MOLECULAR AND CELLULAR BIOLOGY, May 1990, p. 2176-2181 0270-7306/90/052176-06$02.00/0 Copyright C) 1990, American Society for Microbiology
Vol. 10, No. 5
Isolation of Mutant Saccharomyces cerevisiae Strains That Survive without Sphingolipids ROBERT C. DICKSON,* GERALD B. WELLS, ANN SCHMIDT, AND ROBERT L. LESTER Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0084 Received 8 November 1989/Accepted 1 February 1990
Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.
Sphingolipids constitute a diverse group of complex membrane lipids. All contain a long-chain amino alcohol, usually 18 carbons, connected by an amide linkage to a fatty acid to form ceramide. The hydroxyl at the 1 position of ceramide can be linked to various polar head groups, including phosphorylcholine (sphingomyelin), or to glucose units and other moieties (4). Sphingolipids are found mainly in eucaryotes, where they are ubiquitous, with hundreds having been identified and structurally characterized in mammals alone (4). More than a century ago, Thudichum (17) discovered the long-chain-base sphingosine in the human brain and named it after the sphinx because of the riddles it posed. The name remains appropriate. Although sphingolipids are known to play a structural role in membranes, their precise biological function(s) is unknown. Our lack of knowledge is due in part to difficulties in assigning functions which stem from the diversity of sphingolipids present in most cell types. Roles have been proposed in such complex phenomena as cellular differentiation, cell-to-cell recognition, and modulation of growth (4, 10, 12), including involvement in membrane signal transduction pathways (5). We have begun to examine the function of sphingolipids in the yeast Saccharomyces cerevisiae, which is well suited for these studies because sphingolipid diversity is minimized in that S. cerevisiae contains only three abundant sphingolipids, with the following compositions: inositol P-ceramide (IPC), mannose-inositol Pceramide (MIPC) and mannose-(inositol P)2-ceramide [M(IP)2C] (15, 16). Moreover, S. cerevisiae permits full exploitation of molecular genetic techniques which have not been used previously to examine sphingolipid function. Our studies build upon the work of Wells and Lester (18), who isolated a strain of S. cerevisiae that requires a longchain base (Icb, the original allele being Icbl-J) such as phytosphingosine for growth. The absence of serine palmitoyltransferase activity in the lcbJ mutant (W. J. Pinto, G. B. Wells, A. C. Williams, K. A. Anderson, E. C. Teater, and R. L. Lester, Fed. Proc. 45:1826, 1986) suggests that LCBJ codes for this enzyme or a subunit of the enzyme, the first enzyme in the long-chain-base synthetic pathway of yeasts. This mutant provided the first genetic evidence in any *
organism that sphingolipids are essential for normal cellular function. The Icbl-defective strains are a limited subject for the study of sphingolipid function because the mutation is lethal. However, such mutations can be used in an alternative strategy based on the isolation of secondary mutations (7) that suppress or bypass the lcbJ defect and allow growth in the absence of exogenous long-chain base. Suppressor genes could function by enabling the strain to make sphingolipids by a novel synthesis pathway, by creating a lipid(s) that replaces sphingolipids, or by enabling the strain to grow without making sphingolipids. Analysis of the latter class of hypothetical suppressors should reveal specific sphingolipid functions. For example, the product of a suppressor allele, in comparison with the wild-type gene product, may no longer require sphingolipid for function. We report here that the desired hypothetical suppressor strains can be isolated and that they grow while making little if any detectable sphingolipid. MATERIALS AND METHODS Strains. S. cerevisiae SJ21R (MATa ura3-52 leu2-3,112 adel MEL]) (8) was used as the wild-type strain for LCBJ. Strains carrying the lcbl:: URA3 deletion allele were constructed by gene replacement (13). Strain SJ21R was transformed with 10 ,ug of the 3.1-kilobase (kb) NruI-to-StuI DNA fragment carrying the URA3 gene (see Fig. 1C) and Ura+ transformants were selected. Two transformants, strains 1A4 and 1A7, showing a stable Ura+ phenotype and carrying the lcbl:: URA3 deletion allele, were used for further study, including selection of the suppressor strains designated 7R4 and 7R6. Strain YNN27 (MATa trpl-289 ura3-52 gal2) was used as an LCBJ strain to cross with the putative IcbJ suppressor strains. Analysis of long-chain bases. Cells were cultured overnight to A650 values of 1.3 to 6.6 on basal synthetic medium (18) lacking the long-chain-base phytosphingosine. The medium was modified to contain 50 mM sodium succinate (pH 5) instead of glycylglycine and double concentrations of all constituents except tergitol and glucose. Growth was stopped with 5% trichloroacetic acid, and the cells were washed several times with 5% trichloroacetic acid and then with water. To a cell pellet containing 250 absorbance units
Corresponding author. 2176
