within the region of E2TA previously shown to confer cooperative DNA binding by E2TA and TBP and overlaps with the region of E2TA required for DNA binding and dimerization. Our findings, taken in context with previous studies, lead us to conclude that (i) cooperative DNA binding by E2 proteins and TBP is likely mediated by the direct binding of E2 proteins to TBP, (ii) the weak transcriptional transactivation by E2TR and E8/E2TR may result as a consequence of direct TBP and TFIIB binding by these proteins, and (iii) TBP and/or TFIIB binding may be required but is not sufficient for E2TA's strong transactivational activity.