Phosphatidylserine decarboxylase (PSD) ()is an important enzyme in the synthesis of aminophospholipids. In Escherichia coli the decarboxylase functions in the only pathway for phosphatidylethanolamine (PtdEtn) synthesis(1, 2) . However, in eukaryotes, PtdEtn can be synthesized by the action of the decarboxylase or ethanolamine phosphotransferase(3, 4) . In addition, mammalian cells possess a base exchange enzyme whose contribution to PtdEtn synthesis in vivo is poorly understood(5) . Early genetic examination of aminophospholipid synthesis by Atkinson et al.(6) in the yeast Saccharomyces cerevisiae focused on the isolation of mutants requiring ethanolamine or choline for growth. All of the mutants isolated belonged to a single complementation group designated cho1 that had defects in the enzyme phosphatidylserine synthetase. Interestingly, none of the mutants were reported to have defects in PSD.

The auxotrophic requirement of the cho1 mutants is not only fulfilled by ethanolamine and choline, but also by monomethylethanolamine and dimethylethanolamine, indicating that phosphatidylcholine (PtdCho), rather than phosphatidylserine (PtdSer) or PtdEtn, is the essential end product(6, 7, 8) . Mutants having defects in either the first methylation step (pem1/cho2) or second and third methylation steps (pem2/opi3) in the conversion of PtdEtn to PtdCho do not require choline or ethanolamine for growth, presumably because low levels of PtdCho and/or the mono- and dimethyl forms of PtdEtn suffice. Strains with both pem1/cho2 and pem2/opi3 mutations, however, are stringently auxotrophic for choline, indicating that PtdEtn alone will not substitute for all the methylated phospholipids(8) .

Our initial efforts to isolate yeast strains defective in PSD by isolating ethanolamine auxotrophs in a wild type background failed entirely. ()Using alternative methods, we were, however, able to isolate strains defective in PSD activity, clone the gene encoding for the mitochondrial PSD (PSD1), and create yeast strains containing a psd1-1::TRP1 gene disruption(9) . These null mutants possessed <5% wild type PSD activity, yet did not require ethanolamine for growth and had metabolic characteristics similar to wild type cells. Similar findings were reported by Clancey et al.(10) . These observations led us to hypothesize that the yeast S. cerevisiae possesses two functional PSD activities. The goals of the current studies were to: 1) describe the presence of this second PSD activity (PSD2) in psd1-1::TRP1 mutants, 2) isolate mutants lacking PSD2 activity (psd2) by selecting ethanolamine auxotrophs in the psd1-1::TRP1 background, and 3) identify the subcellular location of the PSD2 enzyme activity. The results unambiguously establish the presence of a second, non-mitochondrial PSD activity. In addition, these studies demonstrate that loss of both PSD1 and PSD2 enzyme activities results in ethanolamine auxotrophy and impaired PtdSer turnover.

EXPERIMENTAL PROCEDURES

Chemicals

Yeast Strains and Growth Methods

The growth media for yeast, YPD, minimal medium plus methionine and auxotrophic requirements (SD), and minimal medium containing all amino acids (SC), were prepared by standard methods(14) , except where noted. Adenine and uracil (20 mg/liter) were routinely added to YPD to give YPDAU, and ethanolamine was added where noted to give YPDAUE. Ethanolamine-supplemented media contained 2-5 mM ethanolamine added from a 0.5 M, pH 5-6, sterile-filtered stock solution. Media for subcellular localization experiments, except where noted, was that described by Daum et al.(15) , referred to here as DBS medium: 0.3% yeast extract, 1 g/liter each of glucose, KH PO and NHCl, 0.5 g/liter each of CaCl2HO and NaCl, 0.6 g/liter MgSOHO, 0.3 ml/liter 1% FeCl, and 22 ml of 85% DL-lactic acid, adjusted to pH 5.5.

