Curr Genet (1998) 34: 93–99

© Springer-Verlag 1998

O R I G I N A L PA P E R

James H. Crowley · Shirley Tove · Leo W. Parks

A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae

Received: 19 February / 25 May 1998

Abstract ERG24 is the structural gene for the C14-sterol reductase in yeast. A lack of activity in that enzyme, mediated either by the morpholine fungicides or the insertional inactivation of ERG24, causes the accumulation of the aberrant sterol ignosterol. Cells producing this sterol are unable to grow aerobically in the routine laboratory medium, YPD. However, growth does occur on a synthetic defined medium. A novel calcium-dependent phenotype associated with alterations in the ergosterol biosynthetic pathway in yeast is described. In addition, reduction of yeast growth with an azole inhibitor of the C-14 sterol demethylase was also modulated by an excess of calcium ions in the culture medium. These results define a new effect of ergosterol deficiency and provide important practical implications for utilizing morpholine and azole sterol biosynthetic-inhibiting fungicides. Key words Ergosterol · Antifungal · Calcium · Fenpropimorph · Azole · ERG24 gene

Introduction

Ergosterol is the preferred sterol of the yeast Saccharomyces cerevisiae. Mutations in genes encoding for ergosterol biosynthetic enzymes often have pleiotrophic phenotypes, many of which may result from improper membrane

J. H. Crowley 1 · S. Tove · L. W. Parks (½) Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA Tel.: +1-919-5157860 Fax: +1-919-5157867 e-mail: [email protected] Present address: 1 Nutrasweet-Kelco Co., 8355 Aero Drive, San Diego, CA 92123-1718, USA Communicated by C. P. Hollenberg

function due to the use of aberrant sterols by the mutant yeast cells. Although mutations in early ergosterol biosynthetic genes result in ergosterol auxotrophy (Chambon et al. 1991), mutations in succeeding ERG genes are resistant to nystatin and super-sensitive to cycloheximide. Nystatin binds specifically to ergosterol, causing membrane damage (Norman et al. 1976). Defective synthesis of ergosterol allows resistance to nystatin due to the production of non-ergosterol sterols that bind inefficiently to nystatin (Parks et al. 1985). Sensitivity to cycloheximide may result from increased permeability of erg mutants to this protein-synthesis inhibitor. Mutations in the ERG6 gene (C-24 sterol methyltransferase) result in defects in mating, tryptophan uptake, and permeability to Na+ and Li+ (Gaber et al. 1989; Welihinda et al. 1994). In addition to hypersensitivity to Ca+2 (Taguchi et al. 1994), mutations in the ERG3 gene (C-5 sterol de-saturase) result in defects in the utilization of non-fermentable carbon sources (Smith and Parks 1993) and resistance to environmental stresses such as ultraviolet light and osmotic stress (Hemmi et al. 1995). Although certain phenotypes of strains containing mutations in ergosterol biosynthesis are associated with a particular erg mutation, such as Na+ and Li+ sensitivity in an erg6 mutant, most of these phenotypes can also be found associated with other erg mutations. Thus, an alteration in ergosterol biosynthesis causes a number of effects on the metabolism of yeast; however, the particular ERG gene mutated may dictate the extent of the phenotype resulting from growth on a certain sterols. Treatment of yeast with syringomycin results in an increase in the influx of Ca+2 into the cell associated with membrane potential (Takemoto et al. 1991). This is likely to occur through a membrane disturbance caused by the formation of syringomycin/ergosterol complexes. A syringomycin-resistant yeast strain, R4-3G, was isolated and found to have an increase in Ca+2 influx into the cell. This strain was shown to have a mutation in the ERG3 gene, which conferred sensitivity to high calcium levels (Taguchi et al. 1994). Taken together, these results suggest a defect in the maintenance of normal levels of calcium in cells of a strain without ergosterol as the major sterol.