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at 650 nm was added 4.0 ml of 1.0 N HCl in methanol-H20 (82:18, vol/vol), and then the mixture was heated at 80°C for 21 h; 100 nmol of phytosphingosine and 200 nmol of M(IP)2C were treated similarly for standards. After the addition of 12 ml of concentrated NH40H, long-chain bases were extracted three times with 3 ml of CHC13. The combined CHC13 extracts were washed with 3 ml of concentrated NH40H and evaporated to dryness. The long-chain bases were converted to UV-absorbing biphenylcarbonyl derivatives by treatment (90 min at room temperature) with 10 ,ul of 4-biphenylcarbonyl chloride (Sigma Chemical Co., St. Louis, Mo. (5%, wt/vol) in tetrahydrofuran plus 100 ,ul of triethylamine (0.5%, vol/vol) in tetrahydrofuran. The reaction was terminated, and excess reagent was destroyed by the addition of 10 pul of ethanolamine (20%, vol/vol) in tetrahydrofuran followed by incubation for 30 min at room temperature. Samples (5 to 20 pul) of the reaction mixtures were resolved on an Altex 5-p.m Ultrasphere ODS column (0.46 by 25 cm; Beckman Instruments, Inc., Fullerton, Calif.) with a 5-p.m Lichrosorb RP-18 precolumn (0.32 by 4.5 cm; E. Merck AG, Darmstadt, Federal Republic of Germany) eluted with methanol-H20 (90:10, vol/vol) at a flow rate of 1.5 ml/min and monitored at 280 nm. This procedure was adapted from that of Jungalwala et al. (9). Sphingolipid analysis. Cells were cultured overnight in the same medium used for long-chain-base analysis except that it contained 78 p.M [3H]inositol (400 dpm/pmol). Where appropriate, 25 p.M phytosphingosine was added to the medium. After free inositol was removed by washing with 5% trichloroacetic acid, the lipids were extracted, deacylated, added to a mixture of yeast sphingolipid standards, and resolved by high-pressure liquid chromatography (18). Fatty acid analysis. Samples of the cell cultures used for long-chain-base analysis were also used for analysis of fatty acids. A cell pellet containing 250 absorbance units at 650 nm as well as lipid standards [200 nmol of M(IP)2C and 100 nmol each of cerotic acid and hydroxycerotic acid] was treated with 4 ml of 2 N KOH in methanol-H20 (2:1, vol/vol) at 80°C for 17 to 21 h. After the addition of 4 ml of 10 N HCl, the fatty acids were extracted three times with 3 ml of diethyl ether. The combined ether extracts were washed with 5 ml of 0.5 N HCl and evaporated to dryness. UV-absorbing phenacyl derivatives were prepared by reaction with oa-bromoacetophenone by the method of Wood and Lee (19). The derivatives were dissolved in 250 plI of CHC13, and 10-plI volumes were resolved on an Altex 5-p.m Ultrasphere ODS column (0.46 by 25 cm) with a 5-pum Lichrosorb RP-18 precolumn (0.32 by 4.5 cm) eluted isocratically with methanol-acetonitrile (1:1, vol/vol) at a flow rate of 2.0 ml/min. The column effluent was monitored at 242 nm. Miscellaneous procedures. The PYED medium used for measuring doubling times contained 1% peptone, 1% yeast extract, 50 mM sodium succinate (pH 5), inositol (50 mg/ liter), potassium phosphate monobasic (50 mg/ml), and 2% glucose. For Southern blot analysis, total cellular DNA was isolated (6), 3 ,ug of DNA was cleaved with the restriction
endonucleases NruI and StuI, the DNA fragments were separated on a 0.9% agarose gel, and a Southern blot was prepared by using nitrocellulose paper. DNAs used as hybridization probes were labeled with [32P]dATP (3). RESULTS Isolation of strains that suppress the need for long-chain base. Isolation of secondary suppressor mutations that would alleviate the need to add long-chain base to the culture
YEAST MUTANTS LACKING SPHINGOLIPIDS
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FIG. 1. Southern blot analysis of mutant strains. Southern blots were used to verify that the lcbl::URA3 deletion mutation was present in the two suppressor strains, 7R4 and 7R6, that can bypass the need for exogenous long-chain base. The lcbl::URA3 deletion allele was constructed by gene replacement as described in Materials and Methods. (a, lanes 1 through 5) The blot was hybridized with the indicated 4-kb NruI-to-StuI DNA fragment containing [32P]dATP. (a, lanes 6 through 10) The blot used in lanes 1 through 5 was washed to remove radioactivity and rehybridized with a [32P]dATP-labeled 1.05-kb DNA fragment defined by the PstI sites indicated in panel b. The DNA samples are wild-type strain SJ21R (lanes 1 and 6), strain 1A4 carrying a Icbl :: URA3 deletion mutation (lanes 2 and 7), strain 1A7 carrying a Icbl:: URA3 deletion mutation (lanes 3 and 8), strain 7R4 (lanes 4 and 9), and strain 7R6 (lanes 5 and 10). Restriction endonucleases: B, BamHI; Hp, HpaI; P, PstI; Sm, SmaI; St, StuI; N, NruI.
medium required a nonreverting allele of lcbJ such as a deletion allele. The original lcbl-l allele reverted to wild type at high frequency, thus precluding the isolation of suppressor mutations in other genes. To create a deletion allele, we first isolated and partially characterized LCBJ (unpublished data). We then deleted a portion of the cloned gene between the BamHI and HpaI restriction endonuclease sites and replaced the deleted region with the URA3 gene (Fig. 1). Finally, the Icbl:: URA3 deletion was used to replace the wild-type LCBJ chromosomal allele. Two independently derived Icbl:: URA3 deletion strains, termed 1A4 and 1A7, were used for further experiments. Both were strict auxotrophs for long-chain base, and even in its presence the strains grew slowly. In an attempt to select suppressors or bypass mutant strains that retained the Icbl:: URA3 deletion mutation but that could grow without addition of phytosphingosine to the culture medium, we plated 107 cells per PYED plate lacking long-chain base. After about a week at 30°C, small colonies arose at a frequency of l0-7. Cells from the colonies were purified by streaking and then tested
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DICKSON ET AL.
MOL. CELL. BIOL.
TABLE 1. Doubling times of strains Doubling time (min) in:
Strain
SJ21R (wild type) 1A4 (Icbl::URA3 deletion) 1A7 (Icbl::URA3 deletion) 7R4 (IcbJ suppressor) 7R6 (Icbl suppressor) a
b
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PYED + LCBa
85 NGb NG 262 183
81 85 85 83 75
LCB, 25 FM phytosphingosine. NG, No growth, i.e., less than one doubling in 24 h.
