complex, even under conditions in which the CDP-choline pathway does not contribute to the generation of net cellular

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1994, p Vol. 176, No /94/$ Copyright C 1994, American Society for Microbiology Functional Redundancy of CDP-Ethanolamine and CDP-Choline Pathway Enzymes in Phospholipid Biosynthesis: Ethanolamine- Dependent Effects on Steady-State Membrane Phospholipid Composition in Saccharomyces cerevisiae TODD P. McGEE,t HENRY B. SKINNER, AND VYTAS A. BANKAITIS* Department of Cell Biology, University ofalabama at Birmingham, Birmingham, Alabama Received 7 July 1994/Accepted 7 September 1994 It has been established that yeast membrane phospholipid content is responsive to the inositol and choline content of the growth medium. Alterations in the levels of transcription of phospholipid biosynthetic enzymes contribute significantly to this response. We now describe conditions under which ethanolamine can exert significant influence on yeast membrane phospholipid composition. We demonstrate that mutations which block a defined subset of the reactions required for the biosynthesis of phosphatidylcholine (PC) via the CDP-choline pathway cause ethanolamine-dependent ellects on the steady-state levels of bulk PC in yeast membranes. Such an ethanolamine-dependent reduction in bulk membrane PC content was observed for both choline kinase (cki) and choline phosphotransferase (cptl) mutants, but it was not observed for mutants defective in cholinephosphate cytidylyltransferase, the enzyme that catalyzes the penultimate reaction of the CDP-choline pathway for PC biosynthesis. Moreover, the ethanolamine effect observed for cki and cptl mutants was independent of the choline content of the growth medium. Finally, we found that haploid yeast strains defective in the activity of both the choline and ethanolamine phosphotransferases experienced an ethanolamine-insensitive reduction in steady-state PC content, an effect which was not observed in strains defective in either one of these activities alone. The collective data indicate that specific enzymes of the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis, while able to contribute to PC synthesis when yeast cells are grown under conditions of ethanolamine deprivation, do not do so when yeast cells are presented with this phospholipid headgroup precursor. Phosphatidylcholine (PC) is a major phospholipid (PL) of most eukaryotic cells and is an important structural molecule of both cellular membranes and lung surfactant (19). PC is synthesized either by the incorporation of choline into PC via a cytidine-based process (i.e., the CDP-choline pathway) or via the methylation of phosphatidylethanolamine (PE) by the terminal enzymes of the PE methylation pathway (reviewed in reference 5). The CDP-choline pathway involves three distinct reactions: (i) the phosphorylation of choline by a choline kinase (CKIase), (ii) the production of CDP-choline from phosphorylcholine by the action of the cholinephosphate cytidylyltransferase (CCTase), and (iii) the choline phosphotransferase (CPTase)-catalyzed condensation of CDP-choline with diacylglycerol to form PC (5, 13). In addition to its role in the generation of net PC from exogenously supplied choline, the CDP-choline pathway is also involved in the salvage of cholinecontaining metabolites resulting from PC turnover. A directly analogous pathway for the synthesis of PE from free ethanolamine exists (i.e., the CDP-ethanolamine pathway). Both the CDP-choline and CDP-ethanolamine pathways are operative in the yeast Saccharomyces cerevisiae, and strains defective in CKIase (cki), CCTase (cct), CPTase (cptl), and ethanolamine phosphotransferase (EPTase) (eptl) activity have been isolated (13). An entirely unanticipated cellular involvement of the * Corresponding author. Mailing address: Department of Cell Biology, 668 Basic Health Sciences Building, University of Alabama at Birmingham, Birmingham, AL Phone: (205) Fax: (205) t Present address: Department of Biological Sciences, Stanford University, Stanford, CA CDP-choline pathway has also recently been discovered. Genetic analyses of the yeast Golgi secretory process have demonstrated the activity of the CDP-choline pathway to be intimately related to the essential requirement for phosphatidylinositol/phosphatidylcholine transfer protein function in the maintenance of the secretory competence of the yeast Golgi complex, even under conditions in which the CDP-choline pathway does not contribute to the generation of net cellular PC (4, 12). These findings have raised a number of questions concerning the role of PL metabolism in eukaryotic secretory pathway function, organelle membrane composition, and organelle biogenesis. There exists ample evidence to indicate that the transcription of at least several structural genes for yeast membrane phospholipid biosynthetic enzymes is regulated in response to the inositol and choline content of the growth medium (reviewed in reference 13). In this report, we describe conditions under which ethanolamine exerts clear effects on bulk membrane PL composition. We demonstrate that mutations which block defined reactions in the biosynthesis of PC via the CDP-choline pathway exert variable effects on the steady-state levels of bulk PC in yeast membranes. We show that the differential effects on steady-state PC content resulting from the various CDP-choline pathway defects correlate with the activity of biochemically redundant enzymes whose PC biosynthetic capabilities are inhibited by the presence of ethanolamine in the growth medium. Moreover, we have obtained both biochemical and genetic data to indicate that these biochemical homologs represent structural enzymes of the CDP-ethanolamine pathway. The collective data identify a subset of enzymes of the CDP-ethanolamine pathway that, 6861