94 Table 1 Yeast strains used in this study Strain

Genotypea

Sourceb

JC530 JC482 LPY7 SY114 CJ153

a, leu2, ura3, his4 α, leu2, ura3, his4 a, leu2, ura3, his3 a, ura3, his3, trp1 a/α erg24::LEU2/ERG24 leu2/leu2 ura3/ura3 his4/his4 a, erg3::LEU2, leu2, ura3, his4

K.T. K.T. K.T. This laboratory

This study Smith and Parks 1993 SY13 a, erg3::LEU2, leu2, ura3, his3, trp1 This laboratory This work CJ505 a/α, erg11::URA3/ERG11, erg3::LEU2/ERG3, leu2/leu2, ura3/ura3, his4/his4 CJ515 a, erg11::URA3, erg3::LEU2, This work leu2, ura3, his4 Gaber et al. BKY48-5c α, erg6::LEU2, leu2, ura3 1989 LPY11 a, erg6::LEU2, leu2, ura3, his3 This laboratory LPY30 a, erg5::LEU2, leu2, ura3, his3 This laboratory SCV-1 a, erg24::LEU2, leu2, ura3, his4 This laboratory SY32A

a ERG3, C-5 sterol de-saturase; ERG5, C-22 sterol de-saturase; ERG6, C-24 sterol methyltransferase; ERG11, C-14 sterol de-methylase; ERG24, C-14 sterol reductase b K.T., Kelly Tatchell, Louisiana State University, Shreveport, Louisiana

The structural gene for the C-14 sterol reductase, ERG24, was cloned and sequenced in our laboratory (Lorenz and Parks 1992). The sterol reductase is a target for the morpholine antifungal agents and 15-azasterol (Bottema and Parks 1978; Baloch and Mercer 1987; Lorenz and Parks 1991). The insertional inactivation of ERG24 causes the accumulation of the abnormal sterol, ergosta-8,14-dien-3β-ol (ignosterol), as the primary sterol product. That ERG24 encodes the C-14 sterol reductase has been verified, and the essentiality of that reaction in yeast cells that are growing aerobically in the rich laboratory medium, YPD, has been demonstrated (Marcireau et al. 1992; Lai et al. 1994). This confirms that ignosterol is unsuitable for yeast growing under those conditions. An enormous selective advantage was demonstrated for wildtype yeast over an isogenic strain with an insertional inactivation of the ERG24 gene (Palermo et al. 1997). We describe here that the aerobic growth defect on YPD caused by the ERG24 null mutation can be alleviated by the addition of calcium or magnesium ions. Furthermore, we show that the efficacy of some antimycotic inhibitors of sterol biosynthesis is dependent on the external levels of calcium in the growth medium.

Materials and methods Yeast strains and growth conditions. The yeast strains used in this study are listed in Table 1. Strain CJ505 was isolated by transformation of a diploid cross between JC482 and SY32A with a 2.5-kb BamHI-HindIII fragment from p2500H (Kalb et al. 1987), which contains the erg11::URA3 construct. CJ515 (erg11::URA3, erg3::LEU2) was a Leu+, Ura+ strain isolated from the sporulation of CJ505. Analysis of sterols extracted from CJ515 confirmed the

deletion of the ERG11 gene. LPY11 was isolated from a cross between LPY7 and BKY48–5c (erg6::LEU2). Strains were grown in either a synthetic complete medium (Crowley et al. 1996) or YPD (Sherman 1991). Chloride salts of monovalent and divalent cations were added to media after autoclaving from sterile, concentrated stock solutions (above 1.0 M). Where indicated, EGTA [ethylene glycol bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid] was added to media after autoclaving from a sterile 1.0 M solution (pH 7.0), and trifluoperazine was added from a filter-sterilized 0.5 M stock in water. For studies with sterol biosynthetic inhibitors, fenpropimorph and ketoconazole were added from stock concentrations of 10 mg/ml in 95% ethanol (kept at –20°C). All cultures were grown at 28°C; growth was measured with a Klett-Summerson colorimeter equipped with a green filter. Preparation and analysis of sterols. Cultures were grown overnight in YPD and inoculated into 20 ml of fresh YPD with or without sterol-biosynthesis inhibitors. The cultures were allowed to grow at 28°C for 60 h, growth was measured, and the cells were harvested by centrifugation, washed in distilled water, and lyophilized. The dried cell pellets were weighed and total sterols were liberated by saponification and extracted with hexane according to the method of Parks et al. (1985). Sterols were separated by gas chromatography (isothermal) as described by Fenner and Parks (1989), and the percentages of sterols produced were determined based on the peak area. Materials. Dextrose, YNB (yeast nitrogen base without amino acids), and extraction solvents were from Fisher Scientific. Amino acids, nucleotide bases, EGTA, trifluoperazine, and ketoconazole were from Sigma Chemical Co. Fenpropimorph was a gift from BASF (Ludwigshafen, Germany).

