YEAST
VOL. 8
1015-1 024 ( 1992)
Effect of Sterol Alterations on Conjugation in Saccharomyces cerevisiae MICHELE E. TOMEO, GREGEORY FENNER, SHIRLEY R. TOVE AND LEO W. PARKS*
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, U.S.A. Received 23 March 1992; accepted 28 April 1992
Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols. The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols. After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding. KEYWORDS - Saccharomyces
cerevisiae; mating; conjugation; sterols.
INTRODUCTION Conjugation in Saccharomyces cerevisiae normally requires recognition between two haploid cells of opposite mating types and proceeds through a series of reactions culminating in the formation of a zygote. Specifically, both cells respond to the opposite mating pheromone by producing surface agglutinins, arresting in G1 cell-division phase, and elongating toward the pheromonal signal, forming a structure known as a schmoo. Once the cells have made contact, they undergo cytoplasmic and nuclear fusion. The resulting zygote divides mitotically resultingin the production ofnewdiploidcells(Cross et al., 1988). Our laboratory became interested in a possible function for sterols in the yeast mating process when we observed that sterol auxotrophic strains exhibited poor mating and aberrant budding behavior during mating experiments. Under aerobic conditions, wild-type yeast do not take up exogenously supplied sterol and produce ergosterol endogenously for fulfillment of their cellular requirements (Parks, 1978). The exclusion is precise and without regard to the suitability of the sterol for the various functions in the cell. Sterol auxotrophs, which do take up sterol aerobically, are viable only when they contain a concomitant *To whom correspondence should be addressed. 0749-503X/92/12l015-10 $10.00 0 1992 by John Wiley & Sons Ltd
mutational defect in heme biosynthesis (Gollub et al., 1977; Lewis et al., 1985). In fact, further investigations in our laboratory have found that hemes play a negative role in the regulation of sterol uptake for these mutants (Lorenz and Parks, 1987; Shinabarger et al., 1989).Previously, our laboratory has described multiple functions for sterols in the vegetative growth of S. cerevisiae (Lorenz et al., 1989; Rodriguez and Parks, 1983). Variability in the mating response that we observed appeared to be dependent on the sterol that was available. This was surprising since we had not seen substantial variations in the vegetative growth response to the different sterols that were being supplied. We therefore initiated a study of the effect of substituting aberrant sterols for the natural yeast sterol, ergosterol, on mating in S. cerevisiae. MATERIALS AND METHODS Yeast strains
All strains used in this study are presented in Table 1. Media and growth conditions
The YNB medium used for growth contained 2% glucose, 0.67% yeast nitrogen base w/o amino acids,
1016
M. E. TOME0 ETAL.
Table 1. Strains
FY14 FY24 FY34 Y93 JC482 JCS30-4B
Reference or Source
Genotype
Strain
M A T a . heml,ergl::URA3,ade2,leu2 M A T a , heml, ergl::URA3, ade2, his3-200, trp 1-90 M A T a I M A T a . hemllheml, erg]::URA31ergl:: URA3, ade2/ade2 M A T a , ade2-101, his 3-200,lys2-801,trpl-901, ura3 M A T a . hisl, leu2, ura3-52 MATa, hisl, leu2, ura3-52
Lorenz and Parks (1990) This study This study Snyder and Davis (1988) Cannon and Tatchell(l987) K. Tatchell (North Carolina State University, Raleigh, NC)
Table 2. Description of sterols Sterol Ergosterol Cholesterol 7-Dehydrocholesterol Sitosterol Stigmasterol
IUPAC Name
Features
Ergosta-S,7,22-trien-3Pol Cholesta-5-en-3pol Cholesta-5,7-dien-3Pol 24-a-Ethyl-cholesta-5-en-3~ol 24-a-Ethyl-cholesta-5,22-dien-3pol
AS, 7,22, C-24 P-methyl AS AS, 7 AS, C-24 a-ethyl AS, 22, (2-24 a-ethyl
