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Separation of yeast phospholipids using one-dimensional thin-layer chromatography
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 338 (2005) 162–164 www.elsevier.com/locate/yabio
Notes & Tips
Separation of yeast phospholipids using one-dimensional thin-layer chromatography Deirdre L. Vaden1, Vishal M. Gohil1, Zhiming Gu2, Miriam L. Greenberg¤ Department of Biological Science, Wayne State University, Detroit, MI 48202, USA Received 2 September 2004 Available online 8 December 2004
The yeast Saccharomyces cerevisiae is a valuable eukaryotic model system for studying phospholipid biosynthesis. Most phospholipid biosynthetic enzymes and the genes that encode them have been characterized in yeast [1], which is thus an excellent model for human disorders of phospholipid metabolism. The yeast taz1 mutant, for example, has (CL)3 defects similar to those observed in Barth syndrome [2]. The traditional method of analysis of yeast phospholipids involves separation of phospholipids by twodimensional thin-layer chromatography (2-D TLC), visualization with iodine vapor, scraping of spots, and quantiWcation of inorganic phosphate [3]. This multistep approach requires excessive handling of samples. Furthermore, separation of each sample requires one TLC plate. Thus, this method is not suitable for the large-scale analysis of multiple samples. Leray et al. [4] described a one-dimensional thin-layer chromatography (1-D TLC) method for separation of blood platelet phospholipids. This method does not allow eYcient separation of PG from PE and MLCL from PA. In this
*
Corresponding author. Fax: +1 313 577 6891. E-mail address: [email protected] (M.L. Greenberg). 1 Equal contributors. 2 Present address: 2315 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. 3 Abbreviations used: PC, phosphatidylcholine; PA, phosphatidic acid; PE, phosphatidylethanolamine; CL, cardiolipin; MLCL, monolysocardiolipin; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PMME, phosphatidylmonomethylethanolamine; PDME phosphatidyldimethylethanolamine; CDP-DAG, cytidine diphosphate diacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; 1-D TLC, one dimensional thin-layer chromatography; CSM, complete synthetic medium; YPD, complex medium; WT, wild-type. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.020
report, we describe a modiWed 1-D TLC method for the separation of all major yeast phospholipids and minor phospholipids with a high degree of resolution and reproducibility. Materials. Yeast extract, glucose, and peptone were purchased from Difco Laboratories (Detroit, MI). Primuline and phospholipids PA, PE, CL, PG, PS, PI, and PC were from Sigma (St. Louis, MO). PMME, PDME, and lysophospholipids were from Avanti Polar Lipids (Alabaster, AL). 32Pi was purchased from Perkin–Elmer Life Sciences (Boston, MA). Thin-layer chromatography plates (Cat. No. 4855-820, LK5 silica gel 150 A) were purchased from Whatman (Clifton, NJ). Yeast growth media and steady state labeling of phospholipids. Complex medium (YPD) and complete synthetic medium (CSM) were prepared according to Guthrie and Fink [5]. Yeast strains were inoculated in 2 ml YPD or CSM containing 10 Ci 32Pi ml¡1 at a starting A550 of 0.025 and incubated at 30 °C for at least Wve to six generations to achieve steady state labeling. Phospholipid extraction was carried out as described in Gu et al [2]. BrieXy, cells were harvested, washed once with sterile water, and digested by zymolyase at room temperature for 15 min to yield spheroplasts, which were pelleted by centrifugation. Phospholipids were extracted from spheroplasts in 250 l 2:1 chloroform/methanol under constant shaking for 45 min to 1 h at room temperature. Proteins and other nonlipid cellular debris were partitioned in distilled water by low-speed centrifugation for 1 min. Phospholipids were dissolved in 100 l 2:1 chloroform/methanol. One-dimensional thin-layer chromatography. LK5 silica gel 150 A TLC plates were prewashed in chloroform/ methanol (1/1, v/v) to remove impurities. Before use, plates were completely wetted in either 2.3 or 1.8 % boric
Notes & Tips / Anal. Biochem. 338 (2005) 162–164
163
Fig. 1. EVect of boric acid concentration on separation of yeast phospholipids. Phospholipids from CL pathway mutants (crd1, pgs1, and taz1) grown in YPD were separated on a 2.3% (left) or 1.8% boric-acid-washed TLC plate (right). PA, phosphatidic acid; PE, phosphatidylethanolamine; CL, cardiolipin; MLCL, monolysocardiolipin; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.
