Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers<sup>,</sup>

Sommerhalter, Monika; Voegtli, W.C.; Perlstein, Deborah L.; Ge, Jie; Stubbe, Jo Anne; Rosenzweig, Amy C.

doi:10.1021/bi049510m

Cited by 36 publications

(38 citation statements)

References 33 publications

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“…We measured the effect of the gene deletions on Rnr4 protein abundance and localization. Rnr4 is a ribonucleotide reductase regulatory subunit forming a functional heterodimer with Rnr2 (27). During normal cell growth Rnr4 is localized to the nucleus and partially relocates to the cytoplasm during S-phase.…”

Section: Time (Hours)mentioning

confidence: 99%

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud

Becker

Delgado-Gonzalo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

124

151

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Observing cellular responses to perturbations is central to generating and testing hypotheses in biology. We developed a massively parallel microchemostat array capable of growing and observing 1,152 yeast-GFP strains on the single-cell level with 20 min time resolution. We measured protein abundance and localization changes in 4,085 GFP-tagged strains in response to methyl methanesulfonate and analyzed 576 GFP strains in five additional conditions for a total of more than 10,000 unique experiments, providing a systematic view of the yeast proteome in flux. We observed that processing bodies formed rapidly and synchronously in response to UV irradiation, and in conjunction with 506 deletion-GFP strains, identified four gene disruptions leading to abnormal ribonucleotide-diphosphate reductase (Rnr4) localization. Our microchemostat platform enables the large-scale interrogation of proteomes in flux and permits the concurrent observation of protein abundance, localization, cell size, and growth parameters on the single-cell level for thousands of microbial cultures in one experiment.O bserving proteins in the cellular milieu has been a longstanding technical challenge in biology. One major advance was the development of GFP, enabling the visualization of proteins in vivo (1). High-content imaging has been primarily applied to mammalian cells, using either reverse transfection arrays or microtiter-based systems in which the slow doubling time of mammalian cells enables long-term imaging under static conditions (2, 3).The Saccharomyces cerevisiae GFP fusion library covering 4,159 proteins provided the first static view of global protein abundance, localization, and noise (4, 5). This library was recently used to establish the static differences in protein abundance and localization in response to DNA replication stress induced by methyl methanesulfonate (MMS) and hydroxyurea (HU) (6), in response to DTT, H 2 O 2 , and nitrogen starvation (7), and 800 cytoplasmic proteins were analyzed upon entry into stationary phase (8). These three recent large-scale screens all relied on standard microtiter plates for imaging the yeast strains at a single time point before and after perturbation. Meanwhile, microfluidic devices emerged as powerful tools for conducting complex time-lapse experiments on small to medium scales (9, 10), enabling the analysis of cellular network responses (11) and the implementation of synthetically engineered systems (12). However, it has thus far been technically impossible to interrogate thousands of continuously growing microbial strains with high spatiotemporal resolution in a single experiment.Despite the fact that a wealth of systems-level information is available for S. cerevisiae, the single-cell temporal dynamics of protein abundance and localization has not yet been measured on a systems scale. To enable such analyses we developed a microfluidic platform capable of growing and observing 1,152 yeast strains with a temporal resolution of 20 min. We explored the dynamic behavior of ∼2/3 of the...

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Section: Time (Hours)mentioning

confidence: 99%

A chemostat array enables the spatio-temporal analysis of the yeast proteome

Dénervaud

Becker

Delgado-Gonzalo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

124

151

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“…RNR1 is essential for mitotic growth, while RNR3 is highly inducible after DNA damage but dispensable for cell viability. The yeast R2 is a heterodimer of Rnr2 and Rnr4 (17,36,41). Only Rnr2 is capable of forming the (Fe) 2 -Y ⅐ cofactor (36).…”

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confidence: 99%

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

Huang

2008

Molecular and Cellular Biology

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Fidelity in DNA replication and repair requires adequate and balanced deoxyribonucleotide pools that are maintained primarily by regulation of ribonucleotide reductase (RNR). RNR is controlled via transcription, protein inhibitor association, and subcellular localization of its two subunits, R1 and R2. Saccharomyces cerevisiae Sml1 binds R1 and inhibits its activity, while Schizosaccharomyces pombe Spd1 impedes RNR holoenzyme formation by sequestering R2 in the nucleus away from the cytoplasmic R1. Here we report the identification and characterization of S. cerevisiae Dif1, a regulator of R2 nuclear localization and member of a new family of proteins sharing separate homologous domains with Spd1 and Sml1. Dif1 is localized in the cytoplasm and acts in a pathway different from the nuclear R2-anchoring protein Wtm1. Like Sml1 and Spd1, Dif1 is phosphorylated and degraded in cells encountering DNA damage, thereby relieving inhibition of RNR. A shared domain between Sml1 and Dif1 controls checkpoint kinase-mediated phosphorylation and degradation of the two proteins. Abolishing Dif1 phosphorylation stabilizes the protein and delays damage-induced nucleus-to-cytoplasm redistribution of R2. This study suggests that Dif1 is required for nuclear import of the R2 subunit and plays an essential role in regulating the dynamic RNR subcellular localization.Maintenance of genomic stability depends on faithful replication of DNA and repair of lesions after damage. Fidelity of both DNA replication and repair is influenced by perturbation in the sizes and relative ratios of cellular deoxynucleotide triphosphate (dNTP) pools. Ribonucleotide reductase (RNR) catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms and is largely responsible for maintaining cellular dNTP pools (34). As maximal DNA synthesis requires high concentrations of dNTPs, the RNR activity plays an important role in cell proliferation (31). On the other hand, increased RNR activity has been associated with malignant transformation and resistance to chemotherapy (12,14,25,56).The class I RNR holoenzymes are commonly found in eukaryotes and eubacteria and comprise two subunits, R1 and R2 (34). The active site and multiple binding sites for allosteric effectors reside in R1 (20), which can exist as a dimer, tetramer, and hexamer depending on the nucleotides present and their concentrations (21,37,47). R2 is a homodimer or heterodimer that houses a diferric-tyrosyl radical cofactor [(Fe) 2 -Y ⅐ ] essential for nucleotide reduction (36,40,42). Mammalian genomes contain a single R1 gene and two R2 genes; the cell cycle-regulated RRM2 is responsible for providing dNTPs in actively dividing cells, and the DNA damage-inducible p53R2 is required for replenishing dNTP pools in cells under genotoxic stress (9,22,44). Loss of p53R2 causes mitochondrial DNA depletion and increased apoptosis (22). The budding yeast Saccharomyces cerevisiae has two R1 genes, RNR1 and RNR3. RNR1 is essential for mitotic growth, while RNR3 is high...

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“…Recent studies (26)(27)(28) have established the fact that the active form of R2 is the ␤␤Ј heterodimer (formerly designated Rnr2-Rnr4). Only ␤ is capable of forming the (Fe) 2 -Y⅐ cofactor (26,27), whereas ␤Ј facilitates cofactor assembly and stabilizes the resulting holoheterodimer (26)(27)(28)(29)(30).…”

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confidence: 99%

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

Zhang

Yang

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers^,

Cited by 36 publications

References 33 publications

A chemostat array enables the spatio-temporal analysis of the yeast proteome

A chemostat array enables the spatio-temporal analysis of the yeast proteome

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

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

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Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers,

Cited by 36 publications

References 33 publications

A chemostat array enables the spatio-temporal analysis of the yeast proteome

A chemostat array enables the spatio-temporal analysis of the yeast proteome

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

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

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Structures of the Yeast Ribonucleotide Reductase Rnr2 and Rnr4 Homodimers^,