and 1% casamino acids (Difco Laboratories) and was supplemented with 20 pg/ml each of adenine, methionine, tryptophan, histidine and 30 pg/ml of leucine. The medium was buffered with 0.05 Msuccinic acid and adjusted to pH 5.5. Selective YNB medium contained no casamino acids and only the appropriate amino acids. The rich medium, YPD, contained 0.5% yeast extract, 1% peptone and 2% glucose. Cholesterol and ergosterol were recrystallized and sitosterol, 7-dehydrocholesterol and stigmasterol were purified by high-performance liquid chromatography (HPLC; Rodriguez and Parks, 1982) before being added at a concentration of 5 pg/ml in Tergitol Nonidet P-4&95% ethanol (1 :1 [vol/vol]). Structural descriptions of the different sterols are given in Table 2. Unsaturated fatty acid (UFA) supplementation consisted of a mixture of oleic and palmitoleic acids (4: 1 [vol/vol])at a concentration of 0.01YOin Tergitol Nonidet P-4&95% ethanol (1 :1 [vol/vol]). 6-Aminolevulinic acid (ALA) was dissolved in distilled water and filter sterilized (0.25 pm pore size, type HA; Millipore Corp., Bedford, MA) for use at a concentration of
10 pg/ml. Cultures were grown aerobically at 30°C with constant shaking. Growth was monitored by a Klett-Summerson photoelectric colorimeter equipped with a green filter. HPLC purification
Sterols were purified using a Beckman 110A HPLC equipped with a Beckman Ultrasphere ODS 5 pm semi-preparative C18 reverse-phase 10.0 mm i.d. x 25 cm column. The solvent system was an isocratic mixture of methanol/ethanol/water (1710:200:90) run at 5 ml/min. A single peak was collected from each of the sterols being purified, with absorbance being measured at 210 nm. Sterol analysis
Strains FY14, FY24 and FY34, grown to stationary phase in 10 ml of medium containing the sterols to be tested, were centrifuged and washed twice with distilled water. Cell pellets were acid labilized (Gonzales and Parks, 1977) and alkaline saponified
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SACCHAROMYCES CEREVISIAE
(Bailey and Parks, 1975). Sterols were extracted with n-hexane, evaporated to dryness under N,, resuspended in iso-octane, and analysed by gas chromatography (5890A; Hewlett Packard) equipped with a flame ionization detection system. Sterol separation was achieved using an SPB-1 fused silica column, 30 m x 0.32 mm i.d., 0-25 pm film thickness (Supelco Inc., Bellefonte, PA). An oven temperature of 235°C was maintained and the injector and detector were operated at 280°C. Helium was the carrier gas maintained at 32 psi, and the make-up gas was N,. Data were analysed by a Waters Maxima 820 data acquisition processor (Milford, MA) on an IBM/AT work station. Quantitative mating procedures
Matings were performed using the method of Hartwell (1980) with modifications. The haploids were grown overnight at 30°C and harvested for the mating experiments during exponential growth. Cells (1 x lo7)of each mating type were collected on a nitrocellulose filter (type HA; Millipore Corp., Bedford, MA) and the filter was transferred to a YNB medium plate with the designated sterol, UFA, ALA and amino acid supplementation. After 2, 4, 8 and 12 h, the cells were washed from the filters with 10ml of succinic acid buffer, diluted appropriately, and plated on YPD to determine total cell number and on selective YNB to recover prototrophs. Mating efficiency was determined as the ratio of diploid cell number over total cell number multiplied by 100. Results are averages from triplicate experiments. Rescue of the low mating ejiciency
The cells were grown overnight in liquid media containing UFAs, ALA and amino acids. Sterol supplementation was with stigmasterol alone (at 5 pg/ml), ergosterol alone (5 pg/ml), or with one of the following sterol combinations: ergosterol 0.1 pg/ ml stigmasterol 4.9 pg/ml; ergosterol 0.5 pg/ml+ stigmasterol 4.5 pg/ml; ergosterol 2.5 pg/ml+ stigmasterol 2-5 pg/ml. The YNB plates used to conduct the mating experiments were of the same composition as the liquid growth media. Once these cells were removed from the nitrocellulose filter following the mating interval, all the cells, except those mated solely on ergosterol, were plated onto medium containing only stigmasterol as the sterol supplement.
