The Irr1/Scc3 protein implicated in chromosome segregation in Saccharomyces cerevisiae has a dual nuclear-cytoplasmic localization

Background Correct chromosome segregation depends on the sister chromatid cohesion complex. The essential, evolutionarily conserved regulatory protein Irr1/Scc3, is responsible for the complex loading onto DNA and for its removal. We found that, unexpectedly, Irr1 is present not only in the nucleus but also in the cytoplasm. Results We show that Irr1 protein is enriched in the cytoplasm upon arrest of yeast cells in G1 phase following nitrogen starvation, diauxic shift or α-factor action, and also during normal cell cycle. Despite the presence of numerous Crm1-dependent export signals, the cytoplasmic pool of Irr1 is not derived through export from the nucleus but instead is simply retained in the cytoplasm. Cytoplasmic Irr1 interacts with the Imi1 protein implicated in glutathione homeostasis and mitochondrial integrity. Conclusions Besides regulation of the sister chromatid cohesion complex in the nucleus Irr1 appears to have an additional role in the cytoplasm, possibly through interaction with the cytoplasmic protein Imi1

Nuclear Import and Export Signals of Human Cohesins SA1/STAG1 and SA2/STAG2 Expressed in Saccharomyces cerevisiae

Abstract Background: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. Methodology/Principal Findings: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the Nterminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. Conclusions/Significance: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells

Irr1/Scc3 Cohesin Interacts with Rec8 in Meiotic Prophase of Saccharomyces cerevisiae

The meiotic cohesin complex of S. cerevisiae shares with the mitotic one the Irr1/Scc3, Smc1, and Smc3 subunits, while the meiosis-specific subunit Rec8 re-places mitotic subunit Scc1/Mcd1. We noticed earlier that the irr1-1 mutation (F658G) severely affected meiosis. The irr1-1/IRR1 cells were entering meiosis before having completed mitotic cell division. Using meiotic two-hybrid assay and co-immunoprecipita-tion we show that in cells arrested in pachytene due to a lack of a gene-regulatory factor Ndt80, the Irr1 protein interacts with Rec8p and the irr1-1 mutation abolishes this interaction. These findings indicate an important role of Irr1p in early stages of meiosis

Cohesin Irr1/Scc3 is likely to influence transcription in Saccharomyces cerevisiae via interaction with Mediator complex.

The evolutionarily conserved proteins forming sister chromatid cohesion complex are also involved in the regulation of gene transcription. The participation of SA2p (mammalian ortholog of yeast Irr1p, associated with the core of the complex) in the regulation of transcription is already described. Here we analyzed microarray profiles of gene expression of a Saccharomyces cerevisiae irr1-1/IRR1 heterozygous diploid strain. We report that expression of 33 genes is affected by the presence of the mutated Irr1-1p and identify those genes. This supports the suggested role of Irr1p in the regulation of transcription. We also indicate that Irr1p may interact with elements of transcriptional coactivator Mediator

Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease of the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level

SA2S shuttles between nucleus and cytoplasm in yeast cells.

<p>(A) Subcellular localization of SA2S-GFP was analyzed after addition of LMB (Crm1p inhibitor) to 40 ng/ml to cells in logarithmic phase of growth. Strain <i>CRM1-T539C</i> bears LMB-sensitive version of Crm1p. Fourth column shows a composite of two fields from a single experiment but photographed as separate images, as marked. (B) Localization of SA2S-GFP protein was analyzed in thermo-sensitive <i>crm1-1</i> mutant. Transfer of cells grown at 30°C to 37°C for 30 minutes caused nuclear shift of the fusion protein in 100% of cells. Third and fourth columns show a composite of two fields from a single experiment, as marked. On the right in (A) and (B) control experiments in wild-type yeast are shown. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. (C) Frequencies of cells localized predominantly to the cytoplasm (black) or to the nucleus (gray) in strains bearing <i>CRM1-T539C</i> (LMB-sensitive) or <i>crm1-1</i> (thermo-sensitive) versions of Crm1p, following LMB treatment or temperature shift, respectively. MN47 and ABL10 are corresponding control strains bearing wild type <i>CRM1</i> gene, subjected to the same treatments.</p

SA1 contains NLS functional in yeast between 34K and 53K.

<p>(A) – Cells expressing SA1Δ34–53-GFP. (B) – Cells expressing fusion protein SA1^1–71-GFP. DNA was stained with DAPI, GFP represents fluorescence of fusion proteins, VIS – transmitted light. Column (A) shows a composite of two fields from a single experiment but photographed as separate images, as marked. For subcellular localization of intact.</p

NLS of H2B does not confer nuclear localization on SA2S.

<p>(A) Yeast cells expressing fusion protein H2B^1–62-SA2S-GFP. (B) H2B^1–62-SA2S-GFP protein has predicted molecular weight. Diploid yeast strain <i>irr1Δ</i>/<i>IRR1</i> (lacking one copy of <i>IRR1</i> gene) was transformed with centromeric plasmid pUG35 bearing hybrid gene encoding the fusion protein. Details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038740#pone-0038740-g001" target="_blank">Figure 1</a>.</p

Plasmids used in this study.

<p>Abbreviations for description of plasmids: CEN, centromeric; 2 µ, episomal; MCS, multiple cloning site.</p