Results

Previously, we found that a null mutant of ERG24 was able to grow on defined medium but not on a rich medium (e.g., YPD; Crowley et al. 1996). Supplementation of YPD with the components of synthetic complete (SC) medium allowed for some degree of rescue of the growth inhibition on YPD. These results led us to believe that some component of the SC medium was critical for allowing the ERG24 null mutant to grow. We then began a search for this critical compound(s) by adding the separate ingredients of SC medium to YPD, and haploid spores of CJ153 were then germinated under these different conditions. None of the vitamins, trace elements, ammonium sulfate, or succinate buffer, rescued growth; however, the salt solution did. In analyzing the salt solution further, it was found that calcium and magnesium salts were able to allow growth of the ERG24 null mutant on YPD. Studies in broth culture confirmed that 1.0 mM calcium could effectively relieve YPD growth inhibition, although 10 mM was the optimal concentration (Fig. 1). Magnesium ions also allowed the ERG24 null mutant to grow on YPD; however, a higher concentration of this divalent cation was required for optimal growth. Interestingly, concentrations of calcium higher than 20 mM were not effective in preventing growth inhibition on YPD. The profound effect of calcium on the growth of an ERG24 null mutant, which produces a ∆8,14 sterol (i.e., ignosterol) as its primary sterol, led us to speculate that calcium may also affect the sensitivity of S. cerevisiae to the antifungal agent and C-14 reductase inhibitor, fenpropi-

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Fig. 1 Rescue of an ERG24 null mutant with Ca+2 or Mg+2. S. cerevisiae strain SCV-1 (erg24::LEU2) was grown overnight in synthetic complete medium and inoculated into fresh YPD medium at 2.5×105 cells/ml with increasing concentrations of either CaCl2 or MgCl2, and growth was measured after 24 h

Fig. 2A, B Effects of calcium and the calcium chelator, EGTA, on the sensitivity of S. cerevisiae to fenpropimorph. S. cerevisiae strain JC530 was grown in YPD overnight and inoculated into fresh medium at 2.5×105 cells/ml with increasing concentrations of fenpropimorph. The effects of 10 mM CaCl2 (A) or 10 mM EGTA (B) were determined, and cultures were measured after 48 h. Error bars represent triplicate samples

Fig. 3 Effects of monovalent and divalent cations on the sensitivity of S. cerevisiae to fenpropimorph. S. cerevisiae strain JC530 was grown overnight in YPD and inoculated into fresh medium at 2.5×105 cells/ml with the addition of 2.5 µg/ml of fenpropimorph. Growth was measured after 48-h incubation at 30°C

morph. Calcium was found to be very effective in increasing the resistance of S. cerevisiae to fenpropimorph (Fig. 2A). The addition of 10 mM CaCl2 to YPD allowed S. cerevisiae strain JC530 to grow in concentrations of fenpropimorph up to 10 µg/ml, whereas without additional calcium growth was reduced in as little as 0.5 µg/ml of fenpropimorph. One measure of the specificity of calcium in affecting sensitivity to fenpropimorph is to determine the effect of the calcium-specific chelator, EGTA, on sensitivity to fenpropimorph. The addition of 10 mM of EGTA to fenpropimorph-treated S. cerevisiae resulted in a substantial increase in sensitivity of JC530 to fenpropimorph (Fig. 2B). EGTA treatment increased the sensitivity of JC530 to fenpropimorph by a factor of ten. Implicit in these results is the importance of calcium in strains producing ∆8,14 sterols. To examine cation specificity in sensitivity to fenpropimorph further, a variety of chloride salts of different cations were tested for their ability to affect the resistance of JC530 to 2.5 µg/ml of fenpropimorph (Fig. 3). Neither of the monovalent cations, sodium or potassium, were able to increase resistance. In addition, lithium does not affect resistance (data not shown). The inability of zinc to rescue growth inhibition in fenpropimorph-treated cells shows that only certain divalent cations function in allowing cells to overcome some of the effects of fenpropimorph inhibition. We were next interested in determining if the ability of calcium to increase resistance to fenpropimorph may re-

