Hydroxylation of Saccharomyces cerevisiae Ceramides Requires Sur2p and Scs7p*

The Saccharomyces cerevisiae SCS7 andSUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype ofcsg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 andsur2 mutants is expected to provide insight into the function of ceramide hydroxylation.

The SUR2 gene was initially identified in a screen for suppressors of rvs161 mutants (10). Rvs161p is required for endocytosis (11), correct actin localization (12), and viability upon nitrogen, carbon, or sulfur starvation (13). It is similar to amphiphysin, a neuronal protein found in synaptic vesicles that is the autoantigen in stiff-man syndrome (14,15). The molecular function of Rvs161p and the basis of suppression by mutations in the SUR genes have not been identified. However, other SUR genes have been found to function in sphingolipid synthesis. SUR1 is allelic to CSG1, a gene required for mannosylation of sphingolipids (5). SUR4/ELO3 encodes a fatty acid elongase required for the synthesis of the very long chain fatty acids (VLCFA) 1 found in ceramide and sphingolipid (16). The genetic relationship between SUR2, SUR1, and SUR4 suggests that SUR2 may also be involved in sphingolipid synthesis.
Based on the homology of Sur2p with Scs7p, required for hydroxylation of the fatty acid of sphingolipids (6), and the genetic relationship between SUR2 and other sphingolipid synthesis genes, the possibility that SUR2 is required for hydroxylation of C-4 on the long chain base (LCB) found in sphingolipids was investigated.
Constructing the sur2 Null Mutant-In an unrelated study we isolated a YCp50-based plasmid containing a fragment of yeast DNA that included the amino terminus (through to a Sau3A site at codon 120) of the SUR2 gene. A restriction fragment extending from the HindIII site 367 base pairs upstream of the start codon of the SUR2 gene to the SaII site in YCp50 was subcloned from this plasmid into pUC19. The resulting plasmid was linearized at the PstI site in codon 9 of the SUR2 gene, treated with Bal-31 to remove about 100 base pairs, and incubated with dNTPs, Klenow fragment, ligase, and XhoI linkers. A candidate plasmid with a XhoI linker at the deletion junction that was missing about 50 base pairs from each side of the original PstI site was used to construct the disrupting plasmid. A SalI fragment carrying the TRP1 gene was ligated into the XhoI site, the SUR2-disrupting fragment was cut out of the pUC19 plasmid with PvuII and used in a one-step gene replacement (18). The disruption of SUR2 was confirmed using a polymerase chain reaction.
Ceramide Isolation and Analysis-Cells were grown in synthetic medium at 26°C, spun down, and washed once with H 2 O. The cells were vortexed with glass beads in hexane:EtOH (95:5) at 40 A 600 /ml. The supernatant was transferred to a fresh tube, the pellet and beads were washed with hexane:EtOH, and the pooled extract was dried. The lipids from 60 A 600 units of cells were alkali-treated by suspending in 1 ml of EtOH:H 2 O:Et 2 O:pyridine (15:15:5:1) and adding KOH to 0.1 M followed by incubation at 37°C for 3 h (21). After neutralizing with 1 M AcOH, the sample was dried, BuOH-desalted (20), and dried again. Ceramides were analyzed by TLC on silica gel plates using CHCl 3 : MeOH:AcOH (95:4.5:0.5) as the developing solvent (22). Plates were sprayed with 10% copper sulfate in 8% orthophosphoric acid and heated for 20 min at 180°C to char the ceramides (22). The arsenite and borate treated silica gel plates were supplied by Analtech (Newark, DE).
Isolation of Ceramides by Preparative Silica Gel TLC-Ceramides were purified and separated by TLC as described above. The ceramides were visualized by ultraviolet light after spraying the plates with 0.01% 8-anilino-1-napthalenesulfonic acid. The silica gel was scraped off the plate, and the ceramides were eluted by repeated sonication (five times for 10 min each) in 2 ml of CHCl 3 :MeOH (1:1).