2 6862 McGEE ET AL. while able to effect a significant contribution to the steady-state PC content of yeast cells grown under conditions of ethanolamine deprivation, fail to contribute to bulk membrane PC content when yeast cells are cultured in the presence of exogenously supplied ethanolamine. MATERIALS AND METHODS Yeast strains, media, and reagents. YPD and synthetic defined complete media were prepared as previously described with the exception that the agar concentration was held at 2.5% (2, 14). myo-inositol, choline chloride, and ethanolamine hydrochloride were purchased from Sigma (St. Louis, Mo.) and were added, when appropriate, to a final concentration of 1 mm each. 32Pi was purchased from American Radiolabeled Chemicals (St. Louis, Mo.). Butylated hydroxytoluene (BHT) and Rhodamine 6G were purchased from Sigma. All other chemicals and materials were of reagent grade. Yeast strains used for this study were CTY182 (ALATa ura3-52 Ahis3-200 lys2-801) (10), CTY105 (AL4Ta ura3-52 Ahis3-200 lys2-801 sec14-1ts bsr2-5) (5), CTY392 (ALTa ura3-52 Ahis3-200 lys2-801 sec14-1's cki-284:his3) (5), CTY 434 (AL4Ta ura3-52 his5-519 ade2-101 leu2-3,112 sec14-1ts cptl::leu2) (6, 12), C`TY436 (MATa ura3-52 Ahis3-200 lys2-801 sec14-1's eptl::ura3) (this study), CTY479 (MATa ade2-101 ura3-52 Ahis3-200 bsd2-1) (this study), CTY618 (MATa ura3-52 his5-519 ade2-101 leu2-3,112 sec14-1s cptl::leu2 eptl::ura3) (this study), and CTY394 (A ATa ura3-52 Ahis3-200 lys2-801 secl4-88::ura3 cki-284::his3) (this study). Yeast strains CTY436 and CTY618 were created by direct transformation with the eptl::ura3 disruption cassette (prh511) described and constructed by Hjelmstad and Bell (7). Steady-state PL analysis. To determine the steady-state PL composition of cells grown in synthetic defined complete media (9), cells were grown for five to six generations in the medium of interest supplemented with 32Pi to a final concentration of 10,uCi/ml. Following labeling, cells were harvested by centrifugation at 500 x g and the incorporation of label was terminated by resuspension of the cell pellet in 5% ice-cold trichloroacetic acid. PLs were extracted from the fixed cell pellet by incubation in polar extraction solvent (16) at 65 C for 20 min. Lipid extractable material was separated from the aqueous environment by the addition of CHCl3-CH30H-BHT (2:1:0.0005), and the medium was vortexed and centrifuged to separate the aqueous and organic phases. The organic phase was removed and brought to dryness under a stream of nitrogen gas. Extracted lipid material was resuspended in 60,ul of CHCl3-CH30H-BHT (2:1:0.0005) for separation and identification. PLs were subsequently resolved by two-dimensional paper chromatography with Whatman SG81 chromatography paper which had been treated with 2% EDTA and heated to 100 C for at least 20 min prior to sample application. Firstdimension resolution was achieved by using the solvent system CHCl3-CH3OH-NH4OH-H20 (22:9:1:0.26), while the seconddimension solvent consisted of CHC13-CH30H-CH3COOH-H20 (8:1:1.25:0.25). Resolved PLs were located by autoradiography, and the identified PL spots were excised and individually quantitated by scintillation counting. The PL composition of cells grown in YPD was determined by similar methods, with the exception that individual PL species were quantitated by a nonisotopic method. After growth to mid-logarithmic growth phase in YPD (14), cells were harvested, ice-cold trichloroacetic acid (5% final concentration) was added, and the PLs were extracted from the cell pellet as described above. Following resolution by two-dimensional paper chromatography, extracted PLs were located by J. BACTERIOL. staining the chromatograph with a % solution of Rhodamine 6G and inspecting the chromatograph under UV illumination (3). The regions of the chromatograph containing the major PLs were excised, and the PLs were eluted from the chromatography paper by the addition of 2.0 ml of CHCl3- CH30H-BHT (2:1:0.0005%). The isolated PL species were subsequently dried under gentle vacuum, and the relative amount of each PL was determined by assay for total phosphorous as described by Ames (1). [14C]ethanolamine labeling of yeast lipids. The appropriate yeast strains were grown to mid-logarithmic growth phase in defined medium lacking choline and were presented with [14C]ethanolamine hydrochloride (1,uCi/ml) for 20 min at 25 C with shaking. Incorporation of label was measured by removing 1/10 of the culture, immobilizing the culture aliquot on 0.5-,um-pore glass fiber filters, washing the filters four times with 2.5 volumes of a 10 mm ethanolamine solution per wash, and resuspending the dried cell pellets (including filters) in scintillation fluid for counting. ['C]ethanolamine incorporation values were employed to normalize the loading of lipid samples derived from each of these cultures for further analysis. PLs were extracted from the remainder of each culture, as described above, and the incorporation of radiolabel into normalized amounts of lipid sample from each culture was quantitated by scintillation counting. Assessment of opi phenotype. The inositol excretion capabilities of yeast strains were tested on plates lacking inositol, choline, and ethanolamine (I-C-E- plates) that had a reduced agar concentration (1.2%) relative to our standard solid media. The