to verify that they had the Ura+ Leu- phenotypes of the parent lcbl:: URA3 strain and were potential suppressor
strains rather than contaminants. Southern blot analysis was used to confirm that the suppressor strains had retained the Icbl::URA3 mutation. DNA from the wild-type parental strain SJ21R showed the expected 4-kb band of hybridization when total yeast DNA was cleaved with the restriction endonucleases StuI and NruI (Fig. 1A, lane 1). In the two Icbl::URA3 deletion strains and in the two suppressor strains, termed 7R4 and 7R6, the 4-kb band of hybridization should have been absent because of the deletion mutation, and instead there should have been 1.9- and 1.2-kb bands of hybridization since the URA3 gene contained a StuI site (Fig. 1). All of these strains had the 1.9- and the 1.2-kb bands of hybridization and thus retained the lcbl:: URA3 deletion allele (Fig. la, lanes 2 through 5). The lcbl:: URA deletion strains and the suppressor strains showed a faint 4-kb band of hybridization. This hybridization was a result of DNA sequences bordering the LCBJ gene. When the Southern blot (Fig. la, lanes 1 through 5) was washed and rehybridized with a 1.05-kb 32P-labeled PstI DNA fragment, which lies within the LCBJ coding region, there was no hybridization signal to the 4-kb band in the Icbl:: URA3 deleted strains (Fig. la, lanes 7 and 8) or the suppressor strains (Fig. la, lanes 9 and 10). Furthermore, use of the PstI probe verified that LCBJ was deleted in the Icbl:: URA3 parent and suppressor strains since these strains did not hybridize to the probe, whereas the wild-type strain carrying LCBJ did (Fig. la, lane 6). Even though the IcbJ suppressor strains were able to grow in the absence of phytosphingosine, they were not able to grow as fast as the wild-type strain SJ21R (Table 1). When phytosphingosine was added to the medium, the lcbJ suppressor strains and the lcbl :: URA3 deleted strains grew as well as the wild type, indicating that exogenous phytosphingosine could fulfill the needs of the cell for sphingolipid synthesis. To determine if the suppressor gene had a phenotype of its own and to examine the genetic basis of suppression, we crossed suppressor strains with an LCBJ strain (YNN27). Spores from four-spored tetrads were checked for their Lcb phenotypes. The cross involving 7R4 gave 2 parental ditype: 9 tetratype:6 nonparental ditype tetrads, whereas the 7R6 cross gave 1 parental ditype:5 tetratype:4 nonparental ditype tetrads. The number of tetrads analyzed was too small to rule out any genetic linkage of the suppressor gene to Icbl:: URA3, but it was clear that these genes are not tightly linked. The suppressor gene would be separated from the Icbl:: URA3 gene in nonparental ditype tetrads, with the Lcb- spores carrying lcbl:: URA3 and no suppressor gene and the Lcb+ spores carrying LCBJ and the suppressor gene. There was no reason to think that the suppressor gene by itself would produce a particular phenotype; however, we
did test the Lcb+ spores from the nonparental ditype tetrads and found them normal for heat (37°C) and cold (4°C) sensitivity. Further genetic analyses have been defered until a phenotype can be found or until the suppressor gene has been molecularly cloned. Sphingolipid content of suppressor strains. Have the suppressor strains somehow managed to make sphingolipids when grown in the absence of phytosphingosine or have they managed to grow without making sphingolipids? The two analytical strategies employed to explore this question were chemical analyses of whole cells for free and bound longchain bases and assays for inositol-containing sphingolipids after uniform labeling with [3H]inositol. Whole cells cultured without long-chain base were subjected to hydrolysis with methanolic hydrochloric acid to liberate amide-linked long-chain bases which were extracted into chloroform after the hydrolysate was made basic; the long-chain bases in the extract were converted to UVabsorbing N-biphenylcarbonyl derivatives by a modification of the procedure of Jungalwala et al. (9) and subjected to reversed-phase liquid chromatography with monitoring at 280 nm (see Materials and Methods). Wild-type cells (SJ21R) showed two major high-pressure liquid chromatography peaks having the same retention times (13.1 and 23.0 min) as C-18 and C-20 phytosphingosines in the sphingolipid standard M(IP)2C (Fig. 2). It was previously shown that yeast sphingolipids contain both C-18 and C-20 phytosphingosines (15). Suppressor strains 7R4 and 7R6 showed no peaks containing long-chain bases above the base-line noise (Fig. 2); even when a fourfold-larger sample was used, no peaks were discernible either for C-18 or C-20 phytosphingosine or for those at retention times expected for the phytosphingosine precursors 3-keto-dihydrosphingosine (26.6 min) and D-erythro-dihydrosphingosine (23.0 min). To obtain an upper limit for the long-chain-base content of the suppressor strains, the base-line noise in the region of the C-18 and C-20 phytosphingosines was integrated for each of two separate experiments. By this approach, the two suppressor strains 7R4 and 7R6 averaged 1.4 + 0.5 and 1.5 + 0.04 pmol of phytosphingosines per absorbance unit at 650 nm of cells, respectively, whereas the wild-type strain SJ21R averaged 956 ± 31 pmol of phytosphingosines per absorbance unit at 650 nm of cells. These data strongly suggest that the suppressor strains grow without making sphingolipids. The lack of sphingolipids in suppressor strains was verified by analyzing cells directly for the inositol-containing sphingolipids IPC, MIPC, and M(IP)2C. This was done by labeling growing cells with [3H]myoinosiol, extracting the lipids, and analyzing the products of alkali-catalyzed methanolysis by high-pressure liquid chromatography (18). In contrast to the wild-type strain SJ21R, the IcbJ suppressor strains 7R4 and 7R6 made barely detectable levels of sphingolipids when the cells were cultured in medium lacking phytosphingosine (Table 2). When phytosphingosine was added to the culture medium, the suppressor strains made almost normal levels of sphingolipid. The composition of the sphingolipids made by suppressor strains grown in the presence of phytosphingosine was altered somewhat compared with that made by the wild type (Fig. 3). It is not clear if these alterations are physiologically significant or if they are due to the way the cell metabolizes exogenous phytosphingosine. We conclude from these data that the suppressor strains 7R4 and 7R6 make little if any inositol-containing sphingolipids when grown without phytosphingosine. Analysis of very-long-chain fatty acids in suppressor strains. Yeast sphingolipids contain very-long-chain fatty acids, pri-
YEAST MUTANTS LACKING SPHINGOLIPIDS
VOL. 10, 1990
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RETENTION TIME (MIN) FIG. 2. Long-chain-base content of wild-type and IcbJ suppressor strains. N-Biphenylcarbonyl derivatives prepared from both acid hydrolysates of cells and a M(IP)2C standard were resolved by reversed-phase liquid chromatography as described in Materials and Methods. Samples (5 ,ul) representing 10.4 absorbance units at 650 nm of cells or 8.3 nmol of M(IP)2C were analyzed. PHS, Phytosphingosines.
marily C-26, that are hydroxylated (15). Therefore it was of interest to determine if very-long-chain fatty acids accumulated in suppressor strains even though no sphingolipids were being made. The total amounts of very-long-chain fatty acid in the wild-type and Icbi suppressor strains were similar TABLE 2. Presence of inositol-labeled sphingolipids in wild-type and IcbJ suppressor strains Strain
SJ21R (wild type) 7R4 (lcbJ suppressor) 7R6 (Icbl suppressor)
% Inositol-labeled sphingolipidsa in medium + PHS -PHS
100 0.45 ± 0.16 0.35 + 0.42
100 93.9 ± 31 74.3 + 49
a Compared with amounts in wild-type strain SJ21R. For cells grown without phytosphingosine (- PHS), 100% equals 1,088 pmol of inositol per absorbance unit at 650 nm; for cells grown with phytosphingosine (+ PHS), 100%o equals 1,311 pmol of inositol per absorbance unit at 650 nm. Values are the averages of two separate experiments ± standard errors of the means.