Selection of Phosphatidylserine Decarboxylase 2 Mutants

Cells exhibiting only the psd2 mutation (PTY22), thereby expressing only PSD1 activity, were constructed from the psd1-1::TRP1, psd2 strain described above (PTY18) by mating with the PSD1, PSD2, trp 1 strain (422). After sporulation of the diploid, segregation of the tryptophan (trp) and ethanolamine (etn) auxotrophies were analyzed. Segregation of etn spores indicated that the psd2 allele was not lethal and resulted in no discernable growth phenotype when expressed in the presence of the wild type PSD1 gene. Thus, the strains from spores that were trp and etn were either PSD1, PSD2 or PSD1, psd2. Presence of the psd2 genotype was determined by backcrossing these strains with a psd1::TRP1, PSD2 strain (PTY9.2 or PTY9.4) and identifying ethanolamine auxotrophs among sporulated progeny. The cross with the PSD1, PSD2 strain resulted in no etn spores, while the cross with the PSD1, psd2 strain resulted in about 25% etn spores. This segregation of the etn phenotype in the resulting tetrads allowed detection of the psd2 genotype, since only psd1::TRP1, psd2 strains are auxotrophic for ethanolamine.

Determination of Phosphatidylserine Decarboxylase Activity

Lipid Analysis

Subcellular Fractionation

Fractionation of organelles on sorbitol gradients was performed by a modification of the method described by Cleves et al.(18) . Cells were grown and disrupted as described above, except the final homogenate volume was 10 ml/liter of cells. The resulting S1.5 was then centrifuged for 15 min at 30,000 g in a Beckman JA-20 rotor. The resultant supernatant, S30, was underlayed with 1 ml of 80% sorbitol and 1 ml of 25% sorbitol and centrifuged for 2 h at 280,000 g in a Beckman SW41Ti rotor. The interface between the 25 and 80% sorbitol layers was harvested and layered as the 50% step of a 40-80% sorbitol gradient prepared in 10% increments of 2.5 ml each. The gradient was developed by centrifuging in an SW41 Ti rotor at 280,000 g for 40 h. Fractions were collected by aspiration from the top of the gradient and stored at -20 °C.

Percoll gradient separation was carried out by a modification of the procedure described by Cunningham and Wickner(19) . Cells (1 liter) were grown to approximately 5 10/ml in YPDAUE medium and spheroplasts prepared as described by Daum et al.(15) . Spheroplasts (in 10 ml) were then layered over 10 ml of 1.8 M sorbitol, 10 mM Tris-HCl, pH 7.5, and centrifuged for 5 min at 3000 g. The spheroplast pellet was resuspended in 2.5 ml of 0.45 M mannitol, 10 mM Tris-HCl, pH 7.4, disrupted by 15 strokes of a tight-fitting Dounce homogenizer, and diluted to 5 ml with additional buffer and centrifuged 5 min at 1,200 g. The homogenization procedure was then repeated on the pellet. The final pooled homogenate was cleared of undisrupted cells and debris by two consecutive 1,000 g spins for 15 min each. Then 2 ml of resulting supernatant was layered on a two-step self-forming Percoll gradient consisting of 1 ml of 30% Percoll overlaid with 9 ml of 18% Percoll. Both layers also contained 0.5 M mannitol, 10 mM Tris-HCl, pH 7.5. The Percoll gradients were centrifuged at 4 °C for 90 min at 40,000 g in a Beckman Ti50 rotor. Fractions were collected from the top of the gradient by aspiration and stored at -20 °C.

Measurement of Organelle Markers

RESULTS

Measurement of PSD2 Activity

Isolation of Ethanolamine Auxotrophs Lacking PSD Activity

Figure 1: Double PSD mutants require ethanolamine for growth. Viability of PSD mutant and wild type strains cultured in SD medium lacking ethanolamine (and choline) was determined by spreading culture aliquots on rich plates after the indicated times of incubation at 30 °C. Data are a representative one of four experiments.

Table 2shows the PSD activity in mutant and wild type strains as measured by the standard and NBD-PtdSer assay protocols. By the standard assay, cells expressing only PSD2 have <4% wild type enzyme activity, cells expressing PSD1 have 60% wild type activity, and cells with defects affecting both loci express no detectable PSD activity. When the NBD-PtdSer substrate is used, the relative enzyme activity of the PSD2 strain is increased 1.7-fold, activity in cells expressing PSD1 decreases to <40% of wild type, and PSD activity in the double mutant is undetectable. Using this second method, the enzyme activity in psd1-1::TRP1 PSD2 cells was as high as 12% of wild type. These data indicate that the decarboxylase activity of cells containing wild type PSD2 and the null allele for PSD1 is significant and can account for 4-12% of the activity found in wild type cells. It is not difficult to understand how this second PSD activity was missed previously, since by standard methods the activity accounts for only a very small portion of the wild type activity. These data also demonstrate that the NBD-PtdSer substrate is somewhat preferred by PSD2 over the standard PtdSer substrate. The NBD-PtdSer substrate was utilized in subsequent experiments to perform subcellular localization of the PSD2 activity (discussed later).