yeast strain to be tested was patched onto the I-C-E- tester plate and permitted to grow for 72 h at 25 C. The inositol auxotroph reporter strain was then dilution streaked away from the patch, and crossfeeding was scored after an additional 72-h incubation at 25 C. The inositol auxotroph used in this inositol excretion bioassay was haploid strain CTY479 (bsd2-1 SEC14). The bsd2-1 allele manifests itself in a dominant inositol auxotrophy (20), one that is even tighter than the Ino- phenotype of strains defective in inositol biosynthesis (e.g., strains carrying inol-13). RESULTS AND DISCUSSION Bulk membrane PL composition of CDP-choline pathway mutants as a function of growth medium. We have observed that mutations which block specific reactions of the CDPcholine pathway for PC biosynthesis elicit differential effects on the steady-state membrane PL composition when the corresponding mutants are grown in defined medium supplemented with inositol and choline (I+C+). An example of such an effect is provided by a comparison of the bulk membrane PL compositions of mutant strains bearing individual cki-284:: HIS3, bsr2-5, and cptl::leu2 alleles, i.e., loss-of-function mutations in the CKIase, CCTase, and CPTase structural genes, respectively (Table 1). The cki-284::his3 mutant, when grown in I+C+ medium, displayed a PL composition of 25% phosphatidylinositol (PI), 42% PC, 7% phosphatidylserine (PS), and 12% PE, a profile which was essentially indistinguishable from those of wild-type and cptl::leu2 yeast strains. The bsr2-5 mutation represents a loss-of-function mutation in the CCTase structural gene as evidenced by its very tight linkage to the CCTase structural gene (28 parental ditype asci: 0 nonparental ditype asci: 0 tetratype asci) and the absence of CCTase activity in cell extracts prepared from bsr2-5 yeast strains (not shown), and it results in a CDP-choline pathway PC biosynthetic defect of a magnitude similar to that of the defect elicited by either cki-284::his3 or cptl::leu2 (12). However,

3 VOL. 176, 1994 ETHANOLAMINE EFFECTS ON YEAST MEMBRANE PHOSPHOLIPIDS 6863 TABLE 1. Steady-state PL composition of yeast strains grown in I+C+E- and I+C+E+ defined mediaa Type of medium Strain Relevant genotype Mole fraction of the following PL species (% of total extractable PL): PC PI PS PE I+C+E- C1Y182 WTV 44 ± ± ± ± 0.6 C11Y105 bsr ± ± ± ± 1.4 CTY392 cki-284:jhis3 42 ± ± ± ± 0.4 CTY434 cptl::leu2 41 ± ± ± ± 1.0 CTY436 eptl::urh43 40 ± ± ± ± 1.2 C1Y618 cptl::leu2 eptl::ur,43 30 ± ± ± ± 0.8 I+C+E+ CTY182 WT 42 ± ± ± ± 0.8 CTY105 bsr ± ± ± ± 0.9 C1fY392 cki-284::his3 27 ± ± ± ± 0.5 CTY434 cptl::leu2 34 ± ± ± ± 1.0 C1TY436 eptl::ura3 40 ± ± ± ± 0.4 CTY618 cptl::leu2 eptl::ura3 30 ± ± ± ± 0.8 a The indicated strains were grown for five to six generations in synthetic defined medium containing inositol and choline only (I+C+E-) or inositol, choline, and ethanolamine (I+C+E+) and supplemented with 32Pi to 10,uCiml. The PL headgroup precursors were added to growth medium to a final concentration of 1 mm each. Glycerophospholipids were subsequently extracted, resolved, and quantitated as described in Materials and Methods. The mole fraction of each of the major PLs of yeast cells is shown. Radiolabeled material that failed to migrate from the origin was also counted and included in the total PL value used in the mole percentage calculations for the individual PL species. For this reason, the sum of the PI, PC, PS, and PE mole percentages does not equal 100%. Only trace amounts of phosphatidic acid and monomethylated and dimethylated forms of PC were detected (a composite total of <5% of the total PL content), and these trace values were not included in the mole percentage calculations. The values shown represent the averages of those from three independent trials ± standard deviations. The complete genotype of each yeast strain employed in these experiments is given in Materials and Methods. b WT, wild type. this CCTase defect reduced the steady-state bulk membrane PC level to only 30% of the total PL and increased the bulk membrane PI level to 40% of the total PL (Table 1). Thus, although the bsr2-5, cptl::leu2, and cki-284::his3 mutants are all impaired for the activity of the CDP-choline pathway, only bsr2-5 mutants experienced steady-state reductions in bulk membrane PC when cells were grown in I+C+ medium. The significant elevation in the level of bulk membrane PI was uniquely observed for several bsr2 alleles, including a disruption allele, but it was a function of the growth medium that exhibited no discernible pattern that might provide some clues as to the mechanism of elevation of bulk PI levels (see "Conclusions" below). In contrast to the case which occurred when the cki-284:: HIS3 and cptl: LEU2 mutants were grown in I+C+ medium, the PL composition exhibited by these same mutants (when they were cultured in complex [YPD] medium) revealed a uniformly reduced bulk membrane PC content relative to the bulk membrane PC content recorded for the isogenic wild-type strain. Whereas bulk membranes prepared from wild-type cells consisted of 21% PI, 50% PC, 4% PS, and 20% PE, the cki-284:his3 and cptl: LEU2 mutants displayed abnormal PL compositions of 33% PI, 40% PC, 7% PS, and 20% PE and 29% PI, 42% PC, 6% PS, and 22% PE, respectively (data not shown). The CCTase-deficient bsr2-5 strain again exhibited an abnormal bulk membrane PL composition when grown in YPD medium (36% PI, 30% PC, 10% PS, and 26% PE; data not shown). We have previously shown that, under the growth conditions employed in this study, these various strains exhibit very similar