The Yeast Phospholipid N-Methyltransferases Catalyzing the Synthesis of Phosphatidylcholine Preferentially Convert Di-C16:1 Substrates Both in Vivo and in Vitro

Phosphatidylcholine (PC) is an important and abundant structural component of the membranes of eukaryotic cells. In the yeast Saccharomyces cerevisiae, the primary route for the biosynthesis of PC consists of three consecutive methylation steps of phosphatidylethanolamine (PE) catalyzed by the phospholipid N-methyltransferases Cho2p and Opi3p. To investigate how these biosynthetic enzymes contribute to the composition of the PC species profile, the precursor-product relationships between PE and newly synthesized PC were determined at the level of the molecular species by using electrospray ionization tandem mass spectrometry and stable isotope labeling. In vivo labeling of yeast cells for 10 min with [methyl-D3]methionine revealed the preferential methylation of di-C16:1 PE over a range of PE species compositions. A similar preferential conversion of di-C16:1 PE to PC was found with and were to between the of Cho2p and biosynthetic enzymes were found to the methylation with Cho2p the the methylation of PE species by the an important the of PC molecular species Phosphatidylcholine (PC) is an important and abundant structural component of the membranes of eukaryotic cells. In the yeast Saccharomyces cerevisiae, the primary route for the biosynthesis of PC consists of three consecutive methylation steps of phosphatidylethanolamine (PE) catalyzed by the phospholipid N-methyltransferases Cho2p and Opi3p. To investigate how these biosynthetic enzymes contribute to the composition of the PC species profile, the precursor-product relationships between PE and newly synthesized PC were determined at the level of the molecular species by using electrospray ionization tandem mass spectrometry and stable isotope labeling. In vivo labeling of yeast cells for 10 min with [methyl-D3]methionine revealed the preferential methylation of di-C16:1 PE over a range of PE species compositions. A similar preferential conversion of di-C16:1 PE to PC was found with and were to between the of Cho2p and biosynthetic enzymes were found to the methylation with Cho2p the the methylation of PE species by the an important the of PC molecular species Phosphatidylcholine (PC) phospholipid electrospray mass phospholipid electrospray mass is a phospholipid of eukaryotic a structural component of the PC is a a of In the Saccharomyces cerevisiae, PC for of the phospholipid and is for with PC an important for PC the of In the biosynthesis of PC is the of the the phospholipid biosynthesis of PC yeast three methylation of phosphatidylethanolamine (PE) is PC the route methylation of PE is by the phospholipid N-methyltransferases Cho2p and a Cho2p PE to and the and the methylation to and to the the phospholipid PC is of molecular species with a of with the at the of the of the composition is for the and is to a PC and of biosynthesis of PC a the molecular species of of the methylation of PE and the route at the molecular species level were by labeling yeast cells with and and of the of newly synthesized PC species by the the and PC species the profile, the methylation route PC the species of the newly synthesized PC PE was found to of the PE with the species at the of the is for preferential methylation of the species of the is to investigate how the methylation of PE species vivo is was by of the precursor-product between PC and PE molecular species yeast cells species composition of the PE and In the of the Cho2p and the PC molecular species using and the of the of Cho2p for the species the methylation and the were was the and the to the to to the and was by the with the was to the of the the yeast and of were at of with and of the was with labeling were to were with and of yeast the of the and were with [methyl-D3]methionine for 10 was and the a of and cells were with for min the of [methyl-D3]methionine were with for labeling was by a of and to of to and the cells were to were of and by with three for min with were were cells were and were using were and were the of the of PE was by at a of to and a of 10 min of at the methylation was by of a of and was by phospholipid of was using a the were at phospholipid with to the of were the by an electrospray with a of and were at and and was using and of of of 10 at of phospholipid species was of the PC species and the and of and were of the of species was for the isotope of the species with an of were for the between mass and of the was was to of the these were the at In range the was found to of the the range and of for the of phospholipid molecular of of of of of of of of of a were determined using the the of with were to the of and PE to PC of the of the PE the of the PE methylation the molecular species composition of the PE was of phospholipid yeast by the cells by using phospholipid biosynthetic an of PE by for cells abundant PE species were and of of the cells were of the PE species with the of the species and were for by an of the route by of the an of PE at the of PE with the molecular species composition of PE the and between and of the PE molecular species yeast to the PE molecular species were by for the PE at and were and the with an the to the with a mass of of the of the PE molecular species of cells and and of cells the investigate how the species of PE the species composition of PC newly synthesized by the methylation of and cells were for 10 min with were and by using for to the with three of cells revealed a species composition is the species with the of the PE of conversion of the PE species to PC were by the of the species by of PE species and for and of the species composition of the PE the species was to PC to similar methylation by the of the species and of the of these species the PE of the cells cells to and for and PE to PC to an is of the species composition of the of the newly synthesized molecular species cells by for of the of conversion of the abundant PE species to PC for cells and and for cells were by the of and PC the by the of the PE species the species of PE and newly synthesized PC 10 