Isolation of Sphingolipids by Preparative Silica Gel TLC-Sphingolipids were extracted from 600 A 600 units of cells by vortexing with glass beads in 100 ml of CHCl 3 :MeOH (1:1). The extract was dried, alkalitreated, and BuOH-desalted as described previously (5,19,20). The sample was spotted in a line on a silica gel plate and developed using Hydroxyl groups on C-1 and C-3 are found on all LCBs. Sites labeled I through III are potentially hydroxylated. Site I is on C-4 of the LCB, site II is on C-2 of the VLCFA, and site III is also on the VLCFA, but the position has not been determined. Five species (A, B, BЈ, C, and D) of ceramides, IPCs, or MIPCs, which differ according to which of the three sites are hydroxylated, are synthesized. In this report, it is shown that hydroxylation of site I and site II requires Sur2p and Scs7p, respectively. Ceramide is converted to IPC by IPC synthase (reaction 1), and IPC is converted to MIPC by mannosylation (reaction 2). CHCl 3 :MeOH:AcOH (95:4.5:0.5). In this system, fatty acids and ceramides migrate while sphingolipids remain at the origin. The material left at the origin was subjected to acid methanolysis for analysis of the fatty acid methyl esters (FAMEs) and LCBs as described below.
Acid Methanolysis of Ceramides and Sphingolipids and Isolation of FAMEs and LCBs-Ceramides and sphingolipids were purified by silica gel TLC as described above. The purified ceramides or sphingolipids were subjected to acid methanolysis by resuspending in 2 ml of HCl: MeOH:H 2 O (3:29:4) and incubating at 78°C for 18 h (23). The FAMEs were recovered by extracting 3 times with 2 ml of hexane (24). The extracts were pooled, dried, and subjected to TLC using petroleum ether:Et 2 O (17:3) as the developing solvent (24). Plates were sprayed with 10% copper sulfate in 8% orthophosphoric acid and heated for 20 min at 180°C to char the FAMEs.
The LCBs were recovered from the hydrolyzed ceramides or sphingolipids by adjusting the pH of the acid hydrolysate (after extraction of FAMEs) to 11.5 using 1 M NaOH and extracting three times with 2 ml of Et 2 O (24). The pooled extracts were dried, and the LCBs were separated by silica gel TLC using CHCl 3 , MeOH, 2.5 M NH 4 OH (40:10:1) as developing solvent (24). Plates were sprayed with 0.2% ninhydrin in ethanol and incubated at 100°C for 5-10 min to visualize the aminecontaining LCBs.

Sphingolipid Synthesis Is Altered in sur2⌬ Mutant Cells-
The main purpose of this study was to determine if SUR2 encodes the enzyme that hydroxylates C-4 of the sphingoid moiety of sphingolipids. The sur2⌬ mutant was constructed as described under "Experimental Procedures." Sphingolipid synthesis in a sur2⌬ mutant was compared with that of wild-type. Cells were grown for several generations in synthetic minimal medium containing 12 nM inositol with 1 Ci/ml [ 3 H]inositol. Plates were sprayed with 10% copper sulfate in 8% orthophosphoric acid and heated for 20 min at 180°C to char the FAMEs. Ten g of hydroxylated C18 and C24 FAMEs (lanes 5 and 6) and nonhydroxylated C18 and C24 FAMEs (lanes 7 and 8) standards (Sigma) were spotted. The position of the NVLCFAME and HVLCFAME are labeled in the right margin. The spots that migrate just below the NVLCFAMEs as well as those migrating below the HVLCFAMEs are artifacts generated (even in the absence of added lipid) from the acid methanolysis. After extraction of the FAMEs, the pH of the remaining solution was adjusted to 11.5 with NaOH, and the LCBs were extracted with Et 2 O and analyzed by TLC (panel A). The LCBs were visualized by spraying with 0.2% ninhydrin in ethanol and heating at 100°C for 5-10 min. Ten g of phytosphingosine (PS), dihydrosphingosine (DS), and sphingosine (S) standards (Sigma) were spotted. from sur2⌬csg2⌬ mutants (IPC-A and IPC-BЈ) was determined. Polar lipids were extracted and alkali-treated to hydrolyze the glycerol-based phospholipids, and the sphingolipids were isolated by preparative TLC as described under "Experimental Procedures." The sphingolipids were subjected to acid methanolysis to hydrolyze both the phosphodiester bond between the inositol and the ceramide, and the amide bond between the sphingoid moiety and the VLCFA. The LCB was extracted and analyzed by TLC (Fig. 3A). The LCB in the sphingolipids from the sur2⌬ mutant cells (lanes 3 and 5) is exclusively dihydrosphingosine, while the sphingolipids from cells with a wildtype SUR2 gene (lanes 1, 2, and 4) contain primarily phytosphingosine.
