Ent is in large excess over thiols, so that the reaction

Ent is in large excess over thiols, so that the reaction reaches plateau when all thiols are oxidized. As these plateaus are the same for WT and 123ty cells (S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)), it appears that although 60 of Doxy-treated 123ty cells are unable to resume growth and form a colony, i.e. have ceased to be a CFU, they apparently still maintain a normal redox potential and in this sense are alive. S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB) shows that in all cell types tested 0.005 Digitonin achieved full, but 0.001 only partial reduction of the reagent within 20 min (see the more detailed description of get Actinomycin IV results in S4 Fig). We therefore hoped that substrates for acyltransferases added to minimally permeabilized cells would enter the cytosol and be used for lipid biosynthesis in a still preserved ER membrane without skirting the potential need for physiological ER based lipid flippases in this process. Thus, WT and 123ty cells grown in sorbitol and Doxy were preincubated for 30 min with increasing concentrations of Digitonin as above (S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)) whereupon 10 M C16:0-CoA plus 10 M [14C]-G3P (0.5 Ci) were added (Fig 6). When no detergent was added, small amounts of [14C]-G3P seemed to enter cells and produce mainly lyso-phosphatidic acid (LPA), whereby this phenomenon was more pronounced with 123ty than WT cells (Fig 6A). With 0.001 Digitonin, cells started to make SP600125 biological activity significant amounts of phosphatidic acid (PA), which were similar for WT and 123ty cells. With 0.005 (17 mol ) Digitonin, 123ty cells made significantly more PA and neutral lipids than WT (Fig 6C). PA seemed to be chased with time into DAG or TAG, as if PA synthesis was coming to an early plateau and even early halt. Interestingly, in 123ty but not WT cells, the further increase of Digitonin to 0.02 (44 mol ) led to a further increase of activity (Fig 6D), although already 0.005 fully permeabilizes the plasma membrane of all cell types (S4A and S4B Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)). This suggests a basic difference between 123ty and WT cells in that in the former Digitonin at 0.02 may also affect the permeability of the ER membrane or the enzyme activities in the ER. Importantly, higher GPAT/AGPAT activity in 123ty cells was only seen after prolonged culture of cells in Doxy, whereas cells grown in sorbitol without Doxy had normal GPAT/ AGPAT activity (S5B Fig (123ty mutants have normal GPAT and AGPAT activity when not incubated with Doxy) vs. Fig 6C). This finding suggested that it might be the complete depletion of Flc function that induces these acyltransferases or induces a better accessibility of substrates to the enzymes. We repeated the experiment of Fig 6A, but labeling permeabilized cells with [3H]C16:0-CoA rather than [14C]-G3P. In this setting, in the mere presence of [3H]-C16:0-CoA (at 5 mol ), mainly PI was made, but 123ty made significantly more PI than WT (Fig 7A). This reaction most certainly means that [3H]-C16:0-CoA can enter cells and be used for the acylation of lyso-PI. As soon as [3H]-C16:0-CoA was added together with a 100 fold excess of G3P, cells mainly generated PA, again with 123ty making much more PA than WT (Fig 7B, lanes 1?). (As PI and PA had similar migratio.Ent is in large excess over thiols, so that the reaction reaches plateau when all thiols are oxidized. As these plateaus are the same for WT and 123ty cells (S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)), it appears that although 60 of Doxy-treated 123ty cells are unable to resume growth and form a colony, i.e. have ceased to be a CFU, they apparently still maintain a normal redox potential and in this sense are alive. S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB) shows that in all cell types tested 0.005 Digitonin achieved full, but 0.001 only partial reduction of the reagent within 20 min (see the more detailed description of results in S4 Fig). We therefore hoped that substrates for acyltransferases added to minimally permeabilized cells would enter the cytosol and be used for lipid biosynthesis in a still preserved ER membrane without skirting the potential need for physiological ER based lipid flippases in this process. Thus, WT and 123ty cells grown in sorbitol and Doxy were preincubated for 30 min with increasing concentrations of Digitonin as above (S4 Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)) whereupon 10 M C16:0-CoA plus 10 M [14C]-G3P (0.5 Ci) were added (Fig 6). When no detergent was added, small amounts of [14C]-G3P seemed to enter cells and produce mainly lyso-phosphatidic acid (LPA), whereby this phenomenon was more pronounced with 123ty than WT cells (Fig 6A). With 0.001 Digitonin, cells started to make significant amounts of phosphatidic acid (PA), which were similar for WT and 123ty cells. With 0.005 (17 mol ) Digitonin, 123ty cells made significantly more PA and neutral lipids than WT (Fig 6C). PA seemed to be chased with time into DAG or TAG, as if PA synthesis was coming to an early plateau and even early halt. Interestingly, in 123ty but not WT cells, the further increase of Digitonin to 0.02 (44 mol ) led to a further increase of activity (Fig 6D), although already 0.005 fully permeabilizes the plasma membrane of all cell types (S4A and S4B Fig (Permeabilization of cells with Digitonin and detection of cytosolic thiol groups with DTNB)). This suggests a basic difference between 123ty and WT cells in that in the former Digitonin at 0.02 may also affect the permeability of the ER membrane or the enzyme activities in the ER. Importantly, higher GPAT/AGPAT activity in 123ty cells was only seen after prolonged culture of cells in Doxy, whereas cells grown in sorbitol without Doxy had normal GPAT/ AGPAT activity (S5B Fig (123ty mutants have normal GPAT and AGPAT activity when not incubated with Doxy) vs. Fig 6C). This finding suggested that it might be the complete depletion of Flc function that induces these acyltransferases or induces a better accessibility of substrates to the enzymes. We repeated the experiment of Fig 6A, but labeling permeabilized cells with [3H]C16:0-CoA rather than [14C]-G3P. In this setting, in the mere presence of [3H]-C16:0-CoA (at 5 mol ), mainly PI was made, but 123ty made significantly more PI than WT (Fig 7A). This reaction most certainly means that [3H]-C16:0-CoA can enter cells and be used for the acylation of lyso-PI. As soon as [3H]-C16:0-CoA was added together with a 100 fold excess of G3P, cells mainly generated PA, again with 123ty making much more PA than WT (Fig 7B, lanes 1?). (As PI and PA had similar migratio.