N on TLC, selective transformation of PA into DAG by PX-478 biological activity alkaline phosphatase was used to distinguish PA from PI (Fig 7B, lanes 2B, 4B)). These data indeed argue that the plasma membrane is not an absolute barrier for [3H]C16:0-CoA, nor for G3P if very high concentrations are used, but that in 123ty cells the barrier is significantly weaker, for both [3H]-C16:0-CoA and G3P as had already been suggested by Fig 6A. In summary, flc mutants exhibit a marked deficit in sphingolipid biosynthesis. The higher GPAT and AGPAT activities at 0.02 as compared to 0.005 Digitonin observed in DoxyPLOS Genetics | DOI:10.1371/journal.pgen.July 27,10 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 6. 123ty mutants have unstable membranes and increased GPAT and AGPAT activities. (A?D) WT or 123ty cells grown during 16 h with Doxy in 1.4 M sorbitol, permeabilized for 30 min with indicated concentrations of Digitonin were further incubated with 10 M C16:0-CoA, 10 M [14C]-G3P (0.5 Ci) for 5 to 15 min. Lipids were extracted and separated by TLC on solvent 1. doi:10.1371/journal.pgen.1006160.gtreated 123ty cells but not WT (Fig 6C and 6D) seem to indicate that Digitonin at 0.02 has a different effect on ER membranes from Doxy treated123ty cells, be it that it creates better access of G3P to the ER-based GPATs and AGPATs or better derepression of these enzymes. In this context it is noteworthy that yeast GPATs as well as AGPATs have been found to have their active sites on the lumenal side of the ER by conventional biochemical methods [16,40], but more data are required to support or discredit this somewhat unorthodox hypothesis. While this heightened detergent susceptibility of ER membranes in Doxy treated 123ty cells Quinoline-Val-Asp-Difluorophenoxymethylketone supplier certainly represents no strong argument for a role of Flc proteins in floppingPLOS Genetics | DOI:10.1371/journal.pgen.July 27,11 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 7. Doxycycline treated 123ty mutants have increased GPAT and AGPAT activities. (A, B) WT or 123ty cells grown during 16 h with Doxy in 1.4 M sorbitol were incubated with [3H]-C16:0-CoA (1 Ci, 10 M) for the indicated times at room temperature without (A) or with 1 mM G3P (B). PI was distinguished from PA by scraping the silica from lanes 2 and 4 of TLC plates (except the origin), extracting the lipids from silica, splitting samples in two equal halves (2A, 2B and 4A, 4B) and incubating them without (2A, 4A) or with (2B, 4B) alkaline phosphatase (AP). Products were run on TLC and are shown in each panel to the right of the original TLC. doi:10.1371/journal.pgen.1006160.gGPLs or acyl-CoA across the ER membrane, it cannot exclude these possibilities either and data ask for more direct experiments.Analysis of GPI biosynthesis in flc mutantsGPI lipids are built by stepwise addition of sugars to PI: First, N-Acetyl-Glucosamine (GlcNAc) is added to PI, the resulting PI-GlcNAc is then N-deacetylated to PI-GlcN, a FA is attached to the inositol ring to form GlcN-(acyl)PI, which latter then is mannosylated and further modified. While the formation of PI-GlcN is known to occur on the cytosolic side of the ER membrane, the mannosylation occurs in the ER lumen. As arv1 mutants accumulate GlcN-(acyl) PI, it has been proposed that Arv1, having 4 to 6 predicted TMDs, would serve as a GlcN-(acyl) PI flippase [41]. However, a more recent report demonstrated that Gwt1, the acyltransferase transforming PI-GlcN into GlcN.N on TLC, selective transformation of PA into DAG by alkaline phosphatase was used to distinguish PA from PI (Fig 7B, lanes 2B, 4B)). These data indeed argue that the plasma membrane is not an absolute barrier for [3H]C16:0-CoA, nor for G3P if very high concentrations are used, but that in 123ty cells the barrier is significantly weaker, for both [3H]-C16:0-CoA and G3P as had already been suggested by Fig 6A. In summary, flc mutants exhibit a marked deficit in sphingolipid biosynthesis. The higher GPAT and AGPAT activities at 0.02 as compared to 0.005 Digitonin observed in DoxyPLOS Genetics | DOI:10.1371/journal.pgen.July 27,10 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 6. 123ty mutants have unstable membranes and increased GPAT and AGPAT activities. (A?D) WT or 123ty cells grown during 16 h with Doxy in 1.4 M sorbitol, permeabilized for 30 min with indicated concentrations of Digitonin were further incubated with 10 M C16:0-CoA, 10 M [14C]-G3P (0.5 Ci) for 5 to 15 min. Lipids were extracted and separated by TLC on solvent 1. doi:10.1371/journal.pgen.1006160.gtreated 123ty cells but not WT (Fig 6C and 6D) seem to indicate that Digitonin at 0.02 has a different effect on ER membranes from Doxy treated123ty cells, be it that it creates better access of G3P to the ER-based GPATs and AGPATs or better derepression of these enzymes. In this context it is noteworthy that yeast GPATs as well as AGPATs have been found to have their active sites on the lumenal side of the ER by conventional biochemical methods [16,40], but more data are required to support or discredit this somewhat unorthodox hypothesis. While this heightened detergent susceptibility of ER membranes in Doxy treated 123ty cells certainly represents no strong argument for a role of Flc proteins in floppingPLOS Genetics | DOI:10.1371/journal.pgen.July 27,11 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 7. Doxycycline treated 123ty mutants have increased GPAT and AGPAT activities. (A, B) WT or 123ty cells grown during 16 h with Doxy in 1.4 M sorbitol were incubated with [3H]-C16:0-CoA (1 Ci, 10 M) for the indicated times at room temperature without (A) or with 1 mM G3P (B). PI was distinguished from PA by scraping the silica from lanes 2 and 4 of TLC plates (except the origin), extracting the lipids from silica, splitting samples in two equal halves (2A, 2B and 4A, 4B) and incubating them without (2A, 4A) or with (2B, 4B) alkaline phosphatase (AP). Products were run on TLC and are shown in each panel to the right of the original TLC. doi:10.1371/journal.pgen.1006160.gGPLs or acyl-CoA across the ER membrane, it cannot exclude these possibilities either and data ask for more direct experiments.Analysis of GPI biosynthesis in flc mutantsGPI lipids are built by stepwise addition of sugars to PI: First, N-Acetyl-Glucosamine (GlcNAc) is added to PI, the resulting PI-GlcNAc is then N-deacetylated to PI-GlcN, a FA is attached to the inositol ring to form GlcN-(acyl)PI, which latter then is mannosylated and further modified. While the formation of PI-GlcN is known to occur on the cytosolic side of the ER membrane, the mannosylation occurs in the ER lumen. As arv1 mutants accumulate GlcN-(acyl) PI, it has been proposed that Arv1, having 4 to 6 predicted TMDs, would serve as a GlcN-(acyl) PI flippase [41]. However, a more recent report demonstrated that Gwt1, the acyltransferase transforming PI-GlcN into GlcN.
