Yra1 is required for S phase entry and affects Dia2 binding to replication origins.

The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels. Like Dia2, Yra1 associates with chromatin and binds replication origins, suggesting that they may function together in DNA replication. Consistent with this idea, Yra1 and Dia2 coimmunoprecipitate with Hys2, a subunit of DNA polymerase delta. The C terminus of Yra1 is required to interact with Dia2. A yra1 mutant that lacks this domain is temperature sensitive yet has no apparent defect in RNA export. Remarkably, this mutant also fails to enter S phase at the nonpermissive temperature. Significantly, other mutants in transcription-coupled export do not exhibit S phase entry defects or sensitivity to DIA2 expression levels. Together, these results indicate that Yra1 has a role in DNA replication distinct from its role in mRNA export. Furthermore, Dia2 binding to replication origins is significantly reduced when association with Yra1 is compromised, suggesting that one aspect of the role of Yra1 in DNA replication may involve recruiting Dia2 to chromatin.

The F-box protein Dia2 regulates DNA replication.

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCF(Dia2) activity, as a Dia2 DeltaF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.

Rck2 is required for reprogramming of ribosomes during oxidative stress.

Rck2 is a mitogen-activated protein kinase-activated protein kinase in yeast implicated in translational regulation. rck2Delta mutants are mildly sensitive to oxidative stress, a condition that causes dissociation of actively translating ribosomes (polysomes). In rck2Delta cells, polysomes are lost to an even higher degree than in the wild-type upon stress. Cells overexpressing the catalytically inactive rck2-kd allele are highly sensitive to oxidative stress. In such cells, dissociation of polysomes upon stress was instead greatly delayed. The protein synthesis rate decreased to a similar degree as in wild-type cells, however, indicating that in rck2-kd cells, the polysome complexes were inactive. Array analyses of total and polysome-associated mRNAs revealed major deregulation of the translational machinery in rck2 mutant cells. This involves transcripts for cytosolic ribosomal proteins and for processing and assembly of ribosomes. In rck2Delta cells, weakly transcribed mRNAs associate more avidly with polysomes than in wild-type cells, whereas the opposite holds true for rck2-kd cells. This is consistent with perturbed regulation of translation elongation, which is predicted to alter the ratio between mRNAs with and without strong entry sites at ribosomes. We infer that imbalances in the translational apparatus are a major reason for the inability of these cells to respond to stress.

Degradation of Saccharomyces cervisiae Rck2 upon exposure of cells to high levels of zinc is dependent on Pep4.

In undisturbed cells, the MAPK-activated protein kinase Rck2 of Saccharomyces cerevisiae is a stable protein with a turnover time exceeding 60 min. However, we have found that Rck2 is subject to intracellular degradation after exposure of cells to Zn2+ concentrations of 5 mM or more. In high-zinc medium, most of the Rck2 pool is degraded within 5 min. This degradation is blocked by inhibiting the vacuolar proteolytic pathway with the protease inhibitor phenyl methyl sulphonyl fluoride or by mutation of the PEP4 gene. By contrast, blocking the proteasomal pathway with the inhibitor MG132 does not prevent Rck2 degradation upon addition of Zn2+, nor is degradation inhibited in the proteasomal mutations pre1 pre2, cim3, or cim5. The stability of Rck2 is not affected by any of the other stress conditions examined, or by growth rate. Possible mechanisms of the degradation of Rck2 under high zinc conditions, and its physiological significance, are discussed.

Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance.

We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae. We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances. Hog1 concentrates in the nucleus in oxidative stress. Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells. We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen. We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2. Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents. In a fraction of untreated pbs2 cells, Yap2 is nuclear. Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2. These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.

Evolution and cellular function of monothiol glutaredoxins: involvement in iron-sulphur cluster assembly.

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron-sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron-sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.