+
1017
Light microscopy
The mating cells were photographed using a Zeiss Axioskop microscope equipped with differential interference contrast optics. Cell preparation for electron microscopy
Cells were prepared and mated as above except that liquid media were used instead of plates. At the designated time points, cells were harvested by centrifugation and washed in 0.1 M-sodium cacodylate buffer, pH 6.8. The cells were fixed in 3% glutaraldehyde with added lOmM-Cacl, for 16h at 4°C. Glutaraldehyde was removed by three washes in sodium cacodylate buffer. The cells were treated with a solution containing 0.1 M-P-mercaptoethanol, 0.02 M-EDTA and 0.2 M-Tris-HC1 for 10 min at room temperature. Three more washes, in sodium cacodylate buffer, were followed by postfixation with 2% osmium tetroxide for 2 h at 4°C. Once again, the cells were washed in sodium cacodylate buffer three times. For ease in further handling, the cells were embedded in 2% agarose and 1 mm cubes were cut from this mixture. The tissue was then dehydrated through three changes in each of a graded series of ethanols (30,50,70,95 and 100%) at 4”C, where they remained for at least 8 h. The tissue was then brought to room temperature in 100% ethanol. The cells were embedded in Spurr’s resin (firm recipe) in a series of steps (ethanol/Spurr’s [2:1, 1:1, 1:2, 100% Spurr’s, three changes]) with the full resin infiltration steps being performed under gentle vacuum. Hardening took place in a 70°C oven. Thin sections were prepared on a LKB Nova microtome and placed on 75-mesh grids. After staining with Reynold’s lead citrate for 4 min, the grids were examined on a Jeol 100s electron microscope. Chemicals
Ergosterol, cholesterol, 7-dehydrocholesterol, Tergitol Nonidet P-40, ALA, EDTA, Tris and amino acids were purchased from Sigma Chemical Co. (St Louis, MO). Stigmasterol was from Research Plus (Bayonne, NJ). Sitosterol was a generous gift from Dr David Chitwood (USDA, Beltsville, MD). Glutaraldehyde, sodium cacodylate, osmium tetroxide and Spurr’s resin were purchased from Ladd Research (Burlington, VT). Sodium citrate was received from Mallinckrodt Inc. (Paris, KY). Lead nitrate was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ).
1018 P-Mercaptoethanol and all the solvents used were obtained from Fisher Scientific (Pittsburgh, PA) and were redistilled before use.
M.E. TOME0 ETAL.
.---
1
A
RESULTS
FYI4 and FY24 exhibit similar growth responses to various sterol supplements Despite differences in the lag time for these cells grown on the tested sterols, haploids FY14, FY24 and their diploid, FY34, showed comparable doubling times and final densities in stationary phase (Figure 1, Table 3). The stigmasterol-grown cells of FY34, for example, which appeared to be growing more slowly (Figure lC), possessed a doubling time that was comparable to that of these same cells grown on ergosterol (Table 3). These data demonstrate that during exponential growth the cells are metabolizing at a similar rate regardless of their specific sterol supplement. Also, the sterols were extracted from the same cells used in the growth experiments and analysed for sterols by gas chromatography; it was found that the proffered sterols were recovered unmodified at the end of growth (data not shown). Sterol auxotrophic cells mate more eficiently on ergosterol Consistent with our growth experiments, the mating cells were fed exogenous sterol at a concentration of 5 pg/ml. This amount has been shown previously to satisfy fully the membrane sterol requirement of these cells (Rodriguez et al., 1985). ALA, which was found to increase the mating efficiency of these cells, was also added at a concentration of 10 pg/ml along with the methionine and UFA supplements. Furthermore, we chose to mate cells that were harvested in the exponential phase of growth to ensure the population of yeast tested was fully viable at the start of these experiments. The results of mating the sterol auxotrophs grown on the different sterols are shown in Table 4. Mating in these strains occurs relatively rapidly and can be demonstrated 2 h after the opposite mating types are mixed. In fact, the largest increase in mating efficiency for the sterol auxotrophs (FY 14 and FY24) mated to each other can be seen in the period from 2 to 4 h after mixing. Beyond this time, some outgrowth of the diploids should have occurred, as shown previously in a study using wildtype yeast (Hartwell, 1973). The mating percentages shown do not distinguish between the diploids and their progeny; instead, the numbers reflect all the
0
10
20
30
40
Time (hrs) 1000
E
0
10
20
30
40
50
40
50
Time (hrs) 1000
C
0
10
20
30
60
Time (hrs)
Figure 1. Determination of growth patterns of FY14, FY24 and their diploid under various sterol conditions. Cells were grown on 5 pg/ml of ergosterol supplemented with unsaturated fatty acids at 100 pg/ml and ALA at 10 pg/ml. The exponentially growing cells were harvested by centrifugation and any excess exogenous sterol was removed from the cells using a 0.5% tergito1 wash. After resuspendingthe cells in succinate buffer (pH 5 . 9 , a 10 pl inoculum was placed in 10 ml of media containing either one of the test sterols or no sterol as a control. The growth curves shown are the results from the third transfer onto these test sterols. Data presented are from triplicate experiments. (A) FY14, (B) FY24 and (C) FY34 grown on the followingsterols: 0, ergosterol; 0 , cholesterol; A, sitosterol; A , 7-dehydrocholesterol; 0, stigmasterol; I, no sterol.