Serine Incorporation into Phospholipids by PSD Mutants

Figure 2: PSD mutations affect amino phospholipid metabolism. Incorporation of [H]serine into phospholipids of wild type and PSD mutant strains was monitored for cells incubated 6 h in log phase in SD plus 20 µCi/ml L-[3-H]serine without (A) or with (B) 2 mM ethanolamine and choline. Lipids were extracted and separated as described under ``Experimental Procedures.'' Values are the mean ± StdDev of six total determinations performed in three experiments. Abbreviations are: PtdOH, phosphatidic acid; Etn, ethanolamine; Cho, choline.

Significant serine is incorporated into the PtdCho pool in the double mutant despite the defect in conversion of PtdSer to PtdEtn (Fig. 2) and likely represents incorporation via the one-carbon pool (see ``Discussion''). In addition, there is a measurable amount of serine incorporation into PtdEtn in the double mutant. This could be due to leakiness of the psd2 mutation. Alternatively, serine is incorporated into sphingolipids by the action of the enzyme serine palmitoyltransferase, and sphingolipids are broken down to yield phosphoethanolamine, which can be utilized for PtdEtn synthesis(29) . Fig. 3shows the effect of a serine palmitoyltransferase inhibitor(17) , sphingofungin C (SF-C), on the incorporation of [H]serine into phospholipids by the PSD double mutant. In these experiments, treatment of the cells with SF-C reduces incorporation of the labeled serine into PtdEtn from 11% of the total to less than 3%, a level similar to that in PtdIns. These data indicate that a significant portion of PtdEtn label in the double mutant comes from sphingolipid breakdown rather than a leaky psd2 mutation. In contrast to the effect of SF-C upon [H]serine incorporation into PtdEtn are the effects upon PtdCho labeling. This latter result is expected if the labeling of the PtdCho pool is derived principally from the one carbon pool (see ``Discussion''). Thus, these data clearly demonstrate that the ethanolamine auxotrophy of the psd1-1::TRP1, psd2 mutant is due to a metabolic defect in the ability to convert PtdSer to PtdEtn caused by loss of both PSD1 and PSD2 activities.

Figure 3: Sphingofungin C inhibits [H]serine incorporation into phosphatidylethanolamine by the psd1-1::TRP1 psd2 mutant. Cells were pretreated for 15 min with the serine palmitoyltransferase inhibitor sphingofungin C and then incubated 6 h in L-[3-H]serine as described under ``Experimental Procedures'' and Fig. 2. Data are the mean ± StdDev for two separate experiments performed in duplicate. Abbreviations are as in Fig. 2.

Phospholipid Profile and Turnover in PSD Mutants

Figure 4: The phosphatidylethanolamine content of psd11::TRP1 psd 2 cells is significantly reduced. Cells were grown in log phase for greater than five generations in SD plus 2 mM ethanolamine and choline containing 10 µCi/ml [P]orthophosphate. Lipids were extracted and separated as described under ``Experimental Procedures.'' Data are the mean ± StdDev from a representative two of four experiments performed in duplicate.

The turnover of P-labeled lipids in the presence and absence of 2 mM ethanolamine and 2 mM choline was determined by uniformly labeling log phase cells and then shifting the cultures to unlabeled medium (Fig. 5, A-D). In general, inclusion of ethanolamine and choline reduced turnover of PtdEtn but not PtdIns and reduced the accumulation of PtdCho in both wild type and psd1-1::TRP1, psd2 cells (compare open and filled symbols). In both media PtdSer was readily turned over by wild type cells. In the double mutant, however, PtdSer exhibited very low turnover and, in the presence of ethanolamine and choline, appeared to accumulate (Fig. 5A). PtdEtn was readily turned over in both strains (Fig. 5B) accumulating into PtdCho (Fig. 5C). Note that the lower level of PtdCho accumulation in the double mutant is a result of the lower amount of PtdEtn available for conversion to PtdCho (Fig. 5B). Radiolabel in PtdIns also decreased in both strains, although the level in psd1-1::TRP psd2 cells remained about 40% higher than that in wild type throughout the time course (Fig. 5D). These data clearly demonstrate the defect in conversion of PtdSer into PtdEtn in psd1-1::TRP, psd2 cells as evidenced by both the lower relative level of PtdEtn in these cells as well as the lack of PtdSer turnover.

