| This research contains two different parts. In the first part of this research, we investigated the function of Schizosaccharomycespom.be DJ-1 and Hsp31 proteins, homologs of the Parkinson’s disease protein DJ-1. The second part of this research is to study the mechanisms of flocculation and apoptosis of the pentatricopeptide repeat (PPR) proteins mutant strains.The Parkinson’s disease protein DJ-1 is involved in various cellular functions including detoxification of dicarbonyl compounds, autophagy and oxidative stress response. DJ-1 homologs are widely found in both prokaryotes and eukaryotes, constituting a superfamily of proteins that appear to be involved in stress response. Schizosaccharomyces pombe contains six DJ-1 homologs, designated Hsp3101-Hsp3105 and Sdjl. In the first part of this research, the functional of Schizosaccharomyces pombe DJ-1 homologs and the regulation of these proteins was detected during the stationary phase. The results can be summaried as following:(1) Through detecting whether overexpression of yeast homologs of human DJ-1 under the control of the nmtl promoter could modulate the sensitivity of S. pombe cells and △Spglol cells to methylglyoxal (MG) and glyoxal (GO), we found that Hsp3101 and Hsp3102 and Sdjl as well as their homolog protein HSP31 of Saccharomyces cerevisiae may function as GLOⅢ. Overexpression of these proteins can increase the survival of wild-type pombe cells and △Spglol cells when they grow in the presence of either MG or GO.(2) We employed the CFP-Atg8 processing assay which is widely used to monitor autophagy over time from yeast to human to investigate whether S. pombe homologs of DJ-1 have a role in autophagy. At the same time, we also examined whether S. pombe DJ-1 homologs play a role in the localization of Atg8 at the phagophore assembly site/pre-autophagosomal structure (PAS), which is a unique site where most autophagy-related proteins act together to form the double-membraned vesicles known as autophagosomes. The results indicated that Hsp3104 and Sdj 1 and, a lesser extent, Hsp3102, Hsp3105 are involved in, but not essential for the normal autophagy in prolonged stationary phase. And fluorescence microscopy also revealed that all deletion mutants of S. pombe DJ-1 homologs accumulated more GFP-Atg8 puncta than the wild type cells.(3) To test whether the stress-activated MAP kinase Styl and the basic-region leucine-zipper (bZIP) transcription factor Atfl, which play an essential role in the cellular response to nutrient deprivation and environmental stresses, were required for the induction of S. pombe DJ-1 homologs. We disrupted styl and atfl in the wild-type yHL6381 respectively. We detected the protein levels of S. pombe DJ-1 homologs in △styl and Aatfl during stationary phase by western blot, and found that the deletion of styl almost completely abolished the stationary phase induction of S. pombe Hsp3101-Hsp3105 and Sdjl, and also found the induction of Hsp3102 and Sdj1 expression was essentially abolished in Aatfl cells, whereas induction of Hsp3101 and Hsp3105 was attenuated. Based on these observations, we concluded that the induction of S. pombe DJ-1 homologs during stationary phase depends on Styl and the induction of hsp3101, hsp3102, hsp3105 and sdjl involves the Styl-regulated transcription factor Atf1.PPR proteins are a recently recognized class of RNA-binding proteins that characterized by tandem repeats of a degenerate 35 amino acid motif, and are found in the organelle of nearly all eukaryotes but absent from almost all bacteria. All of the data accumulated to date strongly suggest that most of these proteins have a range of essential functions in posttranscriptional processes (including RNA editing, RNA splicing, RNA stabilization and translation) within mitochondria and chloroplasts. In this study, we have found that disruption of some ppr genes induced both non-sexual flocculation and apoptosis in S. pombe. With the result of realtime PCR, loss ofppr3, ppr4,ppr6, and ppr10 genes results in the upregulation of gsf2+/pfll+, pfl3+,pfl4+, pfl6+, pfl8+, pfl9+and genes encoding cell wall-remodeling enzymes, which contribute to their flocculent phenotype. After survival of all mutant strains was measured by counting CFUs or by spotting assays, nuclear were stained with DAPI, mitochondrial were stained with MitoTracker and the ROS accumulation was determined by using the ROS probe DHR123 and fluorescence microscopy, we found the mitochondrial of Appr3, Appr4, Appr6 and Appr10 was fragmentation and the mutant strains were apotosis as a result of reactive oxygen species accumulation and excess iron ions in these cells.
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