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Fig. 4A, B Effects of calcium and the calcium chelator, EGTA, on the sensitivity of S. cerevisiae to ketoconazole. S. cerevisiae strain JC530 was grown in YPD overnight and inoculated into fresh medium at 2.5×105 cells/ml with increasing concentrations of ketoconazole. The effects of 10 mM CaCl2 (A) or 10 mM EGTA (B) were determined, and cultures were measured after 48 or 24 h, respectively. Error bars represent triplicate samples

Fig. 5A, B Effects of calcium on fenpropimorph-mediated sterol inhibition in S. cerevisiae. Growth was measured after 60 h (A), and cultures were harvested for sterol analysis (B) as described in Materials and methods. In both panels, closed circles represent cultures grown without additional CaCl2, and open circles represent cultures grown with 10 mM of CaCl2 . 14-MF refers to 14α-methyl fecosterol and Ign refers to ignosterol. These two sterols appear as a doublet in GC analysis, and were grouped together this analysis. The results shown here are from a single experiment, and repeated experiments gave similar results

flect a capacity to affect resistance to other sterol biosynthetic inhibitors. Figure 4 shows the ten-fold increase in resistance to the C-14 de-methylase inhibitor, ketoconazole, that results from the addition of 10 mM of calcium to the growth medium. In addition, as was seen for fenpropimorph, the sensitivity of S. cerevisiae strain JC530 to ketoconazole was increased substantially by the addition of the calcium chelator EGTA (Fig. 4). One explanation for the property of calcium in affecting the sensitivity of yeast to sterol biosynthetic inhibitors is the possibility that this cation may somehow affect the action of the drugs on inhibiting sterol synthesis. To address this possibility, we determined the effects of calcium on the ability of fenpropimorph and ketoconazole to alter sterol synthesis. A wild-type S. cerevisiae strain was treated with increasing concentrations of fenpropimorph or ketoconazole, with or without the addition of 10 mM of calcium. Although calcium supplementation resulted in increased resistance to fenpropimorph (Fig. 5A), calcium

had little effect on the ability of fenpropimorph to alter sterol metabolism in yeast (Fig. 5B). One may expect that, if calcium interfered with the action of the inhibitor, the decrease in ergosterol and concomitant increase in aberrant sterols would be diminished with calcium treatment. This does not appear to be the case. Similar results were seen with ketoconazole in that calcium caused little change in the normal interference of ketoconazole in the ergosterol biosynthetic pathway (Fig. 6). In ketoconazole treatment, lanosterol accumulates at the expense of ergosterol depletion. An interesting observation in ketoconazoletreated cultures was that the presumed 14 α-methyl-ergosta-8,24(28)-diene-3,6-diol that has been shown to accumulate in S. cerevisiae treated with fluconazole (Watson et al. 1989) only appeared in the cultures treated with higher concentrations of ketoconazole (5 µg/ml and above). At these concentrations, cultures that were not supplemented with calcium had severely diminished growth (Fig. 6A). Thus, the addition of calcium even allowed for

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Fig. 6A, B Effects of calcium on ketoconazole-mediated sterol inhibition in S. cerevisiae. Growth was measured after 60 h (A), and cultures were harvested for sterol analysis (B) as described in Material and methods. In both panels, closed circles represent cultures grown without additional CaCl2, and open circles represent cultures grown with 10 mM of CaCl2. The diol in panel B refers to 14α-methyl-8,24-dien-3β,6α-diol. The results shown here are from a single experiment, and repeated experiments gave similar results