Figure 5: Phosphatidylserine turnover is decreased in psd11::TRP1 psd2 cells. Cells were labeled with [P]-orthophosphate as described in Fig. 4. They were then diluted into SD plus (solid symbols) or minus (open symbols) 2 mM ethanolamine and choline. Samples were taken at time 0, 1, 3 and 6 h, and lipids extracted and separated as described under ``Experimental Procedures.'' Data are the mean of two experiments performed in duplicate.

Differential Fractionation of PSD2 Enzyme Activity

Figure 6: PSD2 activity localizes to a different subcellular fraction from PSD1. Cell homogenates were prepared and separated into 30,000 g pellet and supernatants, and PSDs or marker enzymes assayed as described under ``Experimental Procedures.'' A, fractionation of PSD1 and PSD2 as compared to the mitochondrial marker cytochrome c oxidase. B, comparison of PSD2 fractionation with that of various organelle markers in psd1-1::TRP1 PSD2 cells. Data are the mean ± StdDev for two to five separate experiments with duplicate determinations. Abbreviations are: PL.MEM, plasma membrane; CCR, cytochrome c reductase; E.R., endoplasmic reticulum.

In an effort to determine the subcellular location of the PSD2 enzyme, its sedimentation characteristics were compared with markers of other organelles: plasma membrane ATPase (plasma membrane), cytochrome c reductase (endoplasmic reticulum), GDPase (Golgi), KEX2 protease (late Golgi), and vacuolar ATPase (vacuoles). Fig. 6Bshows the differential sedimentation of these markers at 30,000 g. The majority of the plasma membrane marker and a little less than half of the endoplasmic reticulum marker sediment into the P30 under these conditions, suggesting they are unlikely candidates for PSD2 localization. However, it is possible that PSD2 resides in a subpopulation of these organelles. In contrast, the Golgi and vacuolar markers fractionate similarly to PSD2, primarily to the S30, making these more likely possibilities.

Fractionation of PSD2 Activity on Sorbitol Gradients

Figure 7: PSD2 activity codistributes with vacuolar and Golgi membranes on sorbitol gradients. 30,000 g supernatants (S30) were prepared and separated on 40-80% sorbitol gradients and assayed as described under ``Experimental Procedures.'' A, distribution of PSD activity from S30s of wild type, PSD2, and PSD1 expressing strains. B and C, distribution of marker molecules on a gradient from psd1-1::TRP1 PSD2 cells; CCR, cytochrome c reductase, as a marker for endoplasmic reticulum; plasma membrane ATPase as a marker for plasma membrane; KEX2, as a Golgi marker; and vacuolar ATPase as a vacuolar marker. Data are a representative one of three separate gradients.

Fractionation of PSD2 Activity on Percoll Gradients

Figure 8: PSD2 activity segregates bimodally with vacuolar and Golgi membranes on Percoll gradients. Post-nuclear supernatant from the psd1-1::TRP1 PSD2 strain was separated on a self-forming Percoll gradient and markers assayed as described under ``Experimental Procedures.'' VacATPase, vacuolar ATPase; GDPase, general Golgi marker; KEX2, marker of late Golgi. Data are a representative one of three gradients.

DISCUSSION

We previously cloned the PSD1 gene encoding the mitochondrial phosphatidylserine decarboxylase enzyme of S. cerevisiae and constructed strains containing a null allele for the gene(9) . We observed that despite the deletion/disruption of the PSD1 gene, cells remain prototrophic for ethanolamine, retain 2-5% of parental PSD activity, and show only minor defects in aminophospholipid metabolism. This led us as well as Clancey et al.(10) to hypothesize the presence of a second PSD activity in S. cerevisiae. The current studies demonstrate the presence of a second, non-mitochondrial PSD enzyme activity (PSD2) in the yeast S. cerevisiae. This PSD2 enzyme activity is sufficient to allow growth of psd1-1::TRP1 null mutants in the absence of exogenous ethanolamine. However, mutations leading to loss of PSD2 activity in psd1-1::TRP1 null mutants result in ethanolamine auxotrophy, altered aminophospholipid metabolism, and significantly decreased levels of cellular phosphatidylethanolamine. In addition, the PSD2 enzyme activity is not localized to the inner mitochondrial membrane, as is PSD1, but to subcellular fractions with properties similar to vacuoles and the Golgi apparatus.