PL mass per unit cell equivalent (12). Thus, quantitation of each individual PL species as mole percent of total PL allows direct comparison of PL values (obtained under a given set of growth conditions) between these various strains. As a result, these data indicate that the cki-284::his3 and cptl::leu2 mutants exhibited growth medium-dependent abnormalities in bulk PL composition, whereas the bsr2-5 strain exhibited constitutive abnormalities. A significant component of these compositional abnormalities was a marked reduction in bulk membrane PC content. Bulk membrane PL composition of CDP-choline pathway mutants is sensitive to ethanolamine. The CKIase, while representing the major CKJase activity of the cell, is not the only source of this activity in yeast cells. Evidence to this effect comes from the demonstration that Acki strains of yeast still exhibit CKlase activity in vivo (8, 12). We therefore considered the possibility that this residual CKIase activity contributed to the normal PL composition of cki mutants in I+C+ medium and that this activity was inhibited by some compound present in YPD medium but not contained in the defined medium. One plausible scenario was that the residual CKIase activity present in cki mutants represented a yeast ethanolamine kinase (EKIase) that, while primarily dedicated to PE synthesis via the CDP-ethanolamine pathway, could also utilize choline as a substrate. Thus, when cki yeast cells are grown in ethanolamine-free defined medium supplemented with choline, the EKIase would utilize choline as a substrate and contribute to the essentially wild-type steady-state PC composition exhibited by cki strains in minimal defined I+C+ medium. However, the availability of ethanolamine in rich growth medium would diminish, by substrate-level competition, the contribution of the EKIase to PC biosynthesis and result in the reduced bulk membrane PC content of cki-284::his3 strains in YPD medium. By direct analogy, as the EPTase of the CDP-ethanolamine pathway utilizes both ethanolamine and choline derivatives as substrates in vitro (6, 7), we considered it likely that the action of EPTase was responsible for the medium effect recorded for the cptl::leu2 strain. To provide experimental support for the hypothesis that enzymes of the CDP-ethanolamine pathway provide an ethanolamine-sensitive contribution to the steady-state bulk PC levels of CDP-choline pathway mutants, it was necessary to establish (i) that ethanolamine was the medium component responsible for the observed medium effects and (ii) that the observed medium effects were eliminated by inactivation of the appropriate enzyme of the CDP-ethanolamine pathway. To test the former point, yeast strains unable to execute defined reactions of the CDP-choline pathway were labeled to steady state in I+C+ synthetic medium containing (E+) or lacking

4 6864 McGEE ET AL. (E-) ethanolamine. Following this steady-state labeling, the PL composition of each strain was determined as a function of the growth medium (see Materials and Methods). In I+C+E+ medium, the bulk PL profiles of the wild-type strain closely resembled those displayed by this same strain when grown in I+C+E- medium (Table 1). This result demonstrated that bulk PC of wild-type yeast was insensitive to the medium effects related to ethanolamine supplementation. In marked contrast, strains bearing either the cki-284::his3 or the cptl:: LEU2 allele experienced a decrease in steady-state bulk PC content when grown in E+ medium, relative to the bulk membrane PC content recorded for these same strains grown in E- medium. Bulk membranes prepared from the cki-284:: HIS3 and cptl::leu2 strains grown in I+C+E+ medium were composed of only 28 and 34% PC, respectively (Table 1), compared with the steady-state PC content of approximately 42% of the total PL content measured for these same strains grown in I+C+E- medium (Table 1). These data clearly demonstrated that ethanolamine was the medium component responsible for effecting the reduction in bulk membrane PC content measured for the cki-284::his3 and cptl::leu2 mutant strains. Moreover, the ethanolamine sensitivity of bulk PC in cki-284::his3 yeast strains was consistent with the idea that the correction of bulk membrane PC content, at steady state, was mediated by an ethanolamine-sensitive CKIase. Ethanolamine elect reflects the differential contributions of CDP-ethanolamine pathway enzymes to PC biosynthesis. We wished to test the hypothesis that the differential intercession of CDP-ethanolamine enzymes in PC biosynthesis accounted for this ethanolamine effect. Unfortunately, a direct test of the prediction that yeast EKIase is responsible for the steady-state correction of CKIase deficiency (at the level of bulk membrane PC) is not yet possible, as the structural gene for the yeast EKIase has not been identified. Thus, to test the question of whether inactivation of the appropriate CDP-ethanolamine pathway enzyme would eliminate the observed ethanolamine effects, we focused our attention on the final reactions of the CDP-choline and CDP-ethanolamine pathways, which are catalyzed by the CPT1 and EPTI gene products, respectively (6, 7). The CPTI and EPTI genes encode aminoalcohol phosphotransferases which are capable of utilizing either choline or ethanolamine as a substrate in vitro, in direct analogy to the scenario considered above for the CKIase and the EKIase. Thus, the intercession hypothesis predicted that cptl eptl double mutants would display a reduced, and ethanolamineinsensitive, bulk membrane PC content at steady state. The data are shown in