(Table 3), indicating that synthesis of very-long-chain fatty acids continues in the absence of sphingolipid synthesis. However, there was a large difference in the degree of fatty acid hydroxylation; most of the very-long-chain fatty acid in the wild-type strain was hydroxylated, whereas in the IcbJ suppressor strains, very little hydroxylation occurred when sphingolipids were not made (i.e., no phytosphingosine in the culture medium). The lack of hydroxylation was not the result of a defect in the ability of the cell to hydroxylate, because hydroxylation was normal when IcbJ suppressor strains made sphingolipids (when phytosphingosine was added to the culture medium). The simplest interpretation of these data is that hydroxylation of very-long-chain fatty acids occurs after ceramide formation. DISCUSSION Isolation of the suppressor mutations described in this paper sets the stage for a detailed analysis of sphingolipid function in yeast cells. Further analysis should reveal how the suppressor gene allows cells to survive without sphingolipids. A suppressor gene could act by coding for a variant
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Fraction Numoen FIG. 3. Sphingolipid content of wild-type and Icbl suppressor strains. The sphingolipid content of cells was determined as described in Materials and Methods by using [3H]myoinositol-labeled cells. The cultures either contained (in panel a, x; in panels b and c, -) or lacked (0) 25 ,uM phytosphingosine. The internal standards were detected with a moving-wire detector and coincided exactly with the tritium peaks measured in each column fraction by scintillation spectrometry; the results are expressed in picomoles of inositol (calculated from the initial specific activity) per absorbance unit at 650 nm of cells for each column fraction. (a) IPC peaks at fractions 27 and 31, representing monoand dihydroxy fatty acid variants, respectively; MIPC peaks at fraction 39; and the major M(IP)2C variant peaks at fraction 81.
protein that no longer requires a sphingolipid(s) for function, by synthesizing a novel lipid(s) that substitutes for sphingolipids, or by changing the amount of one or more normal membrane lipids to compensate for the lack of sphingolipids. We have not yet determined if the suppressor mutations in strains 7R4, 7R6, and other uncharacterized suppressor strains are in the same or in different genes, so it is possible that a multiple mechanism(s) exists for bypassing the need for sphingolipids in yeast. Two other observations on suppressor mutants indicate that the puzzle of sphingolipid function may have multiple solutions. First, the growth rate of suppressor strains is low in the absence of phytospingosine and increases to the same rate as the wild type when
phytosphingosine is added to the culture medium (Table 1). Second, even when suppressor strains are cultured with phytosphingosine, the strains are not healthy and lose viability (data not shown). These observations suggest that sphingolipids may be playing nonvital roles in addition to the vital role(s) detected by our suppressor selection scheme. It may be possible to elucidate the nonvital roles played by sphingolipids by comparing the biochemical properties of cells
or
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phytosphingosine with those grown in its absence, since these two growth conditions provide a source of biological membranes that should differ primarily in their sphingolipid content.
VOL. 10, 1990
YEAST MUTANTS LACKING SPHINGOLIPIDS
TABLE 3. Very-long-chain fatty acid content of Icbl suppressor strains Amt of very-long-chain fatty acidsa Strain
Nonhydroxylated
PHSb
Hydroxylated
3. 4.
+ PHS
- PHS
+ PHS
193 565 152
1,667 166 74
2,409 2,607 2,635
5.
a Data (in picomoles of fatty acid per absorbance unit at 650 nm) are the means of two experiments except for data on cells cultured with phytosphingosine, which are from one experiment. The nonhydroxylated fatty acid is C-26, and the hydroxylated fatty acid is primarily C-26 with much smaller amounts of C-24. b PHS, 25 F.M phytosphingosine in culture medium.
6.
-
SJ21R (wild type) 7R4 (lcbJ suppressor) 7R6 (IcbJ suppressor)
168 1,784 2,031
7.
8.
A reduced rate of phosphosphingolipid synthesis has been noted in a strain of S. cerevisiae that carries a chol suppressor mutation named eam2; the nature of the defect accounting for the partial reduction in sphingolipid synthesis is not known (1). The isolation of IcbJ-suppressor strains may also provide a new avenue for studying hydroxylation of the very-longchain fatty acids found in yeast sphingolipids. The substrate for hydroxylation in other organisms is not well defined. In microsomes of rat brains, there is evidence for hydroxylation of lignoceryl-coenzyme A by a cyanide-sensitive oxygenase (14). In Tetrahymenae, there is evidence for direct hydroxylation of sphingolipids (11). Future results should have wide relevance since S. cerevisiae sphingolipids are related to those found in other fungi (2) as well as those in higher plants (4): all contain an inositol group that is phosphodiester-linked to a ceramide moiety consisting of N-fatty-acylated phytosphingosine. Some animal sphingolipids may perform similar functions even though they lack inositol phosphate, having principally oligosaccharides as polar head groups, and contain predominantly sphingosine instead of phytosphingosine (4).
9. 10. 11.
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ACKNOWLEDGMENTS This study was supported by Public Health Service research grants RO1 A120600 and RO1 GM41302 from the National Institutes of Health. We thank our colleagues J. Lesnaw and C. Waechter for helpful comments on this manuscript. LITERATURE CITED 1. Atkinson, K. D. 1985. Two recessive suppressors of Saccharomyces cerevisiae CHOI that are unlinked but fall in the same complementation group. Genetics 111:1-6. 2. Barr, K., and R. L. Lester. 1984. Occurrence of novel antigenic
17.
