Anna Kurlandzka

The Gef1 protein of Saccharomyces cerevisiae is associated with chloride channel activity.

The Gef1 protein of the yeast Saccharomyces cerevisiae (Gef1p) has amino acid homology to the voltage-gated CLC chloride channel family. It has been postulated that it provides the compensatory transport of Cl- anions to the lumen of the Golgi thereby regulating the pH of this compartment. Using GEF1 fusion with heterologous promoter we obtained a yeast strain highly overproducing Gef1p. The electrophysiological properties of the microsomal fraction obtained from this strain were measured using lipid bilayer system. Our data indicate that Gef1p is associated with the chloride channel activity. This anion-selective channel has a unitary conductance of 42 pS when measured in symmetrical 600/600 mM TEA-Cl solutions, is voltage-dependent, and closes at high negative voltages.

The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p

The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

Proteins interacting with Lin1p, a putative link between chromosome segregation, mRNA splicing and DNA replication in Saccharomyces cerevisiae

Proteins involved in chromosome segregation during mitosis are likely to participate in other cell cycle‐coordinated processes. Using a two‐hybrid screen we identified a novel nuclear protein, Lin1, interacting with Irr1p/Scc3p, a component of the cohesin complex. The second round of two‐hybrid assay with Lin1p as the bait resulted in the identification of six proteins: Prp8, Slx5, Siz2, Wss1, Rfc1 and YIL149w. These proteins have previously been shown to participate in mRNA splicing, DNA replication, chromosome condensation, chromatid separation and alternative cohesion. We propose that Lin1p may constitute a link among these processes. Copyright

Mutants of Saccharomyces cerevisiae Partially Defective in the Last Steps of the Haem Biosynthetic Pathway: Isolation and Genetical Characterization

A novel method for the isolation of Saccharomyces cerevisiae mutants partially defective in haem synthesis is described. Mutant clones were identified by their fluorescence under UV light due to the accumulation of porphyrins in cells, and by their ability to grow on nonfermentable carbon sources due to their preserved haemoprotein synthesis. Thirteen such mutants were obtained by this procedure. The defects in haem synthesis and accumulation of porphyrins in all the mutants were confirmed by spectrophotometric analysis. Complementation tests with biochemically defined, haem-less strains showed that in seven mutants uroporphyrinogen decarboxylase was affected and that in three mutants the defect concerned ferrochelatase. The defects in the remaining three mutants were not defined.

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae

Murine DNA methyltransferases Dnmt1 and Dnmt3a were expressed in the yeast Saccharomyces cerevisiae. Adjustment to yeast preferences of the nucleotide sequences upstream and downstream of the translation initiation sites of both cDNAs was needed to obtain significant levels of the methyltransferases. Both proteins were correctly localized to the nucleus and their presence had no measurable influence on the functioning of yeast cells. Both Dnmt1 and Dnmt3a expressed in yeast cells were enzymatically active in vitro, and in vivo in the genomic DNA of the transgenic S. cerevisiae ca. 0.06% and 0.4%, respectively, of cytosines became methylated. This level of DNA methylation is about 100‐ to 10‐fold less than that observed in mammalian cells. The constructed system may be used to investigate the in vivo specificity of individual mammalian DNA methyltransferases and to search for additional factors needed to allow more efficient in vivo methylation of chromatin‐contained DNA and to study their mechanism of action. Copyright

Nuclear import and export signals of human cohesins SA1/STAG1 and SA2/STAG2 expressed in Saccharomyces cerevisiae.

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 N-terminal 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.

SACCHAROMYCES CEREVISIAE IRR1 PROTEIN IS INDIRECTLY INVOLVED IN COLONY FORMATION

The ability of a microorganism to adhere to a solid support and to initiate a colony is often the first stage of microbial infections. To date, studies on S. cerevisiae cell–cell and cell–solid support interactions concerned only cell agglutination during mating and flocculation. Colony formation has not been studied before probably because this species is not pathogenic. However, S. cerevisiae can be a convenient model to study this process, thanks to well‐developed genetics and the full knowledge of its nucleotide sequence. A preliminary characterization of the recently cloned essential IRR1 gene indicated that it may participate in cell–cell/substrate interactions. Here we show that lowering the level of expression of IRR1 (after fusion with a regulatory catalase A gene promoter) affects colony formation and disturbs zygote formation and spore germination. All these processes involve cell–cell or cell–solid support contacts. The IRR1 protein is localized in the cytosol as verified by immunofluorescence microscopy, and confirmed by cell fractionation and Western blotting. This indicates that Irr1p is not directly involved in the cell–solid support adhesion, but may be an element of a communication pathway between the cell and its surroundings. Copyright

The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.

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).

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 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.