The FAMEs released by methanolysis were also analyzed using a TLC system which resolves unhydroxylated very long chain fatty acid methyl esters (NVLCFAME) and hydroxylated very long chain fatty acid methyl esters (HVLCFAME). The sphingolipids from sur2⌬csg2⌬ mutant cells (lane 3) contain both hydroxylated and unhydroxylated fatty acids (Fig. 3B). Because sur2⌬csg2⌬ mutant cells synthesize two sphingolipids, IPC-A and IPC-BЈ, these results indicate that IPC-A contains unhydroxylated fatty acids, whereas IPC-BЈ contains hydroxylated fatty acids.
In a sur2⌬scs7⌬csg2⌬ triple mutant where hydroxylation of C-4 of the LCB is blocked by deletion of SUR2, hydroxylation of the VLCFA is blocked by deletion of SCS7, and mannosylation is blocked by deletion of CSG2, the only sphingolipid synthesized is the very hydrophobic IPC-A (Fig. 2, lane 8, A). The IPC-A species can be mannosylated if Csg2p is present (Fig. 2,  lane 5, MA). The sur2⌬ mutants accumulate some IPC-A or MIPC-A even when Scs7p is present (Fig. 2, lanes 3 and 6, MA  and A) suggesting that phytosphingosine-containing substrates are preferred by Scs7p over dihydrosphingosine-containing substrates. These data (summarized in the model shown in Fig. 1) support the proposal that Sur2p is the hydroxylase that converts dihydroceramide to phytoceramide.
The Sphingolipid Synthesis Defect Conferred by Deletion of sur2 Is Corrected by Exogenous Phytosphingosine but Not Dihydrosphingosine-S. cerevisiae cells can incorporate exogenous phytosphingosine into sphingolipids (25). As would be predicted if Sur2p is required for hydroxylation of C-4 on the LCB, exogenous phytosphingosine, but not dihydrosphingosine, restores synthesis of IPC-C to a sur2⌬csg2⌬ mutant (Fig. 4, lanes 8 and 9). A sur2⌬scs7⌬csg2⌬ triple mutant, that normally makes IPC-A, makes IPC-B in the presence of phytosphingosine (data not shown). These observations provide further evidence that the altered sphingolipids that accumulate in the sur2⌬csg2⌬ and sur2⌬csg2⌬scs7⌬ mutants (IPC-BЈ and IPC-A, respectively) differ from the sphingolipids in csg2⌬ and csg2⌬scs7⌬ mutants (IPC-C and IPC-B, respectively) in the LCB. Addition of phytosphingosine to the scs7⌬csg2⌬ mutant does not result in any IPC-C synthesis (Fig. 4, lane 11), because the IPC-B that accumulates in the scs7⌬ mutant arises from failure to hydroxylate the VLCFA (6).
Deleting the SUR2 or SCS7 Gene Reduces Hydroxylation of Ceramides-The IPCs are synthesized by the transfer of phosphoinositol from phosphatidylinositol to ceramide. Comparison of ceramides isolated from the sur2⌬csg2⌬, scs7⌬csg2⌬ and the sur2⌬scs7⌬csg2⌬ mutants with ceramides from wild-type or csg2⌬ mutant cells demonstrates that the altered mobility of the sphingolipids arises from differences in the ceramide moiety of the sphingolipids. Yeast ceramides were analyzed by TLC (Fig. 5). The predominant ceramide in wild-type and in csg2⌬ mutant cells (lanes 3 and 4), labeled "C" (for C-ceramide) because it is the ceramide of IPC-C, migrates slower in this TLC system than the bovine hydroxylated ceramide standard (Sigma type IV, lane 2) which consists of sphingosine and a hydroxylated fatty acid. Analysis of the LCB and VLCFA of the C-ceramide by TLC following methanolysis confirms that it contains phytosphingosine (Fig. 6A, lanes 5 and 6) and a HV-LCFA (Fig. 6B, lanes 6 and 7) (1, 26). The slower mobility of yeast ceramide compared with hydroxylated bovine ceramides  8 -12). The hydroxylation states of the ceramide species are shown in Fig. 1. is expected, since it contains an additional hydroxyl group (phytosphingosine versus sphingosine).
