Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein

Abstract

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (␣, oligomeric state unknown) and a small subunit R2 (␤ 2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe) 2-Y⅐] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (␤␤ ), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ␤␤ is redistributed from the nucleus to the cytoplasm in a checkpointdependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ␤␤ nuclear localization: the importin ␤ homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ␤␤ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ␤␤ . Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ␤␤ by Kap122 or as an anchor to retain ␤␤ in the nucleus. DNA-damage checkpoint ͉ subcellular redistributionT he levels and relative ratios of dNTP pools are important for high-fidelity DNA replication and repair (1). Failure to increase dNTP levels at the G 1 -to-S transition of the cell cycle is a lethal event at cellular level (2, 3). Conversely, elevated dNTP pools throughout the cell cycle lead to increased mutation rates (4-6). Imbalance in dNTP pools also contributes to mutagenesis by reducing the fidelity of DNA polymerases (7-9). Eukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to ensure proper dNTP pool sizes during the normal cell-cycle progression and in response to genotoxic stress (3, 10-14). A major target of such checkpoint regulation is ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate, an essential step in de novo biosynthesis of dNTPs (15).Class I RNRs were identified originally in Escherichia coli and are conserved from yeast to mammal (16). The mechanisms of enzymatic catalysis (17) and allosteric regulation (18, 19) have been studied extensively in E. coli and, more recently, in mice (20, 21). The archetype RNR holoenzyme consists of a large subunit R1 (␣, whose oligomeric state in eukaryotes is not completely understood) (22) and a homodimeric small subunit (␤ 2 ) (20). The eukaryotic R1 contains the catalytic site, an effector site that controls substrate specificity, an activity site that controls turnover, and a weak ATP-binding site that cont...

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

“…The two proteins act in the same pathway, as the R2 mislocalization phenotype does not differ between the kap122⌬ wtm1⌬ double mutant and each single mutant (51). Kap122 interacts with Wtm1 in vivo and is required for nuclear localization of Wtm1 (51). To determine whether Dif1 functions in the same pathway as Kap122 and Wtm1 in controlling R2 localization, we constructed dif1⌬ kap122⌬ and dif1⌬1 wtm1⌬ mutants and compared Rnr4 localization patterns of the double mutants to those of the dif1⌬, kap122⌬, and wtm1⌬ single mutants.…”

“…The karyopherin protein Kap122 and WD40 repeat protein Wtm1 have previously been shown to be required for proper nuclear localization of the R2 subunit (26,51). The two proteins act in the same pathway, as the R2 mislocalization phenotype does not differ between the kap122⌬ wtm1⌬ double mutant and each single mutant (51). Kap122 interacts with Wtm1 in vivo and is required for nuclear localization of Wtm1 (51).…”

“…In S. cerevisiae, the heterodimeric R2 subunit Rnr2-Rnr4 is cotransported between the nucleus and the cytoplasm (2). Proper nuclear localization of R2 requires the WD40 protein Wtm1, which acts as a nuclear anchor of R2, and the karyopherin Kap122, which is involved in importing Wtm1 into the nucleus (26,51). Subcellular localization of S. pombe R2 is controlled by the 127-residue protein Spd1 (29).…”

Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency

“…Finally, another checkpoint-dependent mechanism facilitates redistribution of Rnr2 and Rnr4 from the nucleus to the cytoplasm, where Rnr1 resides, in response to genotoxic stress (24). In this case, Dun1 kinase promotes Rnr2-Rnr4 heterodimer dissociation from its nuclear anchor protein Wtm1, and in the meantime prevents Rnr2-Rnr4 nuclear import by phosphorylating its importer protein Dif1 targeting it for degradation (17,(25)(26)(27).…”

Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage

“…However, Spd1 has no affinity to R2 (Suc22p) but instead specifically binds and inhibits R1 (Cdc22p) (14). In budding yeast, the Wtm1 protein instead was reported to act as a nuclear anchor for the small subunit (15,16).…”