Browsing by Author "Ilina, Yulia"

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    Functions of the yeast protein Stm1 and its involvement in apoptotic cell death
    (2005) Ilina, Yulia; Hilt, Wolfgang (PD, Dr)
    Selective proteolysis conducted by the ubiquitin-proteasome system regulates many essential cellular processes. Therefore, identification and characterization of new proteasomal substrates is important to reveal cellular functions of the proteasome system. Using an overexpression screen a new proteasomal substrate, termed Stm1, had been found in the yeast Saccharomyces cerevisiae (Ligr et al., 2001). To better understand the biological role of Stm1, its reasonable contribution to different cellular functions ranging from telomeric silencing, DNA repair to apoptosis were elucidated in this work. Regarding the specific affinity of Stm1 for guanine-rich quadruplex DNA and its localization at the nuclear periphery, the possible role of Stm1 in telomeric silencing, as well as its involvement in non-homologous end joining of double strand breaks (NHEJ) were investigated. The results exclude the direct involvement of Stm1 in such processes. Additionally, a possible role of Stm1 in the regulation of telomere elongation was analyzed. The data showed that in vivo telomere elongation was Stm1 independent. Surprisingly, however, chromosomal cleavage was accelerated in stm1Δ mutants. It had been shown that Stm1 triggers apoptosis-like cell death when overexpressed in yeast mutants with impaired proteasomal activity (Ligr et al., 2001). However, defects in the proteasomal function might lead to the alteration of concentration or activity of some target proteins thereby causing possible side effects on the Stm1 function (Hilt and Wolf, 1996). Thus, in the present studies a mutated version, stm1-9 was generated, which causes cell lethality when overexpressed in yeast cells harbouring intact proteasomal activity. Overexpression of stm1-9 in wild type cells induces apoptotic cell death accompanied by cytochrome c release and extensive nuclear DNA cleavage. In addition, using bivariate flow cytometry, FITC-VAD-FMK staining paralleled by propidium iodide staining, indicating disintegration of the plasma membrane was observed under these conditions. As a further sign of apoptosis, enhanced production of ROS was found after stm1-9 overexpression. Using cycloheximide chase analysis, the hyperactive Stm1-9 protein was proven to be proteolytically stabilized. The pro-apoptotic effect of overexpressed Stm1-9 is, therefore, thought to be the result of the accumulation of the stabilized protein. Investigation of potential suppressors of the stm1-9 triggered cell death identified new downstream targets of this apoptosis pathway. One down-stream target is the caspase-like protease Yca1. This so far unique S. cerevisiae metacaspase is functionally involved in apoptotic cell death in yeast (Madeo et al., 2002b). In this work, it could be shown that cell death and induction of apoptotic phenotypes induced by overexpression of stm1-9 is neutralized in the absence of Yca1. This result proves that the stm1-9 induced cell death is Yca1 dependent uncovering that Yca1 is a down-stream element in the stm1-9 mediated pathway. Similar results were obtained for a recently discovered pro-apoptotic protein, the apoptosis-inducing factor Aif1 (Wissing et al., 2004). Deletion of the yeast AIF1 gene leads to suppression of apoptotic phenotypes and cell death induced by stm1-9 overexpression indicating that Aif1 functions as a down-stream element in the stm1-9 pathway. The suppression effect of yca1Δ and aif1Δ could not be enhanced by combination of both deletions. This behavior of epistasis evidences that Yca1 and Aif1 are both part of the same stm1-9 stimulated cell death pathway. Further suppression studies revealed that Mec3, a protein functioning in DNA repair is also required for stm1-9 triggered apoptosis. Co-immunoprecipitation analysis unraveled an interaction between Stm1 and Mec3. Thus, the present data suggest an additional function of Stm1 in DNA repair. A closer inspection of stm1Δ yca1Δ double mutants revealed enhanced sensitivity to UV-induced mutagenesis. This might mean that Yca1 and Stm1 are required for proper execution of apoptotic cell death after DNA damage. Investigation of a series of other apoptosis inducing genes (HEL genes: NSR1, SAR1, PPA1, YNL208w and YOR309c) uncovered that induction of cell death in these cases is Stm1 independent, placing Stm1 in an independent pathway or at an up-stream position.