Table 1. As expected, the eptl::ura3 mutant, which retains normal CPT1 gene product function, displayed a wild-type PC content (40% of the total PL content) when grown in either E+ or E- medium (Table 1). In agreement with the test prediction, the cptl::leu2 eptl::ura3 double mutant, which should be completely deficient in PL synthesis by both the CDP-choline and CDP-ethanolamine pathways, displayed reduced bulk membrane PC content (only 30% of the total PL content) regardless of the ethanolamine content of the growth medium (Table 1). We interpret these data to indicate that the correction of PC biosynthetic defects at the steady-state level in the cptl mutant was due to the ethanolamine-sensitive contribution to PC synthesis by EPTase. As the eptl::ura3 mutation does not exert a significant effect on PC synthesis through the CDPcholine pathway, as measured by pulse-radiolabeling experiments following the incorporation of either [14C]choline or 32p into PL (12), the demonstration that EPTase can contribute significantly to steady-state bulk membrane PC levels indicated TABLE 2. J. BAC-TERIOL. Steady-state PL composition of strains grown in I-C-E- mediuma Mole fraction of the following PL species Strain Relevant (% of total PL content): genotype PC PI PS PE CTY182 WV 42±2.4 12± ± ± 1.7 CTY105 bsr ± ± ± ± 2.5 CTY434 cptl::leu2 43 ± ± ± ± 1.2 CIY436 eptl::ura3 43 ± ± ± ± 1.5 CfY618 cptl::leu2 37 ± ± ± ± 1.6 eptl::ura3 a The indicated strains were grown for five to six generations in I-C-Emedium supplemented with 32Pi to 10,uCi/ml. Glycerophospholipids were extracted, resolved, and quantitated as described in Materials and Methods. Details regarding the determination of the mole percentage values are presented in footnote a to Table 1. Data are averages of those from three independent trials ± standard deviations. Complete genotypes for the strains are provided in Materials and Methods. b WT, wild type. that in vivo catalysis of PC biosynthesis by this enzyme is inefficient at best. Another noteworthy feature of the data described above was that the CCTase-deficient bsr2-5 mutant consistently displayed reduction in bulk membrane PC content and that this reduction was entirely insensitive to the ethanolamine content of the growth medium. This observation was in complete agreement with biochemical data demonstrating that the corresponding enzyme of the CDP-ethanolamine pathway, the ethanolaminephosphate cytidylyltransferase (ECTase), is incapable of utilizing choline phosphate as a substrate (12, 17, 18). CDP-ethanolamine pathway enzymes contribute to choline salvage. When yeast cells are grown in choline-free media, the activity of the CDP-choline pathway is relegated to a salvage pathway that does not contribute to net PC biosynthesis but serves to recycle soluble choline-containing compounds produced by PC turnover back into PC. Thus, we wished to determine the effect of ethanolamine on bulk membrane composition in CDP-choline pathway mutants grown in the absence of exogenously supplied choline. As shown in Table 2, PC represented 42% of the total PL recovered from bulk membranes prepared from wild-type cells cultured in I-C-Emedium. Under these same growth conditions, CCIase-deficient bsr2-5 mutants experienced a reduction in bulk PC content to only approximately 36% of the total PL content. While neither the cptl:.leu2 mutation nor the eptl::ura3 mutation alone exerted any significant effect on cellular PC levels, the combination of both cpti::leu2 and eptl:ura3 effected a decrease in the steady-state bulk membrane PC content to only 37% of the total PL content (Table 2). Moreover, incubation of the cptl::leu2 and cki-284:.his3 mutants in I-C-E+ medium was sufficient to reduce the PC content of each of these mutants to the levels measured for bsr2-5 mutants and cptl::leu2 eptl::ura3 double mutants (data not shown). We consider the observed reductions in steady-state bulk membrane PC content described above to be significant for two reasons. First, these reductions were highly reproducible, and they were unusual in that the contribution of PC to bulk membrane PL content at steady state was very consistent under any given set of experimental conditions. Second, the reduction in bulk membrane PC content manifested itself in a clear overproduction of the inositol (opi) phenotype. The opi phenotype has been correlated with unbalanced PL biosynthesis in S. cerevisiae (11). This opi effect is revealed by the ability of

5 VOL. 176, 1994 ETHANOLAMINE EFFECTS ON YEAST MEMBRANE PHOSPHOLIPIDS 6865 wildtype eptl::ura3 cptl::leu2 cptl,::leu2, eptl::ura3 Downloaded from bsr2-5 FIG. 1. A subset of CDP-choline pathway mutants excrete inositol. A bioassay for inositol excretion is described in Materials and Methods. Briefly, an inositol-requiring strain, CTY479 (bsd2-1 SEC14), was dilution streaked away from a patch of the strain whose inositol excretion (i.e., opi) phenotype was to be tested. Thus, each streak (from left to right and top to bottom in the figure) of CTY479 exhibited progressively fewer cells. The relevant genotype of each strain tested is shown below the corresponding panel. on March 8, 2019 by guest inositol-overproducing strains to excrete inositol and crossfeed an appropriate inositol auxotroph reporter strain. As demonstrated in Fig. 1, neither wild-type nor cptl::leu2 nor eptl:: URA3 strains exhibited crossfeeding capability. All of these strains had similar wild-type bulk membrane PL profiles (see above). By contrast, both the bsr2-5 strain and the cptl::leu2 eptl::ura3 double-mutant strain not only shared the property of