were determined three of PE to an of the preferential methylation of PE is an of the enzymes the similar were using PE species of cells was similar to of the the with and of with for 10 min at revealed the of conversion of the PE species to were similar to found vivo was the of conversion were by the species of newly synthesized PC vivo to the PE species to the PE species the preferential methylation of PE vivo is of the phospholipid N-methyltransferases for PE is similar vivo and of methylation of and PE were determined the to the were with with the vivo the of Cho2p the at the methylation of PE species the of the three methylation steps a was using yeast To the for the was the is the and methylation and the for the at revealed the of and of the of newly synthesized to of PE of an of species were using the cells these the methylation by Cho2p to an of the methylation of PE to similar for PE vivo and of newly synthesized the were with [methyl-D3]methionine for 10 newly synthesized species were by for the and species of the of conversion of the PE species to vivo and the cells were with the of the of to the methylation of PE to the was the methylation of by cells with [methyl-D3]methionine for to min a with the of to PE the cells were the of to the cells with the the these was the of the of species for the species was determined a for min with was the species of of PE newly synthesized the was determined the cells with In were similar between the and similar of species for the the of and PE and of the the species composition of the species determined the cells was a of the for cells. the species of the the cells was and was found to of PC of the species composition of the for Opi3p. a for the species composition the species of the was determined by the cells with for min and by of similar species the route and cells was by labeling with for species were by for the investigate the of the and methylation steps to the methylation of cells were for min with [methyl-D3]methionine the of synthesized PC species and were by for and and the of conversion to PC was for the for the the methylation of PE vivo is by to a by by the and the methylation of of conversion of the and species to PC were determined by labeling for min with [methyl-D3]methionine the of PC species and were by at and the of the of the abundant phospholipid PC yeast the methylation of PE the of PC and to the of yeast the methylation route is a of the PC molecular species and of labeling yeast with phospholipid stable of the newly synthesized by the of phospholipid biosynthetic enzymes to vivo cells. the PE molecular species to PC yeast by labeling with [methyl-D3]methionine is a to the species of PE of conversion of PE to PC was found to by the methylation of the PE species the the the methylation of PE is the of the preferential conversion of PE to PC is of the species composition of the PE and is similar cells and is the of the preferential methylation of the species methylation is with is to the the and the of for the of the of PE of to the species methylation to is a range of species of PE was by the and by yeast the and and of the is to the of PE to the the of the the the of PE the is yeast cells of conversion of the PE species to PC were found to similar the the preferential methylation is by the PE species composition by the of PE to the the the newly synthesized PE the is to the the to over the of a contribute to the species the methylation of preferential methylation of PE membranes by the of the the methylation the of the PE molecular species to the of the PE species the In the the of PE species the the with the of PE the To between these the of the using of PE to the of the of of enzymes the of between the of the to the species and were with [methyl-D3]methionine the and of revealed the methylation catalyzed by Cho2p of the species of is with and the of to a phospholipid To the of the and to the and the In using revealed the molecular species of using revealed a for PE In the methylation of PE is to the and to PC the the of a for species for PC species and with species is similar to with and of the is to an of the by the species methylation yeast preferential methylation of PE by the Cho2p the yeast with a to the between the species of PC and of the and di-C16:1 PC species the of to membranes to the PC species of PC the and of PC by the of the route to PC biosynthesis by PC of to the PC species composition PC synthesized by the route is the species and with PC of PC by a yeast an route the species the PC and to the of the biosynthetic and the PC species Phosphatidylcholine (PC) phospholipid electrospray mass phospholipid electrospray mass is a phospholipid of eukaryotic a structural component of the PC is a a of In the Saccharomyces cerevisiae, PC for of the phospholipid and is for with PC an important for PC the of In the biosynthesis of PC is the of the the phospholipid biosynthesis of PC yeast three methylation of phosphatidylethanolamine (PE) is PC the route methylation of PE is by the phospholipid N-methyltransferases Cho2p and a Cho2p PE to and the and the methylation to and to the the phospholipid PC is of molecular species with a of with the at the of the of the composition is for the and is to a PC and of biosynthesis of PC a the molecular species of of the methylation of PE and the route at the molecular species level were by labeling yeast cells with and and of the of newly synthesized PC species by the the and PC species the profile, the methylation route PC the species of the newly synthesized PC PE was found to of the PE with the species at the of the is for preferential methylation of the species of the is to investigate how the methylation of PE species vivo is was by of the precursor-product between PC and PE molecular species yeast cells species composition of the PE and In the of the Cho2p and the PC molecular species using and the of the of Cho2p for the species the methylation and the were was the and the to the to to the and was by the with the was to the of the the yeast and of were at of with and of the was with labeling were to were with and of yeast the of the and were with [methyl-D3]methionine