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SACCHAROMYCES CEREVISIAE
Table 3. Growth rates of sterol auxotrophs fed exogenous sterols Doubling time* (h) Sterol
FYI4
FY24
FY34
Ergosterol Cholesterol Sitosterol 7-Deh ydrocholesterol Stigmasterol
2.13 2.37 2.14 2.35 2.66
4.27 3.88 4.04 3.24 3.69
2.18 1.17 1.89 1.89 1.89
*Values represent the doubling times as measured by turbidity using the following formula: 0.3 (time 2-time I)/(log klett 2-log klett 1).
diploids that are present at the designated time points. However, the numbers reported here demonstrate that, especially in the case of stigmasterolmated cells, the mating efficiency does not increase dramatically simply by keeping the cells exposed to each other for longer periods of time. Ergosterol was the best sterol for mating of those tested, followed by cholesterol, 7-dehydrocholesterol, sitosterol and finally stigmasterol. In fact, ergosterol possessed a 30-fold better mating efficiency than stigmasterol at 4 h and continued to demonstrate a 22-fold better mating efficiency than stigmasterol even after 12 h. Sterol prototrophs Y93 and JC482 mated at approximately 70% efficiency after 6 h of exposure (data not shown). In these experiments, crossing either FY14 or FY24 to the wild types JC530-4B and JC482 increased the mating efficiencies for all the sterols tested. Ergosterol, however, remained the best sterol for mating, even under these conditions, where only one partner was a sterol auxotroph. In addition, we recognize that the FY 14 x JC5304B cross generally resulted in more prototrophs than the FY24 x JC482 cross. We are unable to explain this difference. Furthermore, the mating defect seen with stigmasterol-grown cells cannot be completely compensated simply by mating them with a wild-type partner because these crosses show less than half the efficiency of comparable matings on ergosterol. Cells mated on stigmasterol demonstrate a conjugation defect late in the mating process Because stigmasterol elicited the lowest recovery of zygotes, we examined the morphology of the mated
1019
pairs. Cells mating on stigmasterol that appear to be fixed in a prezygotic state contain a prominent septum between the two mating cells. Figure 2 demonstrates the typical appearance of these cells during mating. Unlike the wild type (Figure 2A) and ergosterol-grown auxotrophic cells (Figure 2B) which produced many zygotes within 5 h of mating, the majority of the stigmasterol-grown cells were caught at a precytoplasmic fusion step (Figure 2D). No differencesin the earlier steps ofconjugation such as agglutin production and shmoo formation could be detected; however, many of the stigmasterolmated cells continued to exhibit this cytoplasmic fusion defect even after 12 h of exposure to their opposite mating type. In fact, some of the cells which appeared to be unable to fuse had begun to resume mitotic growth, forming a new haploid bud (Figures 2D and 3C). The apical appearance of this bud is in contrast to the usual emergence of the diploid bud at the isthmus between successfully mated pairs (Figure 2A, B). Electron microscopy shows the septum between the cells mated on stigmasterol
The stigmasterol-mated cells which had arrested in the mating process were better characterized by electron microscopy. Using this technique, we were able to identify that not only the cell membrane but also the cell wall was left undissolved between these non-fusing cells (Figure 3B). Figure 3A shows a normal zygote and its primary bud, which has formed after 4 h of incubation on ergosterol medium. Comparatively, Figure 3C demonstrates the appearance of the majority of the stigmasterol-cultured cells after 12 h ofincubation. Apparently, once these cells are desensitized to mating pheromone, and the mating reaction has been abandoned, these haploid cells begin another round of replication including bud formation. Although some stigmasterol-mated cells examined had successfully fused after this extended period of time, many of these cells showed only partial dissolution of the septum between them, indicating that cytoplasmic fusion had occurred only very recently (Osumi, 1974). The addition of ergosterol to stigmasterol-mated cells successfully rescued mating
Based on the finding that the normal yeast sterol ergosterol allowed for so much greater mating efficiency than stigmasterol, it was suggested that the addition of small amounts of ergosterol to