Fig. 2. Separation of phospholipids from wild-type and mutant yeast strains. Yeast cells were grown in YPD {wild-type, crd1, pgs1, taz1} or CSM {wild-type, cho1, opi3, cdg1, ino1 (strains from S. Henry)} media. For growth of the ino1 mutant, CSM was supplemented with 10 M inositol. The opi3 mutant was supplemented with 1 mM monomethylethanolamine (to cause accumulation of PMME and PDME). Steady state labeling, phospholipid extraction, 1-D TLC, and phosphorimaging were carried out as described under Materials and methods. CDP-DAG, cytidine diphosphate diacylglycerol; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; LPA, lysophosphatidic acid; PMME, phosphatidylmonomethylethanolamine; PDME phosphatidyldimethylethanolamine; ? unidentiWed phospholipid; other abbreviations same as in Fig. 1.
acid prepared in 100% ethanol, dried for 5 min, and baked for 15 min at 100 °C. The concentration zone was divided into several lanes by scraping parallel lines of
1.5 cm width, thus providing many lanes in which to load samples. Lipid samples (20 l) were applied to concentration zone lanes. Samples were dried, and plates were
164
Notes & Tips / Anal. Biochem. 338 (2005) 162–164
placed in the chromatography tank that had been lined with Wlter paper (Whatman No. 3) and equilibrated with the solvent chloroform/ethanol/water/triethylamine (30/ 35/7/35, v/v) [4] for at least 2 h. The migration time was t1 h 45 min. Plates were dried completely in a fume hood and then run for a second time in the same solvent system and in the same direction to achieve higher resolution. Phosphorimaging. Radiolabeled samples were exposed to a phosphorimager screen for 15 min to 1 h and scanned by the Storm scan system (Molecular Dynamics). Individual phospholipid bands can be quantiWed using Image-Quant software. In a previous method [4], TLC plates were washed with 2.3% boric acid prior to application of the samples. However, this technique does not clearly separate PG from PE or MLCL from PA. Reducing the concentration of boric acid to 1.8% greatly improved the separation of these phospholipids without altering the separation of other phospholipids (Fig. 1). Well-characterized yeast phospholipid mutants were analyzed to ascertain the extent of separation of phospholipids in biological samples. As seen in Fig. 2, all major phospholipids were present in wild-type cells (WT). CL was absent and PG accumulated in crd1, which lacks CL synthase (crd1) [6]. CL and PG were absent from the PGP synthase mutant pgs1 (pgs1) [7]. MLCL accumulated in the mutant of TAZ1 (taz1) [2]. PA was reduced in WT cells grown in CSM. PS was absent from cho1 cells (cho1) [8]. An accumulation of PMME and PDME is evident in opi3 [9]. A relative increase in PE with concomitant decrease in CDP-DAG was observed in the cdg1 mutant (cdg1) [10]. The ino1 mutant cells grown in low-inositol supplementation (10 M) have barely detectable PI (ino1). An unidentiWed phospholipid band was observed between CL and MLCL in most strains (upon overexposure of the phosphoimager screen or when high amounts of radiolabeled samples are loaded). The simplicity and rapidity of this method makes it a useful tool for phospholipid separation and for
identiWcation of mutants with abnormal phospholipid metabolism.
Acknowledgments This work was supported by Grants HL62263 and MH56220 from the National Institutes of Health. We are grateful to Susan Henry for yeast strains. We thank Asimur Rahman for help in preparing the Wgures.