constitutively reduced bulk membrane PC content, but they also had unambiguous opi phenotypes. We wish to emphasize that these opi phenotypes, while clearly scorable, were nonetheless qualitatively weaker than those elicited by classical opi mutations, e.g., opil mutations that result in transcriptional derepression of INO1, the structural gene for the key inositol biosynthetic enzyme inositol-1-phosphate synthase (10). Curiously, addition of ethanolamine to the medium failed to elicit an opi phenotype in cptl::leu2 and cki-284::his3 mutants (data not shown), even though ethanolamine supplementation effected reductions in bulk membrane PC content in these strains. We interpret this result to indicate that ethanolamine supplementation of the medium does not lead to a quantitatively equivalent reduction in the involvement of EKIase and EPTase in PC biosynthesis to that elicited by genetic disruption of at least the EPTase structural gene. Thus, the opi phenotype appears to be a less sensitive (or less direct) indicator of PC biosynthetic defects than is reduced bulk membrane PC content itself. These collective data indicate that the salvage

6 6866 McGEE ET AL sla m % 102% 92.1% 14.6% -.7.7%~~~~~~~~~. ~ o 1s s1 FIG. 2. Incorporation of ethanolamine into PL in CDP-choline pathway mutant strains. The indicated strains were grown to midlogarithmic growth phase in 2.0 ml of minimal defined I+C-E- growth medium and pulse-radiolabeled with ['4C]ethanolamine (1 RCi/ml) for 20 min. Aliquots were removed from the labeled culture and evaluated for total uptake of radiolabel into cells, and radiolabeled PLs were extracted from the remainder of the culture. Values shown represent the percentages of total incorporated radiolabel recovered in the PL fraction (determined as described in Materials and Methods). activity of the CDP-choline pathway was required for the maintenance of normal bulk membrane PC content when yeast cells were grown in the absence of exogenously supplied choline and that intercession of specific CDP-ethanolamine pathway enzymes was sufficient to correct PC salvage pathway defects at the level of either CKIase or CPTase when the corresponding mutants were grown in ethanolamine-free medium. PE synthesis in CDP-choline pathway mutants. To further assess the extent of cross-pathway intercession by enzymes of the CDP-choline and CDP-ethanolamine pathways, we wished to determine the extent to which CDP-choline pathway enzymes could contribute to the apparent rate of bulk membrane PE synthesis. Thus, the ability of the CDP-choline pathway mutants to incorporate radiolabeled ethanolamine into lipid was examined. Strains bearing individual cki-284:his3, bsr2-5, cptl::leu2, or eptl::ura3 mutations were grown to midlogarithmic growth phase in I+C-E- medium and pulseradiolabeled with [14C]ethanolamine for 20 min. Radiolabeling was terminated by the addition of trichloroacetic acid, and the lipids were extracted from each culture and analyzed as described in Materials and Methods. Representative data are presented in Fig. 2. In agreement with previous reports that the CKJ gene product contributes to PE synthesis in vivo (18), the cki-284::is3 disruption reduced the efficiency of incorporation of [14C]ethanolamine into lipid by 55% relative to that of the isogenic wild-type strain. The bsr2-5 allele, however, failed to exert a discernible effect on the apparent rate of PE TABLE 3. J. BACTERIOL. Steady-state PL composition of strains grown in I+C-E+ mediuma Mole fraction of the following PL species Strain genotype Relevant (% of total PL content): PE PI PS PC CTY182 WT 24 ± ± ± ± 2.0 CIsY105 bsr ± ± ± ± 1.8 CTY392 cki-284::his3 21 ± ± ± ± 1.5 CT'Y434 cptl::leu2 23 ± ± ± ± 1.6 CIY436 eptl::ura3 18 ± ± ± ± 2.1 CIY618 cptl::leu2 18 ± ± ± ± 1.8 eptl::ura3 athe indicated yeast strains were cultured for five to six generations in choline-free synthetic defined medium supplemented with inositol and ethanolamine (each at a final concentration of 1 mm) and 32p; (10,uCi/ml). Glycerophospholipids were extracted and resolved by paper chromatography, and each individual PL species was quantitated as described in Materials and Methods. Details regarding determination of the mole percentage values are presented in footnote a to Table 1. Values are averages of those from three independent trials ± standard deviations. b WT, wild type. synthesis via the CDP-ethanolamine pathway, as evidenced by the wild-type level of incorporation of [14C]ethanolamine into lipid displayed by the bsr2-5 strain. In contrast, disruption of the EPTI gene resulted in an 85% decrease in the incorporation of [14C]ethanolamine into lipid, while disruption of CPT1 effected only an approximately 8% reduction in the activity of the CDP-ethanolamine pathway (Fig. 2). As expected, the cptl::leu2 eptl::ura3 double mutant was very defective in the incorporation of radiolabeled ethanolamine tracer into PL (92% reduction relative to the wild-type level). Steady-state PE levels in bulk membranes of CDP-choline pathway mutants. Intercession of CDP-ethanolamine enzymes in the CDP-choline pathway is apparent only at the steadystate level in mutant strains that are defective for the corresponding CDP-choline pathway enzyme (see above). Thus, we wished to assess the influence of CDP-choline pathway enzyme activity on bulk membrane PE levels at steady state in a mutant defective for CDP-ethanolamine pathway function. To investigate the extent to which CDP-choline pathway enzymes could contribute to bulk membrane PE composition at the steadystate level, we determined the bulk membrane PL composition of