1020
M. E. TOME0 ET AL.
Table 4. Mating efficiencies in sterol auxotrophic strains fed exogenous sterols. Sterol*? Time Cross
(h)
Ergosterol
Cholesterol
7-Dehydrocholesterol
Sitosterol
Stigmasterol
JC530-4B x FY 14
2 4 8 12
20.0 f 1.58 35.0 f6.70 67.8 f5.40 79.6 f3.32
19.3 f3.91 32.7f 1.91 54.4 f5-68 66.4f4.39
3.7 f0.8 1 25.0 f3.47 38.0f 1.91 67.2 f8.20
2.8 f0.29 10.2 f2.64 26.0 3.87 60.9 f5.12
2.0 f0.60 3.2f0.55 11.0f0.47 30-2f5.19
JC482 x FY24
2 4 8 12
12.0f 1.91 29-9f4.27 55.9 f4.50 72.1 f7.09
4.9 & 0.92 26.4 f 1.40 48.0 f5.57 45.9 f9.27
2.0 0.55 8.4f0.98 18.9f 1.07 42.2f 5.87
1.2& 0.30 11.7f2.12 33.7 f5.79
1.7f 1.14 4.2 f 1-79 10.8f 1.10 13.9 f2.30
2 4 8 12
13.3 f5.25 19.5 f7.1 3 21.6 f2.70 28.7 f 1.50
1.3f0.21 6.7 2.26 11.2f3.50 15.0 & 3.29
0.54 f0.08 3.1 f 1.65 7.3 f2.11 9.0 f0.9 1
0.50 f0.50 2.6 f0.50 6.4f 1.51 8.8f0.91
0.25 f0.14 0.62 f0.29 0.77f0.17 1.30f0.19
FY14 x FY24
*
*
5.5 f2.15
*Sterol was supplied at 5 pg/ml, and the cells used for these mating experiments were taken at exponential phase. tValues represent k the standard deviation for the experiments performed in triplicate expressed as YOmating events based on the following formula: (diploid cells/total cell no.) x 100% =mating efficiency.
stigmasterol-mated cells would increase their mating efficiency. Therefore, cells that had been previously grown on stigmasterol were used to inoculate medium containing either 0.1, 0.5 or 2-5pg/ml ergosterol, along with enough stigmasterol to maintain the total sterol concentration at 5 pg/ml. The mating experiment was conducted using the same procedure as before (see Methods), and the cells were allowed to incubate for 4 h before they were plated to selective medium. It was discovered from this experiment that as little as 0.5pg/ml of ergosterol produced 1.4 0.43% mating, as opposed to approximately 0.70% mating for cells grown only on stigmasterol. Even more convincingly, the addition of equivalent amounts of ergosterol and stigmasterol (2.5 pg/ml) allowed for a mating efficiency that was equal to that of ergosterol alone, 12.2f 1.42% and 16.4f3.43%, respectively. DISCUSSION Several vegetative functions have been proposed for sterol in the yeast S. cerevisiae, with some of these requirements being best fulfilled by the features of the native yeast sterol, ergosterol (Dahl et al., 1987; Dahl and Dahl, 1985; Kawasaki et al., 1985; Pinto and Nes, 1983; Rampogal et al., 1990; Rampogal
and Bloch, 1983; Rodriguez et al., 1982, 1985; Rodriguez and Parks, 1983). We have now shown that sterol also plays a role in the yeast conjugation process. The sterol auxotrophs, FY14 and FY24, which require sterol supplementation in order to grow aerobically, mate more efficiently when they are grown on ergosterol than when they have been grown on cholesterol, 7-dehydrocholesterol, sitosterol or stigmasterol. Interestingly, while ergosterol fulfills this role in mating the most efficiently, all of the other sterols tested do allow for some mating in our experiments. However, the cells mated only very poorly on stigmasterol, and it would appear, based on our observations using light and electron microscopy, that many of these cells were unable to mate due to their failure to undergo cytoplasmic fusion. This phenomenon raises the question of how non-ergosterol sterols substituted into the yeast membranes for ergosterol affect the process of membrane fusion during mating. We have demonstrated in previous research that yeast strain RD5R has the ability to alter its fatty acid and phospholipid composition as an accommodation to different exogenous sterols. These changes have been proposed to modulate the fluid state of the plasma membrane (Low et al., 1985). Papahadjopoulos et al. (1974), have shown
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SA CCHAROMYCES CEREVISIAE
1021
Figure 2. Light micrographs showing the typical appearance of the wild type and the sterol auxotrophs after 4 h of mating on various sterols. Bar indicates 0.3 p. (A) JC482 x JC530-4B mated on stigmasterol; (B) FY 14 x FY24 mated on ergosterol; (C) FY 14 x FY24 mated on 7-dehydrocholesterol; (D) FY 14 x FY24 mated on stigmasterol.