References [1] G.M. Carman, S.A. Henry, Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes, Prog. Lipid Res. 38 (1999) 361–399. [2] Z. Gu, F. Valianpour, S. Chen, F.M. Vaz, G.A. Hakkaart, R.J. Wanders, M.L. Greenberg, Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome, Mol. Microbiol. 51 (2004) 149–158. [3] G.S. Getz, S. Jakovcic, J. Heywood, J. Frank, M. Rabinowitz, A two-dimensional thin-layer chromatographic system for phospholipid separation. The analysis of yeast phospholipids, Biochim. Biophys. Acta 218 (1970) 441–452. [4] C. Leray, X. Pelletier, S. Hemmendinger, J.P. Cazenave, Thin-layer chromatography of human platelet phospholipids with fatty acid analysis, J. Chromatogr. 420 (1987) 411–416. [5] C. Guthrie, G.R. Fink, Methods Enzymol. 194 (1991) 14–15. [6] F. Jiang, H.S. Rizavi, M.L. Greenberg, Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or nonfermentable carbon sources, Mol. Microbiol. 26 (1997) 481–491. [7] Q. Zhong, M.L. Greenberg, Regulation of phosphatidylglycerophosphate synthase by inositol in Saccharomyces cerevisiae is not at the level of PGS1 mRNA abundance, J. Biol. Chem. 278 (2003) 33978–33984. [8] K.D. Atkinson, B. Jensen, A.I. Kolat, E.M. Storm, S.A. Henry, S. Fogel, Yeast mutants auxotrophic for choline or ethanolamine, J. Bacteriol. 141 (1980) 558–564. [9] M.L. Greenberg, L.S. Klig, V.A. Letts, B.S. Loewy, S.A. Henry, Yeast mutant defective in phosphatidylcholine synthesis, J. Bacteriol. 153 (1983) 791–799. [10] L.S. Klig, M.J. Homann, S.D. Kohlwein, M.J. Kelley, S.A. Henry, G.M. Carman, Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis, J. Bacteriol. 170 (1988) 1878– 1886.
Notes & Tips
Separation of yeast phospholipids using one-dimensional thin-layer chromatography Deirdre L. Vaden1, Vishal M. Gohil1, Zhiming Gu2, Miriam L. Greenberg¤ Department of Biological Science, Wayne State University, Detroit, MI 48202, USA Received 2 September 2004 Available online 8 December 2004
The yeast Saccharomyces cerevisiae is a valuable eukaryotic model system for studying phospholipid biosynthesis. Most phospholipid biosynthetic enzymes and the genes that encode them have been characterized in yeast [1], which is thus an excellent model for human disorders of phospholipid metabolism. The yeast taz1 mutant, for example, has (CL)3 defects similar to those observed in Barth syndrome [2]. The traditional method of analysis of yeast phospholipids involves separation of phospholipids by twodimensional thin-layer chromatography (2-D TLC), visualization with iodine vapor, scraping of spots, and quantiWcation of inorganic phosphate [3]. This multistep approach requires excessive handling of samples. Furthermore, separation of each sample requires one TLC plate. Thus, this method is not suitable for the large-scale analysis of multiple samples. Leray et al. [4] described a one-dimensional thin-layer chromatography (1-D TLC) method for separation of blood platelet phospholipids. This method does not allow eYcient separation of PG from PE and MLCL from PA. In this
*