for 10 was and the a of and cells were with for min the of [methyl-D3]methionine were with for labeling was by a of and to of to and the cells were to were of and by with three for min with were were cells were and were using were and were the of the of PE was by at a of to and a of 10 min of at the methylation was by of a of and was by phospholipid of was using a the were at phospholipid with to the of were the by an electrospray with a of and were at and and was using and of of of 10 at of phospholipid species was of the PC species and the and of and were of the of species was for the isotope of the species with an of were for the between mass and of the was was to of the these were the at In range the was found to of the the range and of for the of phospholipid molecular of of of of of of of of of a were determined using the the of with were to the of and and the were was the and the to the to to the and was by the with the was to the of the the yeast and of were at of with and of the was with labeling were to were with and of yeast the of the and were with [methyl-D3]methionine for 10 was and the a of and cells were with for min the of [methyl-D3]methionine were with for labeling was by a of and to of to and the cells were to were of and by with three for min with were were cells were and were using were and were the of the of PE was by at a of to and a of 10 min of at the methylation was by of a of and was by phospholipid of was using a the were at phospholipid with to the of were the by an electrospray with a of and were at and and was using and of of of 10 at of phospholipid species was of the PC species and the and of and were of the of species was for the isotope of the species with an of were for the between mass and of the was was to of the these were the at In range the was found to of the the range and were determined using the the of with were to the of and PE to PC of the of the PE the of the PE methylation the molecular species composition of the PE was of phospholipid yeast by the cells by using phospholipid biosynthetic an of PE by for cells abundant PE species were and of of the cells were of the PE species with the of the species and were for by an of the route by of the an of PE at the of PE with the investigate how the species of PE the species composition of PC newly synthesized by the methylation of and cells were for 10 min with were and by using for to the with three of cells revealed a species composition is the species with the of the PE of conversion of the PE species to PC were by the of the species by of PE species and for and of the species composition of the PE the species was to PC to similar methylation by the of the species and of the of these species the PE of the cells cells to and for and PE to PC to an is of the species composition of the of the newly synthesized molecular species cells by for of the of conversion of the abundant PE species to PC for cells and and for cells were by the of and PC the by the of the PE species the species of PE and newly synthesized PC 10 were determined three of PE to an of the preferential methylation of PE is an of the enzymes the similar were using PE species of cells was similar to of the the with and of with for 10 min at revealed the of conversion of the PE species to were similar to found vivo was the of conversion were by the species of newly synthesized PC vivo to the PE species to the PE species the preferential methylation of PE vivo is of the phospholipid N-methyltransferases for PE is similar vivo and of methylation of and PE were determined the to the were with with the vivo the of Cho2p the at the methylation of PE species the of the three methylation steps a was using yeast To the for the was the is the and methylation and the for the at revealed the of and of the of newly synthesized to of PE of an of species were using the cells these the methylation by Cho2p to an of the methylation of PE to similar for PE vivo and of newly synthesized the were with [methyl-D3]methionine for 10 newly synthesized species were by for the and species of the of conversion of the PE species to vivo and the cells were with the of the of to the methylation of PE to the was the methylation of by cells with [methyl-D3]methionine for to min a with the of to PE the cells were the of to the cells with the the these was the of the of species for the species was determined a for min with was the species of of PE newly synthesized the was determined the cells with In were similar between the and similar of species for the the of and PE and of the the species composition of the species determined the cells was a of the for cells. the species of the the cells was and was found to of PC of the species composition of the for Opi3p. a for the species composition the species of the was determined by the cells with for min and by of similar species the route and cells was by labeling with for species were by for the investigate the of the and methylation steps to the methylation of cells were for min with [methyl-D3]methionine the of synthesized PC species and were by for and and the of conversion to PC was for the for the the methylation of PE vivo is by to a by by the and the methylation of of conversion of the and species to PC were determined by labeling for min with [methyl-D3]methionine the of PC species and were by at and In PE to PC of the of the PE the of the PE methylation the molecular species composition of the PE was of phospholipid yeast by the cells by using phospholipid biosynthetic an of PE by for cells abundant PE species were and of of the cells were of the PE species with the of the species and were for by an of the route by of the an of PE at the of PE with the To investigate how the species of PE the species composition of PC newly synthesized by the methylation of and cells were for 10 min with were and by using for to the with three of cells revealed a species composition is the species with the of the PE of conversion of the PE species to PC were by the of the species by of PE species and for and of the species composition of the PE the species was to PC to similar methylation by the of the species and of the of