that the presence of negatively charged phospholipids in vesicles increases their ability to fuse. Perhaps alterations in either the phospholipids or fatty acids or both of these sterol auxotrophs grown on stigmasterol have deleterious effects on the normal fusigenic properties of their cellular membrane, leading to subsequent difficulties in mating. Alternatively, a second possible explanation for the lowered mating efficiency of the sterol auxotrophs
grown on different sterols is that the sterol molecule interacts with some protein which enables cells to fuse during mating. Several researchers have characterized the FUS genes, FUSI, FUS2 (McCaffrey et al., 1987; Trueheart et ul., 1987) and FUS3 (Elion et al., 1990), whose gene products function specifically in the mating events of cytoplasmic fusion and G1 arrest, respectively, in S. cerevisiue. It has been concluded that FUSl and probably FUS2 act at the
I022
M. E. TOME0 ET AL.
Figure 3. Electron micrographs demonstrating the appearance of the sterol auxotrophic strains mated on ergosterol or stigmasterol. (A) Zygote on ergosterol after 4 h of mating. Bar indicates 1 p. (B) The intraparentaljunction of a prezygote mated on stigmasterol. Bar indicates 0.5 p. (C) Cells mating on stigmasterol for 12 h; the interparental junction is still visible. Bar indicates
point of contact between mating cells and somehow allow for the dissolution and reorganization of the cellular membrane, which leads to zygote formation (Trueheart et al., 1987). Successful mating may require sterol to work in conjunction with these fusion proteins to permit proper membrane reorganization. We have shown that the haploid strain mutant atfusl produces normal yeast sterols (unpublished experiments). Little can be concluded from the results concerning which sterol structural feature is most important in facilitating mating. Cholesterol was found to be the second most efficient sterol for mating, and this sterol lacks the unsaturations at C-7, C-22 and the alkyl group at C-24, indicating that the absence of these features does not preclude efficient mating in
S. cerevisiue. The greatest structural difference between ergosterol and the poorest mating sterol, stigmasterol, is the presence of an ethyl group at C24 with an a orientation. The fact that sitosterol also contains a C-24 a-ethyl group but still allows for four-fold better mating than stigmasterol further complicates these findings. The occurrence of unsaturation at C-5 has been found to be a necessary structural feature for fulfillment of the sparking function in yeast (Rodriguez et ul., 1982, 1985; Rodriguez and Parks, 1983). A sterol lacking the C5 has not been tested for its ability to allow efficient mating. Clearly, a lot more sterols will have to be tested before we can fully understand how sterol structure is affecting this process. Even then, the results may not be unambiguous. We have shown
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SA(XHAROMYCES CEREVISIAE
that the side-chain features of ergosterol appear to contribute cumulatively to feed-back inhibition of sterol synthesis (Casey et al., 1991). A similar situation may exist in the mating reactions. Regardless of which specific feature(s) of the sterol molecule facilitates mating in S. cerevisiae, we have shown that ergosterol plays a role in mating by demonstrating that cells previously growing on stigmasterol can be rescued from their mating deficiency by the introduction of ergosterol to these cells. While addition of only 0.5 pg/ml of ergosterol partially relieved the mating defect of the stigmasterol mating cells, 2.5 pg/ml of ergosterol allowed for complete restoration of mating ability. Working with the yeast strain RDSR, Rodriguez et ul. (1985) have shown that this amount of sterol is enough to fulfill the bulk role of sterol in S. cerevisiae. Probably no other eukaryotic cellular component has as many functional natural analogs as do the sterols. Indeed, attempting to show a precisely defined requirement for a specific feature of the ergosterol molecule can be exacerbated by imposed physiological adjustments in the cell. Yeast have a very efficient mechanism for excluding exogenously supplied sterols aerobically, presumably by allowing for a selective advantage for the use of ergosterol. It is paradoxical, therefore, that a myriad of sterols seem to be accommodated efficiently in the growth of the cells (Figure l), providing suitable mutations are present which permit the uptake of sterols from the media. We have shown here that at least one critical step in the cytoplasmic fusion even in conjugation has a marked preference for ergosterol. Our continuing challenge is to dissect that process and discern the sterol-dependent step. ACKNOWLEDGEMENTS This research was supported by the National Science Foundation (DCB881437), the National Institutes of Health of the U.S. Public Health Service (DK37222), U.S. Army Research Office staff research award to S.R.T. (DAAL03-89-D-003D7), and the North Carolina Agricultural Research Service. The authors gratefully acknowledge the assistance of Dr Kelly Tatchell, who provided cultