Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubations at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. This substitution is positioned in a 1.3 kbp intergenic region containing divergent promoters and it seems unlikely that may affect expression of the IZH4, the upstream gene, which encode a family of four paralogous membrane proteins involved in zinc ion homeostasis and whose expression is regulated by fatty acids and zinc altered levels. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH3 coding regions, obtaining the new SDP8 strain. Our results show that depletion of Pkh results in a differential transcriptional response in front of a heat shock stress. The general transcriptional response was attenuated in the SDP8 when compared when WT cells, but lack of Pkh activity did not affect the repression of the ribosomal proteins.

Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubations at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. This substitution is positioned in a 1.3 kbp intergenic region containing divergent promoters and it seems unlikely that may affect expression of the IZH4, the upstream gene, which encode a family of four paralogous membrane proteins involved in zinc ion homeostasis and whose expression is regulated by fatty acids and zinc altered levels. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH3 coding regions, obtaining the new SDP8 strain. Our results show that depletion of Pkh results in a differential transcriptional response in front of a heat shock stress. The general transcriptional response was attenuated in the SDP8 when compared when WT cells, but lack of Pkh activity did not affect the repression of the ribosomal proteins. Four samples were analyzed: the SDP8 strain, a tetO7 conditional mutant, and its isogenic WT strain both, in the presence and in the absence of doxycyline (for 8 and 24 h). We compared the expression profile of: 1) WT + 16h Dox + 40 min heat shock vs WT + 16h Dox. 2) SDP8 + 16h Dox + 40 min heat shock vs SDP8 + 16h Dox. A Dye-swap was carried out for each comparison of samples. Total number of chips analyzed: 4.

Project description:The yeast proteins Pkh1 and Pkh2 are functional counterparts of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Cells carrying simultaneous deletion in both genes, PKH1 and PKH2 are not viable, at least in some genetic backgrounds (Casamayor et al., 1999; Inagaki et al., 1999). Consequently, a different strategy is needed to study the function of these apparently redundant gene products. The approach that has been successfully used to dissect the functions of these proteins consist in the use of a strain that combine the disruption of PKH2 with the temperature sensitive pkh1D398G allele (Inagaki et al., 1999). Since Pkh1 and Pkh2 phosphorylate Pkc1 in vitro in its activation loop (Thr983), and the cell lysis defects of the pkh1D398G pkh2 strain at the restrictive temperature are partially restored by constitutive activation of the Pkc1-Slt2 MAPK pathway, it has been suggested that the Pkh activity is important for maintenance of the cell wall integrity in S. cerevisiae (Inagaki et al., 1999; Friant et al., 2001). We considered it necessary that a different genetic strategy would be convenient in order to carry out specific studies about the functions of the Pkh proteins. In order to avoid incubation at temperatures that activate the Slt2 pathway, and induce cellular lysis, we decided to use a strategy based on the use of a regulatable promoter. We replaced 487 bp located immediately upstream the start PKH2 codon by a cassette containing the doxycycline-repressed promoter tetO7. This substitution is positioned in a 1.3 kbp intergenic region containing divergent promoters and it seems unlikely that may affect expression of the IZH4, the upstream gene, which encode a family of four paralogous membrane proteins involved in zinc ion homeostasis and whose expression is regulated by fatty acids and zinc altered levels. In this strain (MB002) the PKH2 expression could be switched off by addition of doxycycline. In order to deplete the cell for the Pkh activity, we disrupted both, the PKH1 and PKH1 coding regions, obtaining the new SDP8 strain. Our results show that the long term depletion of Pkh results in a cell transcriptional response that include overexpression of genes typically induced in response to oxidative stress, as well as up-regulation of chaperons and genes that induce the unfolded protein response. Four samples were analyzed: the SDP8 strain, a tetO7 conditional mutant, and its isogenic WT strain both, in the presence and in the absence of doxycyline (for 8 and 24 h). We compared the expression profile of: 1) SDP8 + Dox after 8h vs WT + Dox after 8h 2) SDP8 + Dox after 24h vs WT + Dox after 24h A Dye-swap was carried out for each comparison of samples. Total number of chips analyzed: 4.

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Depletion of yeast PDK1 orthologs triggers a stress-like transcriptional response

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