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phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum. Biochemistry 23:5581-5588. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. Hakomori, S.-I. 1983. Chemistry of glycosphingolipids, p. 1150. In J. N. Kanfer and S.-I. Hakomori (ed.), Sphingolipid biochemistry Plenum Publishing Corp., New York. Hannun, Y. A., and R. M. Bell. 1989. Functions of sphingolipids and sphingolipid breakdown in cellular regulation. Science 243:500-507. Hoffman, C. S., and F. Winston. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267-272. Jarvik, J., and D. Botstein. 1975. Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc. Natl. Acad. Sci. USA 72:2738-2742. Johnston, S. A., and J. E. Hopper. 1982. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc. Natl. Acad. Sci. USA 79: 6971-6975. Jungalwala, F. B., J. E. Evans, E. Bremer, and R. H. McCluer. 1983. Analysis of sphingoid bases by reversed-phase high performance liquid chromatography. J. Lipid Res. 24:1380-1388. Karlsson, K.-A. 1989. Animal glycosphingolipids as membrane attachment sites for bacteria. Annu. Rev. Biochem. 58:309-350. Kaya, K., C. S. Ramesha, and G. A. Thompson. 1984. On the formation of a-hydroxy fatty acids; evidence for a direct hydroxylation of nonhydroxy fatty acid-containing sphingolipids. J. Biol. Chem. 259:3548-3553. Ledeen, R. W. 1989. Biosynthesis, metabolism, and biological effects of gangliosides, p. 43-83. In. R. U. Margolis and R. K. Margolis (ed.), Neurobiology of glycoconjugates. Plenum Publishing Corp., New York. Rudolph, H., I. Koenig-Rauseo, and A. Hinnen. 1985. One-step gene replacement in yeast by cotransformation. Gene 36:87-95. Shigematsu, H., and Y. Kishimoto. 1987. Alpha-hydroxylation of lignoceryl-CoA by a cyanide-sensitive oxygenase in rat brain microsomes. Int. J. Biochem. 19:41-46. Smith, S. W., and R. L. Lester. 1974. Inositolphosphorylceramide, a novel substance and the chief member of a major group of yeast sphingolipids containing a single inositolphosphate. J. Biol. Chem. 249:3395-3405. Steiner, S., S. Smith, C. J. Waechter, and R. L. Lester. 1969. Isolation and partial characterization of a major inositol-containing lipid in baker's yeast, mannosyl-diinositol, diphosphoryl-ceramide. Proc. Natl. Acad. Sci. 64:1042-1048. Thudichum, J. L. W. 1884. A treatise on the chemical constitution of brain. Bailliere, Tindall, and Cox, London. Wells, G. B., and R. L. Lester. 1983. The isolation and characterization of a mutant strain of Saccharomyces cerevisiae that requires a long chain base for growth and for synthesis of phosphosphingolipids. J. Biol. Chem. 258:10200-10203. Wood, R., and T. Lee. 1983. High-performance liquid chromatography of fatty acids: quantitative analysis of saturated, monoenoic, polyenoic and geometrical isomers. J. Chromatogr. 254:237-246.
Vol. 10, No. 5
Isolation of Mutant Saccharomyces cerevisiae Strains That Survive without Sphingolipids ROBERT C. DICKSON,* GERALD B. WELLS, ANN SCHMIDT, AND ROBERT L. LESTER Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536-0084 Received 8 November 1989/Accepted 1 February 1990
Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.
Sphingolipids constitute a diverse group of complex membrane lipids. All contain a long-chain amino alcohol, usually 18 carbons, connected by an amide linkage to a fatty acid to form ceramide. The hydroxyl at the 1 position of ceramide can be linked to various polar head groups, including phosphorylcholine (sphingomyelin), or to glucose units and other moieties (4). Sphingolipids are found mainly in eucaryotes, where they are ubiquitous, with hundreds having been identified and structurally characterized in mammals alone (4). More than a century ago, Thudichum (17) discovered the long-chain-base sphingosine in the human brain and named it after the sphinx because of the riddles it posed. The name remains appropriate. Although sphingolipids are known to play a structural role in membranes, their precise biological function(s) is unknown. Our lack of knowledge is due in part to difficulties in assigning functions which stem from the diversity of sphingolipids present in most cell types. Roles have been proposed in such complex phenomena as cellular differentiation, cell-to-cell recognition, and modulation of growth (4, 10, 12), including involvement in membrane signal transduction pathways (5). We have begun to examine the function of sphingolipids in the yeast Saccharomyces cerevisiae, which is well suited for these studies because sphingolipid diversity is minimized in that S. cerevisiae contains only three abundant sphingolipids, with the following compositions: inositol P-ceramide (IPC), mannose-inositol Pceramide (MIPC) and mannose-(inositol P)2-ceramide [M(IP)2C] (15, 16). Moreover, S. cerevisiae permits full exploitation of molecular genetic techniques which have not been used previously to examine sphingolipid function. Our studies build upon the work of Wells and Lester (18), who isolated a strain of S. cerevisiae that requires a longchain base (Icb, the original allele being Icbl-J) such as phytosphingosine for growth. The absence of serine palmitoyltransferase activity in the lcbJ mutant (W. J. Pinto, G. B. Wells, A. C. Williams, K. A. Anderson, E. C. Teater, and R. L. Lester, Fed. Proc. 45:1826, 1986) suggests that LCBJ codes for this enzyme or a subunit of the enzyme, the first enzyme in the long-chain-base synthetic pathway of yeasts. This mutant provided the first genetic evidence in any *
organism that sphingolipids are essential for normal cellular function. The Icbl-defective strains are a limited subject for the study of sphingolipid function because the mutation is lethal. However, such mutations can be used in an alternative strategy based on the isolation of secondary mutations (7) that suppress or bypass the lcbJ defect and allow growth in the absence of exogenous long-chain base. Suppressor genes could function by enabling the strain to make sphingolipids by a novel synthesis pathway, by creating a lipid(s) that replaces sphingolipids, or by enabling the strain to grow without making sphingolipids. Analysis of the latter class of hypothetical suppressors should reveal specific sphingolipid functions. For example, the product of a suppressor allele, in comparison with the wild-type gene product, may no longer require sphingolipid for function. We report here that the desired hypothetical suppressor strains can be isolated and that they grow while making little if any detectable sphingolipid. MATERIALS AND METHODS Strains. S. cerevisiae SJ21R (MATa ura3-52 leu2-3,112 adel MEL]) (8) was used as the wild-type strain for LCBJ. Strains carrying the lcbl:: URA3 deletion allele were constructed by gene replacement (13). Strain SJ21R was transformed with 10 ,ug of the 3.1-kilobase (kb) NruI-to-StuI DNA fragment carrying the URA3 gene (see Fig. 1C) and Ura+ transformants were selected. Two transformants, strains 1A4 and 1A7, showing a stable Ura+ phenotype and carrying the lcbl:: URA3 deletion allele, were used for further study, including selection of the suppressor strains designated 7R4 and 7R6. Strain YNN27 (MATa trpl-289 ura3-52 gal2) was used as an LCBJ strain to cross with the putative IcbJ suppressor strains. Analysis of long-chain bases. Cells were cultured overnight to A650 values of 1.3 to 6.6 on basal synthetic medium (18) lacking the long-chain-base phytosphingosine. The medium was modified to contain 50 mM sodium succinate (pH 5) instead of glycylglycine and double concentrations of all constituents except tergitol and glucose. Growth was stopped with 5% trichloroacetic acid, and the cells were washed several times with 5% trichloroacetic acid and then with water. To a cell pellet containing 250 absorbance units
