Nuclear transport and transcription.

The compartmentalization of DNA in the nucleus of eukaryotic cells establishes a connection between the nuclear transport machinery and the transcriptional apparatus. General transcription factors, as well as specific transcriptional activators and repressors, such as p53 and NF-AT, need to be imported into the nucleus following their translation. In addition, nuclear transport plays a crucial role in regulating the activity of many transcription factors.

The ins and outs of cell-polarity decisions.

The guanine-nucleotide-exchange factor Cdc24 is a critical regulator of cell polarity. Far1 is a key player in Cdc24 regulation, controlling Cdc24 activity in budding yeast by regulating its subcellular localization in response to two very different signals - one external and one internal.

Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

Roles of phosphorylation sites in regulating activity of the transcription factor Pho4.

Transcription factors are often phosphorylated at multiple sites. Here it is shown that multiple phosphorylation sites on the budding yeast transcription factor Pho4 play distinct and separable roles in regulating the factor's activity. Phosphorylation of Pho4 at two sites promotes the factor's nuclear export and phosphorylation at a third site inhibits its nuclear import. Phosphorylation of a fourth site blocks the interaction of Pho4 with the transcription factor Pho2. Multiple phosphorylation sites provide overlapping and partially redundant layers of regulation that function to efficiently control the activity of Pho4.

Phosphorylation of the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85.

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.

Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81.

A complex consisting of the cyclin-dependent kinase (CDK) PHO85 and the cyclin PHO80 phosphorylates and is thought to inactivate the transcription factor PHO4 when yeast cells are grown in medium containing high concentrations of phosphate. The CDK inhibitor PHO81 inhibits the kinase activity of the PHO80-PHO85 complex when Saccharomyces cerevisiae cells are grown in medium depleted of phosphate. A region of PHO81 with similarity to the mammalian CDK inhibitor p16INK4 is sufficient for inhibition in vitro. These studies demonstrate that CDK inhibitors are used to regulate kinases involved in processes other than cell cycle control and suggest that the ankyrin repeat motif may be commonly used for interaction with cyclin-CDK complexes.

Evidence that the leucine zipper is a coiled coil.

Recently, a hypothetical structure called a leucine zipper was proposed that defines a new class of DNA binding proteins. The common feature of these proteins is a region spanning approximately 30 amino acids that contains a periodic repeat of leucines every seven residues. A peptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 was synthesized and characterized. This peptide associates in the micromolar concentration range to form a very stable dimer of alpha helices with a parallel orientation. Although some features of the leucine zipper model are supported by our experimental data, the peptide has the characteristics of a coiled coil.

Preferential heterodimer formation by isolated leucine zippers from fos and jun.

The products of the nuclear oncogenes fos and jun are known to form heterodimers that bind to DNA and modulate transcription. Both proteins contain a leucine zipper that is important for heterodimer formation. Peptides corresponding to these leucine zippers were synthesized. When mixed, these peptides preferentially form heterodimers over homodimers by at least 1000-fold. Both homodimers and the heterodimer are parallel alpha helices. The leucine zipper regions from Fos and Jun therefore correspond to autonomous helical dimerization sites that are likely to be short coiled coils, and these regions are sufficient to determine the specificity of interaction between Fos and Jun. The Fos leucine zipper forms a relatively unstable homodimer. Instability of homodimers provides a thermodynamic driving force for preferential heterodimer formation.

Sequence requirements for coiled-coils: analysis with lambda repressor-GCN4 leucine zipper fusions.

A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage lambda repressor as a reporter for dimerization. This system was used to analyze the importance of the amino acid side chains at eight positions that form the hydrophobic interface of the leucine zipper dimer from the yeast transcriptional activator, GCN4. When single amino acid substitutions were analyzed, most functional variants contained hydrophobic residues at the dimer interface, while most nonfunctional sequence variants contained strongly polar or helix-breaking residues. In multiple randomization experiments, however, many combinations of hydrophobic residues were found to be nonfunctional, and leucines in the heptad repeat were shown to have a special function in leucine zipper dimerization.

X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil.