a significant accumulation of a sterol thought to be toxic to the growth of S. cerevisiae. The observations that the dependency of yeast altered in ergosterol metabolism on external calcium concentration suggested a relationship between ergosterol and calcium homeostasis. To explore this further, we determined the sensitivity of a variety of ERG mutants to the calciumbinding protein antagonist, trifluoperazine. We reasoned that if a change in ergosterol metabolism within the cell resulted in an inability to maintain a proper intracellular calcium concentration, then these cells would be supersensitive to a drug that interferes with proteins dependent on intracellular calcium levels. In fact, several ERG mutants are super-sensitive to trifluoperazine (Fig. 7). As expected, the erg24 null mutant was particularly sensitive to trifluoperazine. An erg11, erg3 double mutant, which mimics ketoconazole treatment in much the same way as an erg24 mutant mimics fenpropimorph treatment, is also quite sensitive to trifluoperazine. It was evident that the type of ERG mutation governed sensitivity to trifluoperazine. While an erg6 mutant showed intermediate sensitiv-

Fig. 7 Sensitivity of S. cerevisiae ERG null mutants to trifluoperazine. Cultures were grown overnight in synthetic complete medium and inoculated into fresh medium at 2.5×105 cells/ml with increasing concentrations of trifluoperazine. Cultures were allowed to incubate for 24 h, then growth was measured

ity, the erg3 and erg5 mutants were not any more sensitive than a wild-type strain producing ergosterol as the major sterol.

Discussion

We began this study in order to determine why a strain containing a mutation in the ergosterol biosynthetic pathway was not able to grow on the rich, routine laboratory medium, YPD. Strains containing a mutation in the ERG24 gene (C-14 sterol reductase) have a unique phenotype among erg mutants in that erg24 mutants cannot grow aerobically on YPD; however, they can grow on a synthetic medium (Crowley et al. 1996). During the course of our study, not only did we discover why erg24 mutants would not grow on YPD, we also found that the efficacy of antimycotic sterol biosynthetic inhibitors is dependent on external levels of calcium ions. A search for compounds in synthetic media that would rescue erg24 mutants on YPD revealed that strains containing an ERG24 null mutation had an enhanced calcium-

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requiring phenotype. This phenotype was remediable with higher levels of magnesium ions as well. It should be noted that YPD does have a lower concentration of calcium (0.3 mM) than is found in routine synthetic medium (0.68 mM), which, based on the current results, can account for the inability of erg24 mutants to grow on YPD. The sensitivity of wild-type yeast to fenpropimorph, an inhibitor of the ERG24 gene product, was also related to external calcium levels. This was evident from the fact that calcium could increase resistance to fenpropimorph, while the calcium chelator, EGTA, increased the sensitivity of yeast to fenpropimorph. In addition, magnesium could also increase resistance to fenpropimorph; however, other monovalent or divalent cations, such as Na+, K+, Li+, Zn+2, or Mn+2, did not increase resistance to the antifungal agent. Ketoconazole is an inhibitor of the 14-demethylase reaction within the ergosterol biosynthetic pathway that is separate from the target for fenpropimorph. Ketoconazolemediated growth inhibition was also affected by the external calcium concentration, a result which suggests a more general relationship between calcium homeostasis and ergosterol synthesis. The effect of calcium on rescue from fenpropimorph or ketoconazole is most likely due to an accommodation by yeast to the effects of the sterol change, as calcium supplementation had no effect on the inhibition of sterol synthesis by these sterol biosynthesis inhibitors. Moreover, a similar remediation of growth arrest in the ERG24 null mutant argues that calcium ions are somehow able to permit growth of the yeast with an aberrant sterol. Point mutations in the SCS1 and SCS2 genes also result in a calcium-requiring phenotype (Zhao et al. 1994). The SCS1 and SCS2 genes (also called LCB1 and LCB2) (Buede et al. 1991; Nagiec et al. 1994) encode subunits of the serine palmitoyltransferase, the first committed step in sphingolipid biosynthesis (Zhao et al. 1994). Mutations in the SCS1 and SCS2 genes cause a sphingolipid deficiency, which, in turn, results in pleiotrophic phenotypes such as sensitivity to low pH, high temperature, and high salt concentration (Patton et al. 1992). The similarity in some phenotypes of sphingolipid mutants and ergosterol mutants is intriguing, considering that both of these lipid classes are found primarily in the plasma membrane. It is conceivable that these lipids may work synergistically to allow proper plasma-membrane function in yeast. In developing an explanation for how ergosterol may affect calcium ion requirements in yeast, it is worth considering three other genes that, when mutated, result in a similar calcium-requiring phenotype. Mutations in YPT1 (Schmitt et al. 1988), USO1 (Kito et al. 1996), or PMR1 (Rudolph et al. 1989) result in a calcium-dependent phenotype. The YPT1 gene encodes a GTP-binding protein that is similar to ras-related genes in yeast (Baker et al. 1990), and its gene product is found associated with Golgi membranes and is involved in the transport of proteins from the endoplasmic reticulum to the Golgi complex (Segev et al. 1988; Baker et al. 1990). A temperature-sensitive allele of YPT1 will grow at 37°C with the addition of calcium to the growth medium (Schmitt et al. 1988). Although Ca+2 and

