The nuclear Ras-related protein Ran binds guanine nucleotide and is involved in cell cycle regulation. Models of the signal pathway predict Ran to be active as Ran.GTP at the initiation of S phase upon activation by the nucleotide exchange factor RCC1 and to be inactivated for the onset of mitosis by hydrolysis of bound GTP. Here a nuclear homodimeric 65-kDa protein, RanGAP1, is described, which we believe to be the immediate antagonist of RCC1. It was purified from HeLa cell lysates and induces GTPase activity of Ran, but not Ras, by more than 3 orders of magnitude. The Ran mutant Q69L, modeled after RasQ61L, which is unable to hydrolyze bound GTP, is insensitive to RanGAP1.

https://www.pnas.org/content/pnas/91/7/2587.full.pdf

RanGAP1 induces GTPase activity of nuclear Ras-related Ran.

RAN/TC4 is a small nuclear G protein1 that forms a complex with the chromatin-bound guanine nucleotide release factor RCC1 (ref. 2). Loss of RCC1 causes defects in cell cycle progression3,4, RNA export5-7 and nuclear protein import8. Some of these can be suppressed by overexpression of Ran/TC4 (ref. 1), suggesting that Ran/TC4 functions downstream of RCC1. We have searched for proteins that bind Ran/TC4 by using a two-hybrid screen, and here we report the identification of RanBP2, a novel protein of 3,224 residues. This giant protein comprises an amino-terminal 700-residue leucine-rich region, four RanBPl-homologous (refs 9, 10) domains, eight zinc-finger motifs similar to those of NUP153 (refs 11, 12), and a carboxy terminus with high homology to cyclophilin13. The molecule contains the XFXFG pentapeptide motif characteristic of nuclear pore complex (NPC) proteins14, and immunolocalization suggests that RanBP2 is a constituent of the NPC. The fact that NLS-mediated nuclear import can be inhibited by an antibody directed against RanBP2 supports a functional role in protein import through the NPC.

A giant nucleopore protein that binds Ran/TC4.

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8 acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.

Arabidopsis UVR8 Regulates Ultraviolet-B Signal Transduction and Tolerance and Contains Sequence Similarity to Human Regulator of Chromatin Condensation 1

A rapid molecular response is induced in all eukaryotic cells on exposure to adverse environmental conditions. The response invariably involves changes in the level of gene transcription resulting from modulation of the activity of particular transcription factors (Treisman 1996). The link between stress signals and the transcription machinery is provided by certain MAP kinase signaling pathways that are strongly activated by a variety of adverse stimuli including osmotic, oxidative, and heat shock, UV light, and inhibition of protein synthesis. In mammalian cells these pathways are also activated by agonists, such as proinflammatory cytokines, which are released following stress or infection (for review, see Waskiewicz and Cooper 1995; Kyriakis and Avruch 1996). There are two classes of stress-activated MAP kinase pathways in mammalian cells: the stress-activated protein kinases (SAPKs), also known as the c-Jun-amino-terminal kinases (JNKs), and the p38/RK/CSBP kinases (Marshall 1994; Kyriakis and Avruch 1996). These pathways have been implicated in a number of biological processes, many of which are related to the stress response; they include cell transformation (Rodrigues et al. 1997), the promotion of apoptosis, and the synthesis of certain cytokines (for reviews, see Kyriakis and Avruch 1996; Cohen 1997; Karin et al 1997).

An important feature of these kinase pathways is their high degree of conservation: Structurally similar stress-activated kinases have been identified in Drosophila (Riesgo-Escovar et al. 1996; Sluss et al. 1996) as well as in budding (Brewster et al. 1993) and fission yeast (Millar et al. 1995; Shiozaki and Russell 1995; Kato et al. 1996). In fission yeast, the stress activated kinase Sty1 (also known as Spc1 and Phh1) is necessary for the cell to survive a number of different stress conditions including osmotic stress, UV irradiation, and nutrient limitation (Warbrick and Fantes 1991; Millar et al. 1995; Shiozaki and Russell 1995; Degols et al. 1996; Kato et al. 1996; Shiozaki and Russell 1996; Degols and Russell 1997).

