-(acyl)PI has its putative active site on the lumenal

-(acyl)PI has its putative active site on the lumenal side of the ER [22]. This would imply that Arv1 acts after the GPI substrate (PI-GlcN) has already been flipped and suggests that the generation of GlcN-(acyl)PI requires a still unknown flippase other than Arv1, as well as the flipping of acyl-CoA. As flc mutants show a severe cell wall phenotype, characteristic also for mutants with compromised GPI protein biosynthesis, we desired to test whether flc mutants have any difficulty in making GlcN-(acyl)PI. We tried to test this by using a microsomal in vitro assay of GPI biosynthesis with UDP-[3H]-GlcNAc added as the substrate [42]. As shown in Fig 8, microsomes incubated with UDP-[3H]-GlcNAc make GlcN-(acyl)PI when they are allowed to make acylCoA in presence of added ATP and CoA. Similar results were obtained with permeabilized cells. In all cases Doxy treated 123ty cells made significantly more GlcN-(acyl)PI than WT, suggesting that the complete depletion of Flc function induces enzymes for GPI biosynthesis. This finding is reminiscent of the increased GPAT and AGPAT Dactinomycin chemical information activities in these cells (Figs 5 and 6C and 6D). Once more, results are ambiguous and suggest that the flipping of GlcN-PIPLOS Genetics | DOI:10.1371/journal.pgen.July 27,12 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 8. Microsomal GlcN-(acyl)PI synthesis activity in flc mutants is increased. Microsomes were produced from WT or 123ty cells grown for 12 h in the presence of Doxy and sorbitol, and were labeled using 80 nM UDP-[3H]-GlcNAc (4 Ci), 1 mM ATP and 1 mM CoA. In the last lane, to produce GPI (S)-(-)-Blebbistatin cost intermediates without the acyl chain bound to the inositol moiety, CoA and ATP were omitted and 1 mM G3P was added in order to deplete microsomes of preexisting acyl-CoA. Lipids were extracted and separated by TLC. doi:10.1371/journal.pgen.1006160.gand acyl-CoA are normal, but they can’t exclude the possibility that an abnormal permeability or orientation of the ER derived microsomes obviates the need for the corresponding PI-GlcN and acyl-CoA flippases.Cst26 introduces C26 fatty acids into lyso-PIUnrelated to the search of lipid flippases, our attention was caught by the very strong negative interactions of cst26 with elo2 and elo3 deletions in MSP- and MSP/C-E-MAPs (S scores between?9.5 and -11.0). CST26/PSI1 encodes an acyltransferase that transfers stearic acid (C18:0) from C18:0-CoA onto the sn-1 position of lyso-PI [43]. Apart from a further very strong negative interaction with the chitin synthase CHS1, cst26 interacts only with elo2 and elo3, but not elo1 (see below). ELO1 allows elongating FAs up to C18:0. ELO2 and ELO3 are partially redundant, cannot be deleted simultaneously and are required to further elongate FAs up to C26:0. Each of them is required to make C26:0 in sufficient quantity for the ceramide synthases Lag1 and Lac1, for which C26:0-CoA is the preferred substrate [44]. Elo2 and elo3 therefore make markedly reduced amounts of ceramide and mature sphingolipids. However, they grow normally whereas cst26 elo3 cells grow less rapidly (S6 Fig (Comparison of growth rates of elo3, cst26 and elo3 cst26 cells)). While sphingolipids are essential, their presence is dispensable if, due to a gain of function suppressor mutation in SLC1, cells can make PI carrying a very long chain FA in the sn-2 position of the glycerol moiety, even if this form of PI accounts for only a tiny fraction of membrane lipids [45?8]. Moreover, i.-(acyl)PI has its putative active site on the lumenal side of the ER [22]. This would imply that Arv1 acts after the GPI substrate (PI-GlcN) has already been flipped and suggests that the generation of GlcN-(acyl)PI requires a still unknown flippase other than Arv1, as well as the flipping of acyl-CoA. As flc mutants show a severe cell wall phenotype, characteristic also for mutants with compromised GPI protein biosynthesis, we desired to test whether flc mutants have any difficulty in making GlcN-(acyl)PI. We tried to test this by using a microsomal in vitro assay of GPI biosynthesis with UDP-[3H]-GlcNAc added as the substrate [42]. As shown in Fig 8, microsomes incubated with UDP-[3H]-GlcNAc make GlcN-(acyl)PI when they are allowed to make acylCoA in presence of added ATP and CoA. Similar results were obtained with permeabilized cells. In all cases Doxy treated 123ty cells made significantly more GlcN-(acyl)PI than WT, suggesting that the complete depletion of Flc function induces enzymes for GPI biosynthesis. This finding is reminiscent of the increased GPAT and AGPAT activities in these cells (Figs 5 and 6C and 6D). Once more, results are ambiguous and suggest that the flipping of GlcN-PIPLOS Genetics | DOI:10.1371/journal.pgen.July 27,12 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip FlopFig 8. Microsomal GlcN-(acyl)PI synthesis activity in flc mutants is increased. Microsomes were produced from WT or 123ty cells grown for 12 h in the presence of Doxy and sorbitol, and were labeled using 80 nM UDP-[3H]-GlcNAc (4 Ci), 1 mM ATP and 1 mM CoA. In the last lane, to produce GPI intermediates without the acyl chain bound to the inositol moiety, CoA and ATP were omitted and 1 mM G3P was added in order to deplete microsomes of preexisting acyl-CoA. Lipids were extracted and separated by TLC. doi:10.1371/journal.pgen.1006160.gand acyl-CoA are normal, but they can’t exclude the possibility that an abnormal permeability or orientation of the ER derived microsomes obviates the need for the corresponding PI-GlcN and acyl-CoA flippases.Cst26 introduces C26 fatty acids into lyso-PIUnrelated to the search of lipid flippases, our attention was caught by the very strong negative interactions of cst26 with elo2 and elo3 deletions in MSP- and MSP/C-E-MAPs (S scores between?9.5 and -11.0). CST26/PSI1 encodes an acyltransferase that transfers stearic acid (C18:0) from C18:0-CoA onto the sn-1 position of lyso-PI [43]. Apart from a further very strong negative interaction with the chitin synthase CHS1, cst26 interacts only with elo2 and elo3, but not elo1 (see below). ELO1 allows elongating FAs up to C18:0. ELO2 and ELO3 are partially redundant, cannot be deleted simultaneously and are required to further elongate FAs up to C26:0. Each of them is required to make C26:0 in sufficient quantity for the ceramide synthases Lag1 and Lac1, for which C26:0-CoA is the preferred substrate [44]. Elo2 and elo3 therefore make markedly reduced amounts of ceramide and mature sphingolipids. However, they grow normally whereas cst26 elo3 cells grow less rapidly (S6 Fig (Comparison of growth rates of elo3, cst26 and elo3 cst26 cells)). While sphingolipids are essential, their presence is dispensable if, due to a gain of function suppressor mutation in SLC1, cells can make PI carrying a very long chain FA in the sn-2 position of the glycerol moiety, even if this form of PI accounts for only a tiny fraction of membrane lipids [45?8]. Moreover, i.

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