The major ceramide from sur2⌬ mutant cells (BЈ-ceramide) has a mobility similar to the hydroxylated bovine ceramide standard. Ceramides having dihydrosphingosine might be expected to have similar hydrophobicity to those having sphingosine. The LCB from the BЈ-ceramide is dihydrosphingosine (Fig. 6A, lane 7) and the VLCFA is hydroxylated (Fig. 6B,  lane 8).
The B-ceramide that accumulates in the scs7⌬ mutant cells contains phytosphingosine (Fig. 6A, lane 8) and unhydroxylated VLCFA (Fig. 6B, lane 9). The mobility of the ceramide in scs7⌬ mutant cells is quite distinct from that in sur2⌬ mutant cells (Fig. 5, lanes 5 and 6). Either hydroxylation of the VLCFA increases the hydrophilicity of the ceramide less than does the hydroxylation of the LCB, or these species interact differently with the silica gel matrix.
The A-ceramide that is present in the sur2⌬scs7⌬ double mutant (Fig. 5, lane 7) migrates with the unhydroxylated bovine ceramide standards as would be expected if it lacks hydroxyl groups on both C4 of the LCB and on the VLCFA (Fig. 5,  lanes 1 and 7). The LCB of the A-ceramide is dihydrosphingosine (Fig. 6A, lane 9) and the VLCFA is unhydroxylated (Fig.  6B, lane 10). The absence of vicinal hydroxyl groups (C-3 and C-4 of phytosphingosine) on ceramide from sur2⌬ mutants is also indicated by the effect of the glycol-complexing ions arsenite and borate on the chromatographic behavior of the BЈ-and A-ceramides (Fig. 7). The complex between vicinal hydroxyl groups with arsenite increases their mobility on silica gel, while the borate complex decreases their mobility (27,28). The mobility of C-ceramide (from wild-type and csg2⌬ mutant cells) and B-ceramide (from csg2⌬scs7⌬ mutant cells) is greatly increased by addition of NaAsO 2 to the silica gel (compare Fig. 7,  B to A, lanes 1, 2, and 4) and reduced by addition of Na 2 B 4 O 7 (Fig. 7C, lanes 1, 2, and 4), indicating that these ceramides contain the C-3,4 vicinal hydroxyl groups of phytoceramide. The mobility of the BЈ-ceramide (from csg2⌬sur2⌬ mutant cells) and A-ceramide (from csg2⌬sur2⌬scs7⌬ mutant cells) is much less affected by arsenite or borate, consistent with the conclusion that they have dihydrosphingosine instead of phytosphingosine as the LCB.
Deletion of the SUR2 Gene Suppresses Ca 2ϩ Sensitivity of csg2 Mutants-Cells lacking the CSG2 gene are defective in mannosylation of inositolphosphorylceramides and therefore accumulate the inositolphosphorylceramide, IPC-C (Fig. 2, lane 2) (5,19). Overaccumulation of IPC-C or a related metabolite confers Ca 2ϩ -sensitivity. Mutations in a variety of genes required for the synthesis of IPC-C suppress the Ca 2ϩ -sensitive phenotype of the csg2 mutants. For example, deletion of SCS7, which encodes the enzyme that hydroxylates the VLCFA suppresses the Ca 2ϩ sensitivity of the csg2 mutant (Fig. 8) (6, 19). Therefore, the effect of deletion of SUR2 on the Ca 2ϩ sensitivity of the csg2⌬ mutant was investigated. As shown in Fig. 8, deletion of the SUR2 gene reverses the Ca 2ϩ sensitivity of a csg2 mutant.

SUR2 Is Required for the Hydroxylation of C-4 of the LCB and SCS7 Is Required for Hydroxylation of the VLCFA of Ceramides-
The effect of deleting SUR2 on the hydroxylation of C-4 on the LCB of ceramide and sphingolipid and the sequence similarity between Sur2p and a family of desaturases/hydroxylases indicate that Sur2p catalyzes the hydroxylation of C-4. The LCB of ceramides and sphingolipids in sur2⌬ mutants is dihydrosphingosine instead of the phytosphingosine predominantly found in wild-type cells. Exogenous phytosphingosine restores synthesis of sphingolipids with a phytosphingosine LCB in sur2⌬ mutants.