Corresponding author. 2176
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at 650 nm was added 4.0 ml of 1.0 N HCl in methanol-H20 (82:18, vol/vol), and then the mixture was heated at 80°C for 21 h; 100 nmol of phytosphingosine and 200 nmol of M(IP)2C were treated similarly for standards. After the addition of 12 ml of concentrated NH40H, long-chain bases were extracted three times with 3 ml of CHC13. The combined CHC13 extracts were washed with 3 ml of concentrated NH40H and evaporated to dryness. The long-chain bases were converted to UV-absorbing biphenylcarbonyl derivatives by treatment (90 min at room temperature) with 10 ,ul of 4-biphenylcarbonyl chloride (Sigma Chemical Co., St. Louis, Mo. (5%, wt/vol) in tetrahydrofuran plus 100 ,ul of triethylamine (0.5%, vol/vol) in tetrahydrofuran. The reaction was terminated, and excess reagent was destroyed by the addition of 10 pul of ethanolamine (20%, vol/vol) in tetrahydrofuran followed by incubation for 30 min at room temperature. Samples (5 to 20 pul) of the reaction mixtures were resolved on an Altex 5-p.m Ultrasphere ODS column (0.46 by 25 cm; Beckman Instruments, Inc., Fullerton, Calif.) with a 5-p.m Lichrosorb RP-18 precolumn (0.32 by 4.5 cm; E. Merck AG, Darmstadt, Federal Republic of Germany) eluted with methanol-H20 (90:10, vol/vol) at a flow rate of 1.5 ml/min and monitored at 280 nm. This procedure was adapted from that of Jungalwala et al. (9). Sphingolipid analysis. Cells were cultured overnight in the same medium used for long-chain-base analysis except that it contained 78 p.M [3H]inositol (400 dpm/pmol). Where appropriate, 25 p.M phytosphingosine was added to the medium. After free inositol was removed by washing with 5% trichloroacetic acid, the lipids were extracted, deacylated, added to a mixture of yeast sphingolipid standards, and resolved by high-pressure liquid chromatography (18). Fatty acid analysis. Samples of the cell cultures used for long-chain-base analysis were also used for analysis of fatty acids. A cell pellet containing 250 absorbance units at 650 nm as well as lipid standards [200 nmol of M(IP)2C and 100 nmol each of cerotic acid and hydroxycerotic acid] was treated with 4 ml of 2 N KOH in methanol-H20 (2:1, vol/vol) at 80°C for 17 to 21 h. After the addition of 4 ml of 10 N HCl, the fatty acids were extracted three times with 3 ml of diethyl ether. The combined ether extracts were washed with 5 ml of 0.5 N HCl and evaporated to dryness. UV-absorbing phenacyl derivatives were prepared by reaction with oa-bromoacetophenone by the method of Wood and Lee (19). The derivatives were dissolved in 250 plI of CHC13, and 10-plI volumes were resolved on an Altex 5-p.m Ultrasphere ODS column (0.46 by 25 cm) with a 5-pum Lichrosorb RP-18 precolumn (0.32 by 4.5 cm) eluted isocratically with methanol-acetonitrile (1:1, vol/vol) at a flow rate of 2.0 ml/min. The column effluent was monitored at 242 nm. Miscellaneous procedures. The PYED medium used for measuring doubling times contained 1% peptone, 1% yeast extract, 50 mM sodium succinate (pH 5), inositol (50 mg/ liter), potassium phosphate monobasic (50 mg/ml), and 2% glucose. For Southern blot analysis, total cellular DNA was isolated (6), 3 ,ug of DNA was cleaved with the restriction
endonucleases NruI and StuI, the DNA fragments were separated on a 0.9% agarose gel, and a Southern blot was prepared by using nitrocellulose paper. DNAs used as hybridization probes were labeled with [32P]dATP (3). RESULTS Isolation of strains that suppress the need for long-chain base. Isolation of secondary suppressor mutations that would alleviate the need to add long-chain base to the culture
YEAST MUTANTS LACKING SPHINGOLIPIDS
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FIG. 1. Southern blot analysis of mutant strains. Southern blots were used to verify that the lcbl::URA3 deletion mutation was present in the two suppressor strains, 7R4 and 7R6, that can bypass the need for exogenous long-chain base. The lcbl::URA3 deletion allele was constructed by gene replacement as described in Materials and Methods. (a, lanes 1 through 5) The blot was hybridized with the indicated 4-kb NruI-to-StuI DNA fragment containing [32P]dATP. (a, lanes 6 through 10) The blot used in lanes 1 through 5 was washed to remove radioactivity and rehybridized with a [32P]dATP-labeled 1.05-kb DNA fragment defined by the PstI sites indicated in panel b. The DNA samples are wild-type strain SJ21R (lanes 1 and 6), strain 1A4 carrying a Icbl :: URA3 deletion mutation (lanes 2 and 7), strain 1A7 carrying a Icbl:: URA3 deletion mutation (lanes 3 and 8), strain 7R4 (lanes 4 and 9), and strain 7R6 (lanes 5 and 10). Restriction endonucleases: B, BamHI; Hp, HpaI; P, PstI; Sm, SmaI; St, StuI; N, NruI.
medium required a nonreverting allele of lcbJ such as a deletion allele. The original lcbl-l allele reverted to wild type at high frequency, thus precluding the isolation of suppressor mutations in other genes. To create a deletion allele, we first isolated and partially characterized LCBJ (unpublished data). We then deleted a portion of the cloned gene between the BamHI and HpaI restriction endonuclease sites and replaced the deleted region with the URA3 gene (Fig. 1). Finally, the Icbl:: URA3 deletion was used to replace the wild-type LCBJ chromosomal allele. Two independently derived Icbl:: URA3 deletion strains, termed 1A4 and 1A7, were used for further experiments. Both were strict auxotrophs for long-chain base, and even in its presence the strains grew slowly. In an attempt to select suppressors or bypass mutant strains that retained the Icbl:: URA3 deletion mutation but that could grow without addition of phytosphingosine to the culture medium, we plated 107 cells per PYED plate lacking long-chain base. After about a week at 30°C, small colonies arose at a frequency of l0-7. Cells from the colonies were purified by streaking and then tested
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DICKSON ET AL.
MOL. CELL. BIOL.