CDP-choline pathway mutants grown in I+C-E+ medium. These experiments involved the steady-state labeling of the appropriate yeast strains with 32Pi followed by extraction, resolution, and quantitation of bulk membrane PLs (see Materials and Methods). By analogy to the ethanolamine effects characterized above, the I+C-E+ medium cannot serve as a source for the PL headgroup precursor (i.e., choline) that might interfere with the involvement of CDP-choline pathway enzymes in the CDP-ethanolamine pathway. As a result, the I+C-E+ growth medium was expected to represent the most permissive condition for detecting potential CDP-choline pathway enzyme-mediated correction of reduced bulk membrane PE content resulting from CDP-ethanolamine pathway dysfunction. The most satisfactory system to analyze this question again involved CPTase and EPTase, as these represent the only corresponding pair of CDP-choline and CDPethanolamine enzymes for which loss-of-function mutants affecting both enzymes presently exist. The representative data presented in Table 3 show that PE accounted for approximately 24% of the total PL in wild-type yeast membranes at steady state under these growth conditions. As expected, while bsr2-5 and cptl::leu2 mutants also

7 VOL. 176, 1994 ETHANOLAMINE EFFECTS ON YEAST MEMBRANE PHOSPHOLIPIDS 6867 exhibited wild-type bulk membrane PE profiles, disruption of CDP-ethanolamine pathway function by the eptl::ura3 mutation elicited a reproducible reduction in bulk membrane PE content, to approximately 18% of the total PL content. If CPTase contributed to bulk membrane PE synthesis in a manner analogous to the contribution of EPTase to bulk membrane PC synthesis (Table 1), the cptl::leu2 eptl::ura3 double mutant would exhibit an even greater reduction in bulk membrane PE content than that measured for the eptl::ura3 single mutant. The data show that the cptl::leu2 eptl::ura3 double mutant exhibited a bulk membrane PE content that was indistinguishable from that recorded for the eptl::ura3 single mutant (Table 3). These results indicate that CPTase did not contribute to bulk membrane PE synthesis at steady state when eptl::ura3 yeast cells were grown in choline-free medium. Two possible explanations for this result are offered. First, as CDP-ethanolamine is a poor substrate for CPTase in vitro (6), it is possible that the lack of contribution by CPTase to PE synthesis simply reflects this aspect of CPTase substrate specificity. Alternatively, the flux of choline through the CDPcholine pathway via salvage may be sufficiently high, even when cells are grown in choline-free medium, to effectively preclude participation of CPTase in CDP-ethanolamine pathway function by virtue of substrate-level competition. Consistent with the latter possibility, we have demonstrated that the activity of the CDP-choline pathway in PC synthesis via choline salvage contributes significantly to the apparent rate of PC synthesis in yeast cells cultured in choline-free medium (12). As CKIase significantly contributes to bulk cellular EKIase activity in yeast cells (Fig. 2) (8, 12), we expected this enzyme to contribute to bulk membrane PE synthesis at steady state. Analysis of bulk membrane PE in cki-284::his3 mutants revealed only a very modest, but reproducible, reduction in bulk membrane PE content (21% of the total PL) relative to that measured for wild-type cells (24% of the total PL; Table 3). This marginal reduction in bulk membrane PE content in the cki-284::his3 mutant likely reflected not only the loss of CKIase-associated EKIase activity, but also the continued involvement of the dedicated EKIase. Cellular requirement for SEC14p is not related to the ethanolamine effect. We had previously demonstrated that inactivation of the CDP-choline pathway is sufficient to completely abrogate the normally essential cellular requirement for SEC14p function (4). These data were interpreted as suggesting that Golgi membranes required a particular PL composition in order to execute Golgi secretory functions and that it was the purview of SEC14p to maintain such an appropriate Golgi membrane PL composition. An extended interpretation of those data that could be considered is that inactivation of the CDP-choline pathway might result in alterations in bulk membrane PL composition that are compatible with a membrane PL composition potentially required for yeast Golgi secretory function. The imposition of such altered bulk membrane composition on Golgi membranes (by bulk membrane flow) might then obviate the requirement for SEC14p. Our demonstration that cki and Cpt mutants showed reductions in bulk membrane PC content when grown in the presence of ethanolamine, and that cct mutants exhibited constitutively reduced bulk membrane PC content, was at least superficially consistent with such an idea. Therefore, we tested whether the "bypass SEC14p" phenotype associated with cki mutations required the presence of ethanolamine in the growth medium. These bypass SEC14p mutants were initially isolated on YPD, an ethanolamine-containing medium (4). The isogenic strains CTY182 (wild type), CTY1-1A (sec14-1j), and CTY394 (secl4-88::ura3 cki-284:wiis3) were streaked out for isolation on I+C+E+ and I+C+E- plates. The abilities of these strains to form isolated colonies on E+ and E- media at 30 C, a permissive temperature for sec14-1ts strains, were assessed. The secl4-88::ura3 allele is normally a haploid lethal mutation under all growth conditions, but in strain CTY394, viability is conferred by the cki-284::his3 allele. As expected, both the wild-type and sec14-1's strains grew under