these species the PE of the cells cells to and for and of PE to an of the preferential methylation of PE is an of the enzymes the similar were using PE species of cells was similar to of the the with and of with for 10 min at revealed the of conversion of the PE species to were similar to found vivo was the of conversion were by the species of newly synthesized PC vivo to the PE species to the PE species the preferential methylation of PE vivo is of Cho2p the at the methylation of PE species the of the three methylation steps a was using yeast To the for the was the is the and methylation and the for the at revealed the of and of the of newly synthesized to of PE of an of species were using the cells these the methylation by Cho2p to an of the of the of to the methylation of PE to the was the methylation of by cells with [methyl-D3]methionine for to min a with the of to PE the cells were the of to the cells with the the these was the of the of species for Opi3p. To the species was determined a for min with was the species of of PE newly synthesized the was determined the cells with In were similar between the and similar of species for the the of and PE and of the the species composition of the species determined the cells was a of the for cells. the species of the the cells was and was found to of PC investigate the of the and methylation steps to the methylation of cells were for min with [methyl-D3]methionine the of synthesized PC species and were by for and and the of conversion to PC was for the for the the methylation of PE vivo is by to a by by the of the of the abundant phospholipid PC yeast the methylation of PE the of PC and to the of yeast the methylation route is a of the PC molecular species and of labeling yeast with phospholipid stable of the newly synthesized by the of phospholipid biosynthetic enzymes to vivo cells. the PE molecular species to PC yeast by labeling with [methyl-D3]methionine is a to the species of PE of conversion of PE to PC was found to by the methylation of the PE species the the the methylation of PE is the of the preferential conversion of PE to PC is of the species composition of the PE and is similar cells and is the of the preferential methylation of the species methylation is with is to the the and the of for the of the of PE of to the species methylation to is a range of species of PE was by the and by yeast the and and of the is to the of PE to the the of the the the of PE the is yeast cells of conversion of the PE species to PC were found to similar the the preferential methylation is by the PE species composition by the of PE to the the the newly synthesized PE the is to the the to over the of a contribute to the species the methylation of preferential methylation of PE membranes by the of the the methylation the of the PE molecular species to the of the PE species the In the the of PE species the the with the of PE the To between these the of the using of PE to the of the of of enzymes the of between the of the to the species and were with [methyl-D3]methionine the and of revealed the methylation catalyzed by Cho2p of the species of is with and the of to a phospholipid To the of the and to the and the In using revealed the molecular species of using revealed a for PE In the methylation of PE is to the and to PC the the of a for species for PC species and with species is similar to with and of the is to an of the by the species methylation yeast preferential methylation of PE by the Cho2p the yeast with a to the between the species of PC and of the and di-C16:1 PC species the of to membranes to the PC species of PC the and of PC by the of the route to PC biosynthesis by PC of to the PC species composition PC synthesized by the route is the species and with PC of PC by a yeast an route the species the PC and to the of the biosynthetic and the PC species In the of the of the abundant phospholipid PC yeast the methylation of PE the of PC and to the of yeast the methylation route is a of the PC molecular species and of labeling yeast with phospholipid stable of the newly synthesized by the of phospholipid biosynthetic enzymes to vivo cells. the PE molecular species to PC yeast by labeling with [methyl-D3]methionine is a to the species of PE of conversion of PE to PC was found to by the methylation of the PE species the the the methylation of PE is the of the preferential conversion of PE to PC is of the species composition of the PE and is similar cells and is the of the preferential methylation of the species methylation is with is to the the and the of for the of the of PE of to the species methylation to is a range of species of PE was by the and by yeast the and and of the is to the of PE to the the of the the the of PE the is yeast cells of conversion of the PE species to PC were found to similar the the preferential methylation is by the PE species composition by the of PE to the the the newly synthesized PE the is to the the to over the of a contribute to the species the methylation of preferential methylation of PE membranes by the of the the methylation the of the PE molecular species to the of the PE species the In the the of PE species the the with the of PE the To between these the of the using of PE to the of the of of enzymes the of To between the of the to the species and were with [methyl-D3]methionine the and of revealed the methylation catalyzed by Cho2p of the species of is with and the of In to a phospholipid To the of the and to the and the In using revealed the molecular species of using revealed a for PE In the methylation of PE is to the and to PC the the of a for species for PC species and with species is similar to with and of the is to an of the by the species methylation yeast preferential methylation of PE by the Cho2p the yeast with a to the between the species of PC and of the and di-C16:1 PC species the of to membranes to the PC species of PC the and of PC by the of the route to PC biosynthesis by PC of to the PC species composition PC synthesized by the route is the species and with PC of PC by a yeast an route the species the PC and to the of the biosynthetic and the PC species and for with the mass spectrometry