Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
Mutation of the cohesin related gene PDS5 causes cell death with predominant apoptotic features in Saccharomyces cerevisiae during early meiosis
Introduction
Apoptosis is an active form of cell death that functions to rapidly remove infected, mutated or other potentially dangerous cells. Constant turnover of cells driven by apoptosis is important for homeostasis and embryonic development. Apoptosis is characterized by certain cellular and biomedical changes of the cell, such as, externalization of phosphatidylserine on cell surface, condensation and fragmentation of chromatin, fragmentation of nuclei and reduction of mitochondrial membrane potential [1].
Because of its altruistic functions, it seemed that apoptosis would only offer the advantage to multicellular systems. Unicellular organisms, like yeast would not need this suicide program. Furthermore, no obvious homologues of the major apoptotic regulators described in metazoan (e.g. caspases, Bax, Bak, Bcl-2 and p53) have been identified in yeast [2]. However, cell death with apoptotic characteristics has been reported in yeast in recent years. In Saccharomyces cerevisiae, for example, apoptosis can be induced by mutation of CDC48 [3], oxidative stress [4], aging [5], [6] and other external signals, including acetic acid [7] and salt [8]. Expression of several mammalian pro-apoptotic genes also caused apoptotic cell death in yeast [2], [9]. Recent studies of YCA1/MCA1 in yeast provided direct evidence for the existence of a caspase-related gene in unicellular organisms [10], [11]. Based on the study of the histone chaperone ASF1/CIA1, Yamaki et al. suggested that yeast might undergo a prototypical active cell death, which possesses characteristics of both apoptosis and necrosis [12].
Cohesin is a protein complex that regulates sister chromatid cohesion and is required for ordered chromosome segregation. Cohesin is also involved in chromosome condensation and DNA break repair. In mitosis, cohesin contains at least four protein subunits, Smc1p, Smc3p, Mcd1p/Scc1p and Scc3p [13], [14]. Cohesin is also required in meiosis. REC8, the meiosis-specific homolog of mitotic cohesin MCD1/SCC1, is essential in meiosis, and is required for sister chromatid cohesion, axial element formation, chromosome pairing and turnover of DNA double-strand breaks toward completion of reciprocal recombination [15], [16], [17], [18]. Spo76p, a cohesin-related protein in Sordaria, has been shown to be essential for sister chromatid cohesion, and chromatin condensation in both mitosis and meiosis [19].
PDS5, the SPO76 homolog in S. cerevisiae, is an essential gene for both mitosis [20], [21] and meiosis (Zhang, unpublished data). Pds5p localizes to chromosomes in a cell cycle-dependent manner and is required for sister chromatid cohesion, chromosome condensation, DNA repair and proper chromosome segregation. Its binding to chromosomes is dependent on Mcd1p function [20] in mitosis and Rec8p in meiosis.
In addition to the function in chromatid cohesion, recent studies show that cohesin has a role in apoptosis [22], [23]. Human RAD21/MCD1/SCC1 is found as a nuclear caspase target. Induction of apoptosis by diverse stimuli causes the cleavage of hRad21. The cleaved C-terminal product of hRAD21 is translocated from the nucleus to cytoplasm and acts as a nuclear signal for apoptosis. In C. elegans, apoptotic nuclei, as indicated by DAPI staining, were observed in the gonads of adult evl-14/pds-5 and scc-3 mutants, suggesting a role of these cohesin related genes in apoptotic cell death [24].
Here we report that mutation of PDS5 causes cell death in yeast S. cerevisiae that displays characteristics of both apoptosis and necrosis, including externalization of phosphatidylserine in the cytoplasmic membrane, accumulation of DNA breaks, condensation and fragmentation of chromatin, fragmentation of nuclei and cell size enlargement. Our study suggests that the defect of DNA repair and the production of reactive oxygen species in pds5-1 mutant are involved in the cell death.
Section snippets
Yeast strains and culture conditions
The wild-type strains used in this study are MATa ADE2 ade5 can1R CYH2s his72 leu1-d lys2-1 met13-d trp1-63 tyr1-1 ura3-13 and MATα ade2 ADE5 CAN1s cyh2R his7-1 leu2 lys2-2 met13-c trp1-63 tyr1-2 ura3-1. Plasmid of pds5-1 was provided by Guacci and co-workers [20]. The plasmid was transformed into the wild-type strains, forming two haploid pds5-1 strains (MATa and MATα). Diploids of wild-type or pds5-1 was constructed by mating the two haploid wild-type or pds5-1 strains. All experiments
Phosphatidylserine exposes at the cytoplasmic membrane in pds5-1 mutant during meiosis
One of the markers of early apoptosis is the translocation of phosphatidylserine from the inner leaflet to the outer leaflet of the plasma membrane. When sporulated at non-permissive temperature (34 °C), annexin V staining revealed a high percentage of apoptotic cell death in pds5-1 mutant, but less than 5% in wild-type (Fig. 1). As seen in Fig. 1B, number of early apoptotic cells reached its peak after 5 h in sporulation and decreased dramatically afterwards, while late apoptotic and necrotic
Discussion
Apoptotic cell death in yeast has been shown to share many features of apoptosis in mammals and other eukaryotes, such as chromatin condensation and DNA fragmentation, production of ROS, exposure of phosphatidylserine on the cells surface [9]. Yeast also provides a unique model system to study the functions of mammalian proteins involved in apoptosis, due to its lack of obvious orthologs of mammalian apoptosis-related proteins, and can therefore be considered a “clean” system for studying the
Acknowledgments
This research was supported by University of Wyoming's Center of Biomedical Research Excellence in Cellular Signaling and the NIH Biomedical Research Infrastructure Network grant (NIH/NCRR grant no. RR-16474).
References (40)
- et al.
Apoptotic detection methods-from morphology to gene
Prog. Histochem. Cytochem.
(2003) - et al.
Apoptosis-like yeast cell death in response to DNA damage and replication defects
Mutat. Res.
(2003) - et al.
A caspase-related protease regulates apoptosis in yeast
Mol. Cell
(2002) - et al.
A metacaspase of Trypanosoma brucei causes loss of respiration competence and clonal death in the yeast Saccharomyces cerevisiae
FEBS Lett.
(2002) - et al.
A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis
Cell
(1999) - et al.
Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs
Cell
(1999) - et al.
Pds5 cooperates with cohesin in maintaining sister chromatid cohesion
Curr. Biol.
(2000) - et al.
Caspase proteolysis of the cohesin component Rad21 promotes apoptosis
J. Biol. Chem.
(2002) - et al.
Role of the C-terminal domain of Bax and Bcl-XL in their localization and function in yeast cells
FEBS Lett.
(1999) - et al.
Mammalian Bax triggers apoptotic changes in yeast
FEBS Lett.
(1998)