Ypt1p act independently to regulate protein transport, studies on Ypt1p function have supported a role for Ca+2 in controlling secretory movement (Baker et al. 1990). The Uso1p protein, also involved in the secretory pathway, is functionally connected with the Ypt1p in that SLY genes, originally cloned as suppressors to YPT1, which also function in the suppression of mutations in USO1 (Kito et al. 1996). SLY genes have homology to synaptobrevins, and may function as components of the protein transport machinery (Dascher et al. 1991). The PMR1 gene is a member of the P-type ATPases and shares homology with known mammalian Ca+2 pumps (Rudolph et al. 1989). Pmrlp localizes to the Golgi complex and is essential for normal Golgi function (Antebi and Fink 1992). Studies with PMR1 provide strong evidence for the role of calcium in protein transport (Rudolph et al. 1989; Antebi and Fink 1992). This calcium pump may act to sequester calcium from the cytosol to vesicles where calcium may be required for one or more essential processes in protein transport through the secretory system (Antebi and Fink 1992). Although YPT1, USO1, and PMR1 encode different kinds of proteins, they have in common that they are all involved in the process of protein translocation in the secretory pathway in yeast. As such, in addition to having a calcium-remedial phenotype, strains containing mutations in YPT1, USO1, or PMR1 display defects in protein translocation and post-translocational modification (i.e., glycosylation) within the cell. Does ergosterol play a role in the secretory process? It may be more than coincidence that disruption of normal ergosterol synthesis also results in calcium dependence. It is conceivable that ergosterol may be required for the normal function of the Pmr1p calcium pump. In this scenario, a decrease in membrane ergosterol would lead to defective deposition of calcium into Golgi and secretory lumens. Supplementation with high levels of external calcium would increase passive transport of calcium into these compartments where the affected essential processes could continue. The super-sensitivity of erg mutants to the calcium-binding protein agonist, trifluoperazine, is consistent with the idea that some compartment in the cell may be deficient in the calcium levels necessary to support one or more calcium-dependent functions. This model predicts that alteration of sterol synthesis would cause proteintranslocation defects similar to those caused by mutations in the PMR1 gene. This has yet to be explored. In addition, there is at least one other calcium pump, Pmc1p (PMC1), which is involved in calcium homeostasis. Pmc1p is a vacuolar calcium ATPase that sequesters calcium to the vacuole (Cunningham and Fink 1994). Mutations in PMC1, unlike mutations in PMR1, result in sensitivity to calcium, which may result from an inability to confine excess cytosolic calcium to the vacuole. We have described here a novel calcium-dependent phenotype associated with alterations in the ergosterol biosynthetic pathway in yeast. Either a mutation in ERG24 or treatment of yeast cells with two different inhibitors of sterol biosynthesis induced calcium dependency, confirming that a disruption in ergosterol synthesis induces a

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change in the normal requirements of calcium in yeast. Our results provide the groundwork for future studies designed to examine how ergosterol may influence calcium homeostasis in S. cerevisiae. Acknowledgements This research was supported in part by grants from the U. S. Army Research Office (DAAAH04-97-0003), the National Institutes of Health (DK 37222), and the North Carolina Agricultural Research Service. Additional funding was provided by the NutraSweet Kelco Corp. We thank Dr. Kelly Tatchell for yeast strains, BASF for supplies of fenpropimorph, Frank Leak for technical assistance, and Kevin Shianna for invaluable help in manuscript preparation.

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