Corresponding author. Fax: +1 313 577 6891. E-mail address: [email protected] (M.L. Greenberg). 1 Equal contributors. 2 Present address: 2315 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. 3 Abbreviations used: PC, phosphatidylcholine; PA, phosphatidic acid; PE, phosphatidylethanolamine; CL, cardiolipin; MLCL, monolysocardiolipin; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PMME, phosphatidylmonomethylethanolamine; PDME phosphatidyldimethylethanolamine; CDP-DAG, cytidine diphosphate diacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; 1-D TLC, one dimensional thin-layer chromatography; CSM, complete synthetic medium; YPD, complex medium; WT, wild-type. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.020
report, we describe a modiWed 1-D TLC method for the separation of all major yeast phospholipids and minor phospholipids with a high degree of resolution and reproducibility. Materials. Yeast extract, glucose, and peptone were purchased from Difco Laboratories (Detroit, MI). Primuline and phospholipids PA, PE, CL, PG, PS, PI, and PC were from Sigma (St. Louis, MO). PMME, PDME, and lysophospholipids were from Avanti Polar Lipids (Alabaster, AL). 32Pi was purchased from Perkin–Elmer Life Sciences (Boston, MA). Thin-layer chromatography plates (Cat. No. 4855-820, LK5 silica gel 150 A) were purchased from Whatman (Clifton, NJ). Yeast growth media and steady state labeling of phospholipids. Complex medium (YPD) and complete synthetic medium (CSM) were prepared according to Guthrie and Fink [5]. Yeast strains were inoculated in 2 ml YPD or CSM containing 10 Ci 32Pi ml¡1 at a starting A550 of 0.025 and incubated at 30 °C for at least Wve to six generations to achieve steady state labeling. Phospholipid extraction was carried out as described in Gu et al [2]. BrieXy, cells were harvested, washed once with sterile water, and digested by zymolyase at room temperature for 15 min to yield spheroplasts, which were pelleted by centrifugation. Phospholipids were extracted from spheroplasts in 250 l 2:1 chloroform/methanol under constant shaking for 45 min to 1 h at room temperature. Proteins and other nonlipid cellular debris were partitioned in distilled water by low-speed centrifugation for 1 min. Phospholipids were dissolved in 100 l 2:1 chloroform/methanol. One-dimensional thin-layer chromatography. LK5 silica gel 150 A TLC plates were prewashed in chloroform/ methanol (1/1, v/v) to remove impurities. Before use, plates were completely wetted in either 2.3 or 1.8 % boric
Notes & Tips / Anal. Biochem. 338 (2005) 162–164
163
Fig. 1. EVect of boric acid concentration on separation of yeast phospholipids. Phospholipids from CL pathway mutants (crd1, pgs1, and taz1) grown in YPD were separated on a 2.3% (left) or 1.8% boric-acid-washed TLC plate (right). PA, phosphatidic acid; PE, phosphatidylethanolamine; CL, cardiolipin; MLCL, monolysocardiolipin; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine.
Fig. 2. Separation of phospholipids from wild-type and mutant yeast strains. Yeast cells were grown in YPD {wild-type, crd1, pgs1, taz1} or CSM {wild-type, cho1, opi3, cdg1, ino1 (strains from S. Henry)} media. For growth of the ino1 mutant, CSM was supplemented with 10 M inositol. The opi3 mutant was supplemented with 1 mM monomethylethanolamine (to cause accumulation of PMME and PDME). Steady state labeling, phospholipid extraction, 1-D TLC, and phosphorimaging were carried out as described under Materials and methods. CDP-DAG, cytidine diphosphate diacylglycerol; LPC, lysophosphatidylcholine; LPI, lysophosphatidylinositol; LPA, lysophosphatidic acid; PMME, phosphatidylmonomethylethanolamine; PDME phosphatidyldimethylethanolamine; ? unidentiWed phospholipid; other abbreviations same as in Fig. 1.