Atkinson et al.(7) previously isolated ethanolamine auxotrophs from mutagenized yeast. All of the mutants described carried mutations in the cho1 gene encoding phosphatidylserine synthase. Interestingly, none of the mutants had defects in PSD. Similar approaches in our laboratory led to similar results. In fact, mutants in PSD1 were only isolated when a brute force screen was used to identify strains with low PSD activity(9) . The results presented in the current study indicate that the likely reason for these previous findings was the presence of a second PSD which is not easily detected by standard assay methods. Yet, as demonstrated before (9, 10) , the activity (PSD2) is sufficient to meet the requirement for cell growth without ethanolamine. Mutagenesis of cells expressing only PSD2 readily results in the creation of strains requiring ethanolamine for growth (Fig. 1), some of which lack any measurable PSD activity (Table 2). The mutation leading to loss of PSD2 activity segregates independently of PSD1, indicating that the genes are not linked. It is also noteworthy that the ethanolamine requirement of strains containing both the psd1::TRP1 and psd2 alleles is also satisfied by choline (data not shown), supporting previous reports (6, 7, 8) that PtdCho, and not PtdEtn, is the essential aminophospholipid end product.

Strains containing the psd1-1::TRP1 allele and lacking measurable PSD2 activity (psd2) express readily observable defects in aminophospholipid metabolism (Fig. 2). In contrast to strains expressing one or the other of the two PSD activities, the double mutants show significant accumulation of [H]serine into PtdSer and very low levels of precursor incorporation into PtdEtn. It was expected that since PSD activity was completely lost in the psd1-1::TRP1, psd2 mutants that undetectable amounts of [H]serine would be incorporated into PtdEtn. However, as much as 12% of the total H recovered in lipids was associated with PtdEtn ( Fig. 2and 3B), indicating either a leaky psd2 mutation or an alternative pathway for radiolabeling the PtdEtn. This alternative pathway was likely to be [H]serine incorporation into sphingolipids via the action of serine palmitoyltransferase, and catabolism of the [H]serine labeled sphingolipids to yield endogenous [H]ethanolamine available for incorporation into PtdEtn (29) . Treatment of psd1-1::TRP1 psd2 cells with sphingofungin C, a drug which specifically blocks serine palmitoyltransferase(17) , reduces incorporation of [H]serine into PtdEtn to essentially background levels (Fig. 3B). Thus, the incorporation of [H]serine into PtdEtn by the double PSD mutant appears to be primarily a result of endogenous ethanolamine production via sphingolipid turnover. Atkinson (31) reported that the cho1 phenotype can be suppressed by increased production of endogenous ethanolamine most likely via sphingolipid breakdown in eam1 mutants. Our data indicate that the normal level of endogenous ethanolamine production is insufficient to repress the ethanolamine auxotrophy of the PSD double mutant. However, the eam1 mutation might be expected to complement our psd1-1::TRP1 psd2 mutant.

Incorporation of [H]serine into PtdCho was comparable among the various psd strains. Treatment of cells with SF-C had little effect on [H]serine incorporation into PtdCho by the double mutant (Fig. 3C), suggesting that the label in PtdCho is not likely to originate from PtdEtn labeled via the sphingolipid pathway. This is not surprising, since Atkinson (31) reports that although significant [H]serine is incorporated into PtdEtn by strains with a bypass of defects in phosphatidylserine synthase (cho1 eam1), labeling of PtdCho is much lower than in ethanolamine-supplemented cho1 EAM1 strains. Thus, the H observed in PtdCho is not likely to originate from [H]PtdEtn synthesized by endogenously produced [H]ethanolamine.