The x-ray crystal structure of a peptide corresponding to the leucine zipper of the yeast transcriptional activator GCN4 has been determined at 1.8 angstrom resolution. The peptide forms a parallel, two-stranded coiled coil of alpha helices packed as in the "knobs-into-holes" model proposed by Crick in 1953. Contacts between the helices include ion pairs and an extensive hydrophobic interface that contains a distinctive hydrogen bond. The conserved leucines, like the residues in the alternate hydrophobic repeat, make side-to-side interactions (as in a handshake) in every other layer of the dimer interface. The crystal structure of the GCN4 leucine zipper suggests a key role for the leucine repeat, but also shows how other features of the coiled coil contribute to dimer formation.

Regulation of PHO4 nuclear localization by the PHO80-PHO85 cyclin-CDK complex.

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.

Signaling phosphate starvation.

Phosphate starvation induces the transcription of several genes involved in phosphate metabolism in the budding yeast Saccharomyces cerevisiae. The signal transduction pathway that mediates this response consists of components that resemble those used to regulate the eukaryotic cell cycle; these include a cyclin-dependent kinase or CDK (Pho85), a cyclin (Pho80) and a CDK inhibitor (Pho81). The possibility that this pathway mediates cell-cycle responses to phosphate starvation is discussed.

Peptide 'Velcro': design of a heterodimeric coiled coil.

BACKGROUND: The leucine zipper is a protein structural motif involved in the dimerization of a number of transcription factors. We have previously shown that peptides corresponding to the leucine-zipper region of the Fos and Jun oncoproteins preferentially form heterodimeric coiled coils, and that simple principles involving electrostatic interactions are likely to determine the pairing specificity of coiled coils. A critical test of these principles is to use them as guidelines to design peptides with desired properties. RESULTS: Based on studies of the Fos, Jun and GCN4 leucine zippers, we have designed two peptides that are predominantly unfolded in isolation but which, when mixed, associate preferentially to form a stable, parallel, coiled-coil heterodimer. To favor heterodimer formation, we chose peptide sequences that would be predicted to give destabilizing electrostatic interactions in the homodimers that would be relieved in the heterodimer. The peptides have at least a 10(5)-fold preference for heterodimer formation, and the dissociation constant of the heterodimer in phosphate-buffered saline is approximately 30 nM at pH 7 and 20 degrees C. Studies of the pH and ionic strength dependence of stability confirm that heterodimer formation is favored largely as a result of electrostatic destabilization of the homodimers. CONCLUSIONS: Our successful design strategy supports previous conclusions about the mechanism of interaction between the Fos and Jun oncoproteins. These results have implications for protein design, as they show that it is possible to design peptides with simple sequences that have a very high preference to pair with one another. Finally, these sequences with 'Velcro'-like properties may have practical applications, including use as an affinity reagent, in lieu of an epitope tag, or as a way of bringing together two molecules in a cell.

An in vitro system recapitulates chromatin remodeling at the PHO5 promoter.

The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of the PHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5 minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5 promoter chromatin structure is changed.

Functional analysis of the cyclin-dependent kinase inhibitor Pho81 identifies a novel inhibitory domain.

In response to phosphate limitation, Saccharomyces cerevisiae induces transcription of a set of genes important for survival. A phosphate-responsive signal transduction pathway mediates this response by controlling the activity of the transcription factor Pho4. Three components of this signal transduction pathway resemble those used to regulate the eukaryotic cell cycle: a cyclin-dependent kinase (CDK), Pho85; a cyclin, Pho80; and a CDK inhibitor (CKI), Pho81. Pho81 forms a stable complex with Pho80-Pho85 under both high- and low-phosphate conditions, but it only inhibits the kinase when cells are starved for phosphate. Pho81 contains six tandem repeats of the ankyrin consensus domain homologous to the INK4 family of mammalian CKIs. INK4 proteins inhibit kinase activity through an interaction of the ankyrin repeats and the CDK subunits. Surprisingly, we find that a region of Pho81 containing 80 amino acids C terminal to the ankyrin repeats is necessary and sufficient for Pho81's CKI function. The ankyrin repeats of Pho81 appear to have no significant role in Pho81 inhibition. Our results suggest that Pho81 inhibits Pho80-Pho85 with a novel motif.