The substrate (dihydrosphingosine or dihydroceramide) for Sur2p has not been identified. Since hydroxylation of C-4 is not required for ceramide or sphingolipid synthesis, either dihydrosphingosine or phytosphingosine can serve as substrate for ceramide synthase, and either dihydroceramide or phytoceramide can serve as substrate for IPC synthase. S. cerevisiae cells contain both dihydrosphingosine and phytosphingosine, and inhibition of ceramide synthase by fumonisin B 1 causes the accumulation of both LCBs (29). However, it is not known whether phytosphingosine comes from de novo synthesis or from turnover of ceramide and sphingolipid.
Scs7p is also a member of this family of hydroxylases/desaturases, but it is responsible for hydroxylation of the VLCFA rather than the LCB. Failure to hydroxylate C4 of the LCB FIG. 6. The LCBs and the FAMEs from A-, B-, B-, and C-ceramides were analyzed by silica gel TLC. Ceramides purified from 100 A 600 units of cells (see Fig. 5) were subjected to acid methanolysis, and FAMEs and LCBs were extracted for analysis as described in Fig. 3. A, the LCBs were separated by silica gel TLC. Standards were the LCB derived from acid methanolysis of 4 g of Sigma type III bovine ceramide (lane 1) and 10 g of sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (PS). The LCBs were visualized by spraying with 0.2% ninhydrin in ethanol and heating at 100°C for 5-10 min. B, the FAMEs were also separated by TLC. Standards were the FAMEs derived from acid methanolysis of Sigma type III and type IV bovine ceramides (4 g) (lanes 1 and 2), and 10 g of C18 FAME, C24 FAME, and hydroxylated C24 FAME (lanes [3][4][5]. The plate was sprayed with 10% copper sulfate in 8% orthophosphoric acid and heated for 20 min at 180°C to char the FAMEs. decreases the Scs7p-catalyzed hydroxylation of the VLCFA, indicating hydroxylation occurs subsequent to ceramide formation. Furthermore, the ceramides from SCS7 ϩ cells are hydroxylated, while those from scs7⌬ mutant cells are not suggesting that the substrate for Scs7p is ceramide. However, it is not yet known whether most of the free ceramides in the cell arise from de novo synthesis or from turnover of sphingolipid, so it remains to be determined whether the substrate for Scs7p is free ceramide or inositolphosphorylceramide. Martin and co-workers (16) recently reported that cells lacking the elongase encoded by ELO3/SUR4 accumulate relatively high levels of hydroxylated C16 fatty acids. We have found that elo3 mutant cells incorporate fatty acids with shorter than normal chain lengths into ceramide. 2 Therefore, it will be interesting to determine whether the hydroxylated C16 fatty acids in the mutants arise from Scs7p-catalyzed hydroxylation of the (shorter than normal) fatty acids on the ceramide.
Sur2p and Scs7p Are Members of a Family of Cytochrome b 5 -dependent Enzymes Located in the Endoplasmic Reticulum-Ceramide and IPC-C are synthesized in the endoplasmic reticulum (4) which appears to be the location of Scs7p and Sur2p as well. Both Scs7p and Sur2p contain C-terminal sequences (KMKYE and VKKEK), matching a consensus sequence specifying retention in the endoplasmic reticulum (6,30). In S. cerevisiae, all five proteins that are members of the oxo-diiron family appear to reside in the endoplasmic reticulum. Along with Sur2p and Scs7p, these are ␦-9 fatty acid desaturase (Ole1p), C-4 sterol methyl oxidase (Erg25p), and C-5 sterol desaturase (Erg3p). The oxo-diiron centers in these enzymes are believed to receive electrons from either cytochrome b 5 or a cytochrome b 5 -like domain. Scs7p and Ole1p contain cytochrome b 5 -like domains at their N and C termini respectively (6,31). Cytochrome b 5 may function to transfer electrons to Sur2p and the other two enzymes. Cytochrome b 5 reductase may catalyze the reduction of both cytochrome b 5 and the cytochrome b 5 lanes 1 and 2)-, B (lane 3)-, B (lane  4)-, and A (lane 5)-ceramides. The isolated ceramides used in the experiment described in Fig. 6 were analyzed by TLC on silica gel plates without (panel A) or with either 1% sodium meta arsenite (panel B) or FIG. 8. Suppression of the Ca 2؉ -sensitive phenotype of csg2⌬ mutants by deletion of SUR2 or SCS7. Cells were grown in synthetic medium to an OD 600 of 0.1 and serially diluted (left to right, 1:5) into the wells of a microtiter plate. Cells were transferred by metal prong to SD agar plates without (ϪCa) or with an additional 50 mM CaCl 2 (ϩCa). The plates were incubated at 26°C for three days.