Similar transcription factors have been found to be substrates for the stress activated kinases in mammalian cells, Drosophila, and fission yeast. Two transcription factor targets for the mammalian stress activated kinases are c-Jun (Hibi et al. 1993; Derijard et al. 1994; Kyriakis et al. 1994) and ATF2 (Gupta et al. 1995; Livingstone et al. 1995; Raingeaud et al. 1995; van Dam et al. 1995), both of which contain bZip DNA binding/dimerization domains. In both cases, phosphorylation enhances the transcriptional activity of the factors, the important phosphorylation sites being within the functionally defined transcriptional activation domains. The factors also contain a high affinity binding site for the kinases that is distinct from the phosphoacceptor sites. These interaction domains are crucial for phosphorylation and provide a mechanism for determining kinase specificity. A conserved link between kinase and transcription factor is found in Drosophila in which genetic evidence suggests that Drosophila Jun (DJun) is a critical target for DJNK (for review, see Noselli 1998). In the case of fission yeast, both biochemical and genetic evidence has demonstrated that one target of the Sty1 kinase is the bZip containing transcription factor Atf1, which shares homology to the mammalian factor ATF2 (Shiozaki and Russell 1996; Wilkinson et al. 1996). In addition, Sty1 and Atf1 can form a stable complex.

Inactivation of the fission yeast Sty1 kinase, or upstream components of the signaling pathway, results in a pleiotropic phenotype. sty1− cells are sensitive to osmotic stress, heat stress, oxidative stress, and UV irradiation; additionally they are defective in their response to nutrient deprivation (Millar et al. 1995; Shiozaki and Russell 1995; Kato et al. 1996; Degols and Russell 1997). When starved for nutrients, fission yeast cells exit the cell cycle and enter a metabolically inert stationary phase or, if mating partners are available, undergo sexual development culminating in meiosis and spore formation. However, sty1− cells are profoundly sterile and rapidly lose viability on growth to saturation indicating a failure to enter stationary phase. These defects emphasize the pivotal role the Sty1 kinase plays in the stress response of fission yeast cells. Sty1 also has a role in the regulation of cell cycle progression. Cells lacking Sty1 are substantially longer at cell division and show synthetic lethality with loss of the Cdc25 tyrosine phosphatase, a known regulator of entry into mitosis (Warbrick and Fantes 1991; Millar et al. 1992, 1995; Shiozaki and Russell 1995). To date, the only known substrate for the Sty1 kinase is the transcription factor Atf1. Cells lacking Atf1 show some, but not all of the phenotypes demonstrated by sty1− cells: They are sterile and sensitive to osmotic stress (Kanoh et al. 1996; Shiozaki and Russell 1996; Wilkinson et al. 1996) but are not sensitive to oxidative stress or to UV irradiation (Degols and Russell 1997). Furthermore, atf1− cells show no obvious cell cycle defects (Kanoh et al. 1996; Shiozaki and Russell 1996; Wilkinson et al. 1996). These observations suggest that alternative Sty1 substrates exist including additional transcription factor(s) and a regulator of mitotic initiation.

In this report, we identify the transcription factor Pap1 as a downstream target of Sty1. Pap1 is a bZip containing protein that has homology and similar DNA binding specificity to the mammalian c-Jun protein (Toda et al. 1991). Previous studies have identified a role for Pap1 in multidrug resistance, its deletion resulting in sensitivity to a variety of toxic compounds (Toda et al. 1991; Kumada et al. 1996). We show that sty1− cells have a similar multidrug-sensitive phenotype and that two genes encoding members of the ABC transporter family are expressed in a Sty1- and Pap1-dependent manner. We also show that cells lacking Pap1 are sensitive to oxidative stress but not to osmotic stress or nutrient deprivation. This sensitivity is caused by the role that Pap1 plays in regulating the expression of multiple genes involved in the oxidative stress response. Thus pap1− cells and atf1− cells show differing and nonoverlapping subsets of the phenotypes observed in sty1− cells. Our data demonstrate that Pap1 is regulated at the level of nuclear localization in a stress- and Sty1-dependent manner. Furthermore, regulated localization appears to be mediated at the level of nuclear export rather than import, in a mechanism that is dependent on the nuclear export factor Crm1 (Exportin). Because Pap1 and Atf1 have homology to c-Jun and ATF2 respectively, their identification as transcription factor targets of Sty1 highlights a remarkable level of conservation in the stress response between fission yeast and mammalian cells.

/pdf/regulation-of-the-fission-yeast-transcription-factor-pap1-by-163lou5wbz.pdf

Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold. It may also bind to DNAvia a protein-protein complex. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

The 1.7 Å crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller

The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis. The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1. A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene. These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch. The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant. Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1994.tb06298.x

A fission yeast RCC1-related protein is required for the mitosis to interphase transition.

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A fission yeast RCC1-related protein is required for the mitosis to interphase transition.