acid prepared in 100% ethanol, dried for 5 min, and baked for 15 min at 100 °C. The concentration zone was divided into several lanes by scraping parallel lines of
1.5 cm width, thus providing many lanes in which to load samples. Lipid samples (20 l) were applied to concentration zone lanes. Samples were dried, and plates were
164
Notes & Tips / Anal. Biochem. 338 (2005) 162–164
placed in the chromatography tank that had been lined with Wlter paper (Whatman No. 3) and equilibrated with the solvent chloroform/ethanol/water/triethylamine (30/ 35/7/35, v/v) [4] for at least 2 h. The migration time was t1 h 45 min. Plates were dried completely in a fume hood and then run for a second time in the same solvent system and in the same direction to achieve higher resolution. Phosphorimaging. Radiolabeled samples were exposed to a phosphorimager screen for 15 min to 1 h and scanned by the Storm scan system (Molecular Dynamics). Individual phospholipid bands can be quantiWed using Image-Quant software. In a previous method [4], TLC plates were washed with 2.3% boric acid prior to application of the samples. However, this technique does not clearly separate PG from PE or MLCL from PA. Reducing the concentration of boric acid to 1.8% greatly improved the separation of these phospholipids without altering the separation of other phospholipids (Fig. 1). Well-characterized yeast phospholipid mutants were analyzed to ascertain the extent of separation of phospholipids in biological samples. As seen in Fig. 2, all major phospholipids were present in wild-type cells (WT). CL was absent and PG accumulated in crd1, which lacks CL synthase (crd1) [6]. CL and PG were absent from the PGP synthase mutant pgs1 (pgs1) [7]. MLCL accumulated in the mutant of TAZ1 (taz1) [2]. PA was reduced in WT cells grown in CSM. PS was absent from cho1 cells (cho1) [8]. An accumulation of PMME and PDME is evident in opi3 [9]. A relative increase in PE with concomitant decrease in CDP-DAG was observed in the cdg1 mutant (cdg1) [10]. The ino1 mutant cells grown in low-inositol supplementation (10 M) have barely detectable PI (ino1). An unidentiWed phospholipid band was observed between CL and MLCL in most strains (upon overexposure of the phosphoimager screen or when high amounts of radiolabeled samples are loaded). The simplicity and rapidity of this method makes it a useful tool for phospholipid separation and for
identiWcation of mutants with abnormal phospholipid metabolism.
Acknowledgments This work was supported by Grants HL62263 and MH56220 from the National Institutes of Health. We are grateful to Susan Henry for yeast strains. We thank Asimur Rahman for help in preparing the Wgures.
References [1] G.M. Carman, S.A. Henry, Phospholipid biosynthesis in the yeast Saccharomyces cerevisiae and interrelationship with other metabolic processes, Prog. Lipid Res. 38 (1999) 361–399. [2] Z. Gu, F. Valianpour, S. Chen, F.M. Vaz, G.A. Hakkaart, R.J. Wanders, M.L. Greenberg, Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome, Mol. Microbiol. 51 (2004) 149–158. [3] G.S. Getz, S. Jakovcic, J. Heywood, J. Frank, M. Rabinowitz, A two-dimensional thin-layer chromatographic system for phospholipid separation. The analysis of yeast phospholipids, Biochim. Biophys. Acta 218 (1970) 441–452. [4] C. Leray, X. Pelletier, S. Hemmendinger, J.P. Cazenave, Thin-layer chromatography of human platelet phospholipids with fatty acid analysis, J. Chromatogr. 420 (1987) 411–416. [5] C. Guthrie, G.R. Fink, Methods Enzymol. 194 (1991) 14–15. [6] F. Jiang, H.S. Rizavi, M.L. Greenberg, Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or nonfermentable carbon sources, Mol. Microbiol. 26 (1997) 481–491. [7] Q. Zhong, M.L. Greenberg, Regulation of phosphatidylglycerophosphate synthase by inositol in Saccharomyces cerevisiae is not at the level of PGS1 mRNA abundance, J. Biol. Chem. 278 (2003) 33978–33984. [8] K.D. Atkinson, B. Jensen, A.I. Kolat, E.M. Storm, S.A. Henry, S. Fogel, Yeast mutants auxotrophic for choline or ethanolamine, J. Bacteriol. 141 (1980) 558–564. [9] M.L. Greenberg, L.S. Klig, V.A. Letts, B.S. Loewy, S.A. Henry, Yeast mutant defective in phosphatidylcholine synthesis, J. Bacteriol. 153 (1983) 791–799. [10] L.S. Klig, M.J. Homann, S.D. Kohlwein, M.J. Kelley, S.A. Henry, G.M. Carman, Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis, J. Bacteriol. 170 (1988) 1878– 1886.