SUR2. Other mutations in sphingolipid biosynthetic genes (subunits of serine palmitoyltransferase, LCB1, SCS1/LCB2; ceramide hydroxylase, SCS7; fatty acid elongases, ELO2/ SUR5/FEN1, ELO3/SUR4; and fatty acid synthetase, FAS2) also suppress the Ca 2ϩ sensitivity of csg mutants. These mutations either decrease the rate of sphingolipid synthesis or alter the sphingolipids that are synthesized. The CSG2 and CSG1 genes are required for mannosylation of IPC to form MIPC. In the absence of mannosylation, IPC-C overaccumulation is observed. It appears that decreasing the accumulation of IPC-C or a related metabolite or altering its structure (to IPC-B, IPC-BЈ, or IPC-A?) reverses the Ca 2ϩ sensitivity. It is hoped that continued analysis of suppressor mutants will identify more genes that function in sphingolipid synthesis and identify the Ca 2ϩ target that triggers cell death.
The genetic relationship between suppressors of the csg2⌬ mutant and suppressors of the rvs161 mutant suggest a role for sphingolipid in some Rvs161p-dependent process. Three genes (SUR1, SUR2, and SUR4) (12) that mutate to suppress rvs161 mutants (10) are related to CSG1 and CSG2 or to genes that mutate to suppress the Ca 2ϩ -sensitive phenotype of csg1⌬ and csg2⌬ mutants. The sur1, sur2, and sur4 mutants have altered phospholipid compositions and abnormal morphologies in stationary phase (10). SUR1, which is allelic to CSG1 (5), is a high copy suppressor of csg2⌬ mutants (5,32). Both SUR1/CSG1 and CSG2 are required for mannosylation of IPC (5,19). SUR2 encodes the enzyme that hydroxylates C-4 of dihydroceramide. SUR4/ELO3 and SUR5/ELO2/FEN1 encode fatty acid elongases required for the synthesis of the C26 fatty acids found in ceramide and sphingolipids (16).
S. cerevisiae Cells Do Not Require Sur2p-or Scs7p-mediated Hydroxylation for Growth or Synthesis of Mature Sphingolipid-The physiological function of Sur2p-and Scs7p-mediated hydroxylation is not known. Growth of S. cerevisiae cells does not depend on hydroxylation of either the C-4 of the LCB or the VLCFA moieties of ceramides and sphingolipids. Cells lacking serine palmitoyltransferase can utilize exogenous dihydrosphingosine or phytosphingosine but not sphingosine, the main long chain base in mammals (25). Since sur2⌬ mutants do not synthesize phytosphingosine, it is not the lack of a C-4 hydroxyl group that precludes sphingosine from substituting as the LCB in ceramide synthesis.
Deletion of SUR2 greatly increases the resistance of cells to the Pseudomonas syringae cyclic lipodepsipeptide syringomycin (33) and to the morpholine fungicide fenpropimorph, 2 an inhibitor of several enzymes in the ergosterol synthesis pathway. As discussed above, deletion of SUR2 also suppresses the Ca 2ϩ -sensitive phenotype of csg2⌬ mutants and the pleiotropic effects of rvs161 mutations. Elucidation of the mechanism by which blocking hydroxylation of the LCB C-4 increases the ability of cells to tolerate syringomycin, fenpropimorph, and high Ca 2ϩ concentrations after CSG2 deletion, and RVS161 deletion may provide clues as to how C-4 hydroxylation affects the functional properties of the LCB, ceramide, and sphingolipids. In addition, the sur2⌬ mutant can be used to identify genetically related genes that encode proteins whose functional properties are effected by C-4 hydroxylation.