both the E+ and E- conditions. Growth of the secl4-88::ura3 cki-284::his3 double mutant was similarly insensitive to the ethanolamine content of the medium (data not shown). Thus, the presence of ethanolamine in the medium was not obligatorily required for cki-mediated suppression of the normally lethal secl4-88::ura3 allele. Conclusions. Collectively, the data presented in this report demonstrate an interrelationship between the CDP-choline and CDP-ethanolamine PL biosynthetic pathways in yeast cells and demonstrate that, in the face of a dysfunctional CDPcholine pathway, biochemically redundant enzymes of the CDP-ethanolamine pathway can significantly contribute to bulk membrane PC biosynthesis-as long as ethanolamine is absent from the growth medium. Although this ethanolaminesensitive contribution to PC biosynthesis is relatively inefficient, as indicated by the lack of effect of the eptl::ura3 allele on apparent rates of PC synthesis via the CDP-choline pathway in in vivo pulse-radiolabeling experiments (12), it is significant that it is sufficient to correct, at steady state, the PC biosynthetic defects of cki-284::his3 and cptl::leu2 mutant strains. By contrast, mutants defective in CCTase activity (i.e., bsr2-5 mutants) experienced an ethanolamine-insensitive reduction in bulk membrane PC content. This finding is readily explained by the demonstration that the phosphoaminoalcohol cytidylyltransferases display tight lipid headgroup precursor specificities. CCTase does not utilize ethanolaminephosphate as a substrate in vitro (17) or in vivo (Fig. 2), while the ECTase was not capable of correcting steady-state PC defects resulting from bsr2-5 defects. On the basis of the collective data reported here, we make the following two predictions. First, we predict that yeast strains defective for both CKIase activity and EKIase activity will exhibit an ethanolamine-insensitive reduction in bulk membrane PC content of the order exhibited by the cptl::leu2 eptl::ura3 double mutant. Second, we predict that ECTase defects will have no effect on the bulk membrane PC of otherwise wild-type strains, regardless of the ethanolamine content of the medium in which such strains are grown, and that inactivation of ECTase will not further exacerbate the ethanolamine-insensitive reduction in bulk membrane PC content exhibited by CCTase-deficient strains. One puzzling feature of the data was our demonstration that bsr2-5 mutants uniquely exhibited elevated PI levels, relative to those of the other strains employed in these studies, when grown in I+C+E- and I-C-E- media (Tables 1 and 2) but not when grown in I+C+E+ or I+C-E+ medium (Tables 1 and 3). The basis for this phenomenon remains unclear. Finally, the data reported here provide additional insight into the relationship between CDP-choline pathway function and the normally essential involvement of a phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p) in the stimulation of Golgi secretory function in S. cerevisiae (2, 4). We proposed that SEC14p functions to control the PI and/or PC content of yeast Golgi membranes so that Golgi secretory function can be maintained. Two possible mechanisms by which inactivation of the CDP-choline pathway could bypass SEC14p function were entertained: (i) that SEC14p exerted some local control of PL composition in the Golgi membranes that was in principle antagonistic to CDP-choline pathway function, a local control that was in effect reimposed in

8 6868 McGEE ET AL. SEC14p-deficient yeast cells by specific inactivation of the CDP-choline pathway, or (ii) that inactivation of the CDPcholine pathway effected a bulk membrane PL composition that was compatible with Golgi secretory function and, upon imposition of this bulk PL composition on Golgi membranes by bulk membrane flow, that rendered SEC14p-mediated control of Golgi membrane PL composition unnecessary (4). Consideration of the latter possibility was prompted by our finding that CDP-choline pathway mutants exhibited reduced bulk membrane PC content when grown in YPD medium. Our demonstration that this reduction in bulk membrane PC content is a simple function of the ethanolamine content of the growth medium, when coupled with our observation that suppression of SEC14p defects by loss of CDP-choline pathway activity does not obligatorily require ethanolamine in the growth medium (see above), provides a strong argument against the latter proposal for how CDP-choline pathway dysfunction effects bypass of the SEC14p requirement for Golgi function and cell viability. Rather, the data are most consistent with a local relationship between SEC14p and CDP-choline pathway function in Golgi membranes. This conclusion is further supported by (i) quantitative analyses of Golgi membrane PL composition, as a function of SEC14p activity, in yeast strains in which Golgi secretory function is uncoupled from its usual SEC14p requirement (12) and (ii) in vivo and in vitro data suggesting that SEC14p represses CDP-choline pathway activity in a PL-ligand-modulated fashion by downregulating CCTase, the rate-determining enzyme of this pathway (15). ACKNOWLEDGMENT This work was supported by grant GM44530 from the National Institutes of Health to V.A.B. REFERENCES 1. Ames, B. N Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8: Bankaitis, V. A., D. E. Malehorn, S. D. Emr, and R. Greene The Saccharomyces cerevisiae SEC14 gene product encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108: Christie, W. W Chromatographic and spectroscopic analysis of lipids, p In W. W. 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