Multi-site phosphorylation of Pho4 by the cyclin-CDK Pho80-Pho85 is semi-processive with site preference.

As part of a nutrient-responsive signaling pathway, the budding yeast cyclin-CDK complex Pho80-Pho85 phosphorylates the transcription factor Pho4 on five sites and inactivates it. Here, we describe the kinetic reaction between Pho80-Pho85 and Pho4. Through experimentation and computer modeling we have determined that Pho80-Pho85 phosphorylates Pho4 in a semi-processive fashion that results from a balance between kcat and k(off). In addition, we show that Pho80-Pho85 phosphorylates certain sites preferentially. Phosphorylation of the site with the highest preference inhibits the transcriptional activity of Pho4 when it is in the nucleus, while phosphorylation of the lowest-preference sites is required for export of Pho4 from the nucleus. This method of phosphorylation may allow Pho80-Pho85 to quickly inactivate Pho4 in the nucleus and efficiently phosphorylate Pho4 to completion.

Phosphate transport and sensing in Saccharomyces cerevisiae.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.

Genetic evidence for a morphogenetic function of the Saccharomyces cerevisiae Pho85 cyclin-dependent kinase.

The Saccharomyces cerevisiae PHO85 gene encodes a nonessential cyclin-dependent kinase that associates with 10 cyclin subunits. To survey the functions provided by Pho85, we identified mutants that require PHO85 for viability. We identified mutations that define seven Pho Eighty-Five Requiring or Efr loci, six of which are previously identified genes-BEM2 (YER155C), SPT7 (YBR081C), GCR1 (YPL075W), SRB5 (YGR104C), HFI1 (YPL254W), and BCK1 (YJL095W)-with one novel gene (YMR212C). We found that mutations in the EFR genes involved in morphogenesis are specifically inviable when the Pho85-associated G1 cyclins encoded by PCL1 and PCL2 are absent. pcl1 Delta bem2, pcl1 Delta pcl2 Delta cla4 Delta, and pcl1 Delta pcl2 Delta cdc42-1 strains are inviable. pcl1 Delta pcl2 Delta mpk1 Delta, pcl1 Delta pcl2 Delta bck1, and pcl1 Delta pcl2 Delta cln1 Delta cln2 Delta strains are also inviable, but are rescued by osmotic stabilization with 1 m sorbitol. We propose that the G1 cyclins encoded by PCL1 and PCL2 positively regulate CDC42 or another morphogenesis promoting function.

A genetic study of signaling processes for repression of PHO5 transcription in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, transcription of a secreted acid phosphatase, PHO5, is repressed in response to high concentrations of extracellular inorganic phosphate. To investigate the signal transduction pathway leading to transcriptional regulation of PHO5, we carried out a genetic selection for mutants that express PHO5 constitutively. We then screened for mutants whose phenotypes are also dependent on the function of PHO81, which encodes an inhibitor of the Pho80p-Pho85p cyclin/cyclin-dependent kinase complex. These mutations are therefore likely to impair upstream functions in the signaling pathway, and they define five complementation groups. Mutations were found in a gene encoding a plasma membrane ATPase (PMA1), in genes required for the in vivo function of the phosphate transport system (PHO84 and PHO86), in a gene involved in the fatty acid synthesis pathway (ACC1), and in a novel, nonessential gene (PHO23). These mutants can be classified into two groups: pho84, pho86, and pma1 are defective in high-affinity phosphate uptake, whereas acc1 and pho23 are not, indicating that the two groups of mutations cause constitutive expression of PHO5 by distinct mechanisms. Our observations suggest that these gene products affect different aspects of the signal transduction pathway for PHO5 repression.

New perspectives on nuclear transport.

A central aspect of cellular function is the proper regulation of nucleocytoplasmic transport. In recent years, significant progress has been made in identifying and characterizing the essential components of the transport machinery. Despite these advances, some facets of this process are still unclear. Furthermore, recent work has uncovered novel molecules and mechanisms of nuclear transport. This review focuses on the unresolved and novel aspects of nuclear transport and explores issues in tRNA, snRNA, and mRNA export that highlight the diversity of nuclear transport mechanisms.