TABLE 1. Doubling times of strains Doubling time (min) in:
Strain
SJ21R (wild type) 1A4 (Icbl::URA3 deletion) 1A7 (Icbl::URA3 deletion) 7R4 (IcbJ suppressor) 7R6 (Icbl suppressor) a
b
PYED
PYED + LCBa
85 NGb NG 262 183
81 85 85 83 75
LCB, 25 FM phytosphingosine. NG, No growth, i.e., less than one doubling in 24 h.
to verify that they had the Ura+ Leu- phenotypes of the parent lcbl:: URA3 strain and were potential suppressor
strains rather than contaminants. Southern blot analysis was used to confirm that the suppressor strains had retained the Icbl::URA3 mutation. DNA from the wild-type parental strain SJ21R showed the expected 4-kb band of hybridization when total yeast DNA was cleaved with the restriction endonucleases StuI and NruI (Fig. 1A, lane 1). In the two Icbl::URA3 deletion strains and in the two suppressor strains, termed 7R4 and 7R6, the 4-kb band of hybridization should have been absent because of the deletion mutation, and instead there should have been 1.9- and 1.2-kb bands of hybridization since the URA3 gene contained a StuI site (Fig. 1). All of these strains had the 1.9- and the 1.2-kb bands of hybridization and thus retained the lcbl:: URA3 deletion allele (Fig. la, lanes 2 through 5). The lcbl:: URA deletion strains and the suppressor strains showed a faint 4-kb band of hybridization. This hybridization was a result of DNA sequences bordering the LCBJ gene. When the Southern blot (Fig. la, lanes 1 through 5) was washed and rehybridized with a 1.05-kb 32P-labeled PstI DNA fragment, which lies within the LCBJ coding region, there was no hybridization signal to the 4-kb band in the Icbl:: URA3 deleted strains (Fig. la, lanes 7 and 8) or the suppressor strains (Fig. la, lanes 9 and 10). Furthermore, use of the PstI probe verified that LCBJ was deleted in the Icbl:: URA3 parent and suppressor strains since these strains did not hybridize to the probe, whereas the wild-type strain carrying LCBJ did (Fig. la, lane 6). Even though the IcbJ suppressor strains were able to grow in the absence of phytosphingosine, they were not able to grow as fast as the wild-type strain SJ21R (Table 1). When phytosphingosine was added to the medium, the lcbJ suppressor strains and the lcbl :: URA3 deleted strains grew as well as the wild type, indicating that exogenous phytosphingosine could fulfill the needs of the cell for sphingolipid synthesis. To determine if the suppressor gene had a phenotype of its own and to examine the genetic basis of suppression, we crossed suppressor strains with an LCBJ strain (YNN27). Spores from four-spored tetrads were checked for their Lcb phenotypes. The cross involving 7R4 gave 2 parental ditype: 9 tetratype:6 nonparental ditype tetrads, whereas the 7R6 cross gave 1 parental ditype:5 tetratype:4 nonparental ditype tetrads. The number of tetrads analyzed was too small to rule out any genetic linkage of the suppressor gene to Icbl:: URA3, but it was clear that these genes are not tightly linked. The suppressor gene would be separated from the Icbl:: URA3 gene in nonparental ditype tetrads, with the Lcb- spores carrying lcbl:: URA3 and no suppressor gene and the Lcb+ spores carrying LCBJ and the suppressor gene. There was no reason to think that the suppressor gene by itself would produce a particular phenotype; however, we
did test the Lcb+ spores from the nonparental ditype tetrads and found them normal for heat (37°C) and cold (4°C) sensitivity. Further genetic analyses have been defered until a phenotype can be found or until the suppressor gene has been molecularly cloned. Sphingolipid content of suppressor strains. Have the suppressor strains somehow managed to make sphingolipids when grown in the absence of phytosphingosine or have they managed to grow without making sphingolipids? The two analytical strategies employed to explore this question were chemical analyses of whole cells for free and bound longchain bases and assays for inositol-containing sphingolipids after uniform labeling with [3H]inositol. Whole cells cultured without long-chain base were subjected to hydrolysis with methanolic hydrochloric acid to liberate amide-linked long-chain bases which were extracted into chloroform after the hydrolysate was made basic; the long-chain bases in the extract were converted to UVabsorbing N-biphenylcarbonyl derivatives by a modification of the procedure of Jungalwala et al. (9) and subjected to reversed-phase liquid chromatography with monitoring at 280 nm (see Materials and Methods). Wild-type cells (SJ21R) showed two major high-pressure liquid chromatography peaks having the same retention times (13.1 and 23.0 min) as C-18 and C-20 phytosphingosines in the sphingolipid standard M(IP)2C (Fig. 2). It was previously shown that yeast sphingolipids contain both C-18 and C-20 phytosphingosines (15). Suppressor strains 7R4 and 7R6 showed no peaks containing long-chain bases above the base-line noise (Fig. 2); even when a fourfold-larger sample was used, no peaks were discernible either for C-18 or C-20 phytosphingosine or for those at retention times expected for the phytosphingosine precursors 3-keto-dihydrosphingosine (26.6 min) and D-erythro-dihydrosphingosine (23.0 min). To obtain an upper limit for the long-chain-base content of the suppressor strains, the base-line noise in the region of the C-18 and C-20 phytosphingosines was integrated for each of two separate experiments. By this approach, the two suppressor strains 7R4 and 7R6 averaged 1.4 + 0.5 and 1.5 + 0.04 pmol of phytosphingosines per absorbance unit at 650 nm of cells, respectively, whereas the wild-type strain SJ21R averaged 956 ± 31 pmol of phytosphingosines per absorbance unit at 650 nm of cells. These data strongly suggest that the suppressor strains grow without making sphingolipids. The lack of sphingolipids in suppressor strains was verified by analyzing cells directly for the inositol-containing sphingolipids IPC, MIPC, and M(IP)2C. This was done by labeling growing cells with [3H]myoinosiol, extracting the lipids, and analyzing the products of alkali-catalyzed methanolysis by high-pressure liquid chromatography (18). In contrast to the wild-type strain SJ21R, the IcbJ suppressor strains 7R4 and 7R6 made barely detectable levels of sphingolipids when the cells were cultured in medium lacking phytosphingosine (Table 2). When phytosphingosine was added to the culture medium, the suppressor strains made almost normal levels of sphingolipid. The composition of the sphingolipids made by suppressor strains grown in the presence of phytosphingosine was altered somewhat compared with that made by the wild type (Fig. 3). It is not clear if these alterations are physiologically significant or if they are due to the way the cell metabolizes exogenous phytosphingosine. We conclude from these data that the suppressor strains 7R4 and 7R6 make little if any inositol-containing sphingolipids when grown without phytosphingosine. Analysis of very-long-chain fatty acids in suppressor strains. Yeast sphingolipids contain very-long-chain fatty acids, pri-
YEAST MUTANTS LACKING SPHINGOLIPIDS
VOL. 10, 1990
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marily C-26, that are hydroxylated (15). Therefore it was of interest to determine if very-long-chain fatty acids accumulated in suppressor strains even though no sphingolipids were being made. The total amounts of very-long-chain fatty acid in the wild-type and Icbi suppressor strains were similar TABLE 2. Presence of inositol-labeled sphingolipids in wild-type and IcbJ suppressor strains Strain
SJ21R (wild type) 7R4 (lcbJ suppressor) 7R6 (Icbl suppressor)
% Inositol-labeled sphingolipidsa in medium + PHS -PHS
100 0.45 ± 0.16 0.35 + 0.42
100 93.9 ± 31 74.3 + 49
a Compared with amounts in wild-type strain SJ21R. For cells grown without phytosphingosine (- PHS), 100% equals 1,088 pmol of inositol per absorbance unit at 650 nm; for cells grown with phytosphingosine (+ PHS), 100%o equals 1,311 pmol of inositol per absorbance unit at 650 nm. Values are the averages of two separate experiments ± standard errors of the means.