Atkinson et al.(7) reported significant PtdCho labeling by [H]serine in cho1 strains supplemented with ethanolamine and suggested that the label was likely to be incorporated via one-carbon metabolism and methylation by S-adenosylmethionine. When [H]serine incorporation by the psd1-1::TRP1 psd2 mutant was examined in medium lacking methionine (not shown), nearly 2-fold more radioactivity was incorporated into PtdCho as compared to wild type cells, suggesting that the mutant may, in the absence of an active PtdSer PtdEtn pathway, readily shunt serine into S-adenosylmethionine biosynthesis and thus into PtdCho. Additionally, since the cells are grown in the presence of ethanolamine, a substantial pool of PtdEtn would be available for methylation to PtdCho. Finally, since the [H]serine is labeled on the hydrogens of the 3-carbon, incorporation into PtdCho via one-carbon metabolism (i.e. the methyl groups of aminophospholipids) would be 3-fold over-represented on a molar basis as compared to that labeled via the common ethyl moiety of PtdSer, PtdEtn, and PtdCho.

Loss of all PSD activity has a notable effect on cellular phospholipid composition despite the presence of exogenous ethanolamine and choline. Steady-state labeling of cellular phospholipids showed that the double mutant contains only 30% of the wild type level of PtdEtn, but comparable levels of PtdCho (Fig. 4). This result occurs in the presence of 2 mM ethanolamine and choline, allowing synthesis of PtdEtn and PtdCho via the CDP base pathway. These data suggest that PtdEtn synthesized via the CDP base pathway may be more readily methylated than that produced by the PSDs. Alternatively, since choline is present and methylation may be down-regulated (see Fig. 5), it may indicate that the PSDs are important in producing a separate pool of cellular PtdEtn.

An important issue in the characterization of the PSD2 enzyme was its subcellular localization. Fig. 6clearly differentiates its localization from that of PSD1, which resides in the inner mitochondrial membrane. These data also suggest that PSD2 is unlikely to be located in peroxisomes, which have a density similar to mitochondria(32) . Fractionation of PSD2 on gradients ( Fig. 7and 8) reveals that the enzyme distribution overlaps primarily with the marker for vacuoles (>65%), but a significant proportion (<35%) co-distributes with the markers for the Golgi. At face value it may be concluded that PSD2 is localized to both the Golgi and vacuolar compartments of the cell. Alternatively, PSD2 may reside in another compartment for which there is no standardized marker, and the activity found in the more dense fractions may be this same compartment associated with some other subcellular component of higher density. Subcellular localization experiments performed in mutants in vacuolar assembly (33) may aid in discerning if PSD2 truly resides in the vacuoles.

A question which remains unanswered is the physiological role of the PSD2 enzyme. The different subcellular locations of PSD1 and PSD2, however, indicate that they play disparate roles in aminophospholipid metabolism. As described previously by us(9) , strains containing the psd1-1:TRP1 allele (i.e. expressing only PSD2 activity), exhibit a greater tendency to produce petite, rho cells. Additional data from our laboratory (not shown) demonstrate that cells expressing only PSD2 have significantly less PtdEtn in the mitochondria, indicating that PtdEtn formed by PSD2 is not efficiently transported to the mitochondria and may account for their dysfunction. Thus, although PSD2 activity is sufficient to meet the biosynthetic needs for cell growth, it clearly cannot completely fulfill the requirements for organelle specific membrane biogenesis.

These studies demonstrate the presence of a second, non-mitochondrial phosphatidylserine decarboxylase (PSD2) activity in the yeast S. cerevisiae. Loss of PSD2 activity, alone, does not result in any detectable phenotype, but loss of PSD2 as well as PSD1 results in ethanolamine auxotrophy and impaired aminophospholipid metabolism. The subcellular location of PSD2 is clearly distinguishable from the mitochondrial PSD1 and has fractionation characteristics which overlap with both vacuolar and Golgi markers. Further understanding of PSD2 awaits the isolation and characterization of the gene encoding the enzyme, examination of strains containing the null allele for the gene, and a more detailed subcellular localization utilizing immunohistochemistry. The cloning and characterization of the structural gene encoding PSD2 is described in the accompanying manuscript(34) .

FOOTNOTES

*

This work was supported by National Institutes of Health Grants GM32453 (to D. R. V.) and GM16701 (to P. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Medicine, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1300; Fax: 303-398-1806.

()

The abbreviations used are: PSD, phosphatidylserine decarboxylase; ER, endoplasmic reticulum; NBD, 1-acyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; SD, minimal medium plus methionine and auxotrophic requirements; SF-C, sphingofungin C; StdDev, standard deviation.

()

D. R. Voelker, unpublished observations.

ACKNOWLEDGEMENTS

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