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

Slow Adaptive Response of Budding Yeast Cells to Stable Conditions of Continuous Culture Can Occur without Genome Modifications

Continuous cultures assure the invariability of environmental conditions and the metabolic state of cultured microorganisms, whereas batch-cultured cells undergo constant changes in nutrients availability. For that reason, continuous culture is sometimes employed in the whole transcriptome, whole proteome, or whole metabolome studies. However, the typical method for establishing uniform growth of a cell population, i.e., by limited chemostat, results in the enrichment of the cell population gene pool with mutations adaptive for starvation conditions. These adaptive changes can skew the results of large-scale studies. It is commonly assumed that these adaptations reflect changes in the genome, and this assumption has been confirmed experimentally in rare cases. Here we show that in a population of budding yeast cells grown for over 200 generations in continuous culture in non-limiting minimal medium and therefore not subject to selection pressure, remodeling of transcriptome occurs, but not as a result of the accumulation of adaptive mutations. The observed changes indicate a shift in the metabolic balance towards catabolism, a decrease in ribosome biogenesis, a decrease in general stress alertness, reorganization of the cell wall, and transactions occurring at the cell periphery. These adaptive changes signify the acquisition of a new lifestyle in a stable nonstressful environment. The absence of underlying adaptive mutations suggests these changes may be regulated by another mechanism

Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)

Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a “DNA cloud assay”. We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP

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

Communication YOR129c, a new element interacting with Cnm67p, a compo- nent of the spindle pole body of Saccharomyces cerevisiae

The Saccharomyces cerevisiae spindle pole body (SPB) consists of numerous pro-teins forming the outer, inner and central plaques. The protein Cnm67 is an important component of the outer plaque. The C-terminus of this protein contains a determinant important for its SPB localization. We identified a protein encoded by YOR129c which interacts with this C-terminus in the two-hybrid system. YOR129c and CNM67 exhibit weak genetic interaction. The double deletion strain yor129c cnm67 exhibits mod-erately increased resistance to 0.1 M LiCl and hygromycin B compared with the cnm67 single mutant. We propose that the YOR129c protein is an accessory factor associated with the cytoplasmic face of SPB and plays a role in cation homeostasis and/or multidrug resistance. The yeast spindle pole body (SPB) is the functional equivalent of the centrosome of an-imal cells and organizes microtubules by forming two poles of the mitotic spindle. The SPB is a multilaminar cylindrical structure and is anchored in the nuclear envelope via el-ements that project from its central plaque. The outer plaque of this organellum faces the cytoplasm, and the inner plaque is directed to the nucleus. Since there is no disassembly of the yeast nucleus during mitosis, the localiza-tion of the inner and outer plaques in relation to the nuclear envelope remains unchanged during the cell cycle (for a recent review see Vol. 50 No. 3/200

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

Additional copies of the NOG2 and IST2 genes suppress the deficiency of cohesin Irr1p/Scc3p in Saccharomyces cerevisiae.

The protein encoded by the IRR1/SCC3 gene is an element of the cohesin complex of Saccharomyces cerevisiae, responsible for establishing and maintaining sister chromatid cohesion during mitotic cell division. We noticed previously that lowering the level of expression of IRR1/SCC3 affects colony formation on solid support. Here we describe two dosage suppressors (IST2, NOG2) overcoming the inability to form colonies of an Irr1p-deficient strain. Ist2 is probably involved in osmotolerance, Nog2p is a putative GTPase required for 60S ribosomal subunit maturation, but may also participate in mRNA splicing

A compound C-terminal nuclear localization signal of human SA2 stromalin

Stromalins are evolutionarily conserved multifunctional proteins with the best known function in sister chromatid cohesion. Human SA2 stromalin, likely involved in the establishment of cohesion, contains numerous potential nuclear localization (NLS) and nuclear export signals (NES). Previously we have found that the C-terminus of SA2 contains NLS(s) functional in human cells. However, the identity of this signal remained unclear since three NLS-like sequences are present in that region. Here we analyzed the functionality of these putative signals by expressing GFP-tagged C-terminal part of SA2 or its fragments in a human cell line and in the yeast Saccharomyces cerevisiae. We found that in human cells the nuclear import is dependent on a unique compound di- or tripartite signal containing unusually long linkers between clusters of basic amino acids. Upon expression of the same SA2 fragment in yeast this signal is also functional and can be easily studied in more detail

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