(Table 3), indicating that synthesis of very-long-chain fatty acids continues in the absence of sphingolipid synthesis. However, there was a large difference in the degree of fatty acid hydroxylation; most of the very-long-chain fatty acid in the wild-type strain was hydroxylated, whereas in the IcbJ suppressor strains, very little hydroxylation occurred when sphingolipids were not made (i.e., no phytosphingosine in the culture medium). The lack of hydroxylation was not the result of a defect in the ability of the cell to hydroxylate, because hydroxylation was normal when IcbJ suppressor strains made sphingolipids (when phytosphingosine was added to the culture medium). The simplest interpretation of these data is that hydroxylation of very-long-chain fatty acids occurs after ceramide formation. DISCUSSION Isolation of the suppressor mutations described in this paper sets the stage for a detailed analysis of sphingolipid function in yeast cells. Further analysis should reveal how the suppressor gene allows cells to survive without sphingolipids. A suppressor gene could act by coding for a variant
2180
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Fraction Numoen FIG. 3. Sphingolipid content of wild-type and Icbl suppressor strains. The sphingolipid content of cells was determined as described in Materials and Methods by using [3H]myoinositol-labeled cells. The cultures either contained (in panel a, x; in panels b and c, -) or lacked (0) 25 ,uM phytosphingosine. The internal standards were detected with a moving-wire detector and coincided exactly with the tritium peaks measured in each column fraction by scintillation spectrometry; the results are expressed in picomoles of inositol (calculated from the initial specific activity) per absorbance unit at 650 nm of cells for each column fraction. (a) IPC peaks at fractions 27 and 31, representing monoand dihydroxy fatty acid variants, respectively; MIPC peaks at fraction 39; and the major M(IP)2C variant peaks at fraction 81.
protein that no longer requires a sphingolipid(s) for function, by synthesizing a novel lipid(s) that substitutes for sphingolipids, or by changing the amount of one or more normal membrane lipids to compensate for the lack of sphingolipids. We have not yet determined if the suppressor mutations in strains 7R4, 7R6, and other uncharacterized suppressor strains are in the same or in different genes, so it is possible that a multiple mechanism(s) exists for bypassing the need for sphingolipids in yeast. Two other observations on suppressor mutants indicate that the puzzle of sphingolipid function may have multiple solutions. First, the growth rate of suppressor strains is low in the absence of phytospingosine and increases to the same rate as the wild type when
phytosphingosine is added to the culture medium (Table 1). Second, even when suppressor strains are cultured with phytosphingosine, the strains are not healthy and lose viability (data not shown). These observations suggest that sphingolipids may be playing nonvital roles in addition to the vital role(s) detected by our suppressor selection scheme. It may be possible to elucidate the nonvital roles played by sphingolipids by comparing the biochemical properties of cells
or
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phytosphingosine with those grown in its absence, since these two growth conditions provide a source of biological membranes that should differ primarily in their sphingolipid content.
VOL. 10, 1990
YEAST MUTANTS LACKING SPHINGOLIPIDS
TABLE 3. Very-long-chain fatty acid content of Icbl suppressor strains Amt of very-long-chain fatty acidsa Strain
Nonhydroxylated
PHSb
Hydroxylated
3. 4.
+ PHS
- PHS
+ PHS
193 565 152
1,667 166 74
2,409 2,607 2,635
5.
a Data (in picomoles of fatty acid per absorbance unit at 650 nm) are the means of two experiments except for data on cells cultured with phytosphingosine, which are from one experiment. The nonhydroxylated fatty acid is C-26, and the hydroxylated fatty acid is primarily C-26 with much smaller amounts of C-24. b PHS, 25 F.M phytosphingosine in culture medium.
6.
-
SJ21R (wild type) 7R4 (lcbJ suppressor) 7R6 (IcbJ suppressor)
168 1,784 2,031
7.
8.
A reduced rate of phosphosphingolipid synthesis has been noted in a strain of S. cerevisiae that carries a chol suppressor mutation named eam2; the nature of the defect accounting for the partial reduction in sphingolipid synthesis is not known (1). The isolation of IcbJ-suppressor strains may also provide a new avenue for studying hydroxylation of the very-longchain fatty acids found in yeast sphingolipids. The substrate for hydroxylation in other organisms is not well defined. In microsomes of rat brains, there is evidence for hydroxylation of lignoceryl-coenzyme A by a cyanide-sensitive oxygenase (14). In Tetrahymenae, there is evidence for direct hydroxylation of sphingolipids (11). Future results should have wide relevance since S. cerevisiae sphingolipids are related to those found in other fungi (2) as well as those in higher plants (4): all contain an inositol group that is phosphodiester-linked to a ceramide moiety consisting of N-fatty-acylated phytosphingosine. Some animal sphingolipids may perform similar functions even though they lack inositol phosphate, having principally oligosaccharides as polar head groups, and contain predominantly sphingosine instead of phytosphingosine (4).
9. 10. 11.
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13. 14.
15.
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ACKNOWLEDGMENTS This study was supported by Public Health Service research grants RO1 A120600 and RO1 GM41302 from the National Institutes of Health. We thank our colleagues J. Lesnaw and C. Waechter for helpful comments on this manuscript. LITERATURE CITED 1. Atkinson, K. D. 1985. Two recessive suppressors of Saccharomyces cerevisiae CHOI that are unlinked but fall in the same complementation group. Genetics 111:1-6. 2. Barr, K., and R. L. Lester. 1984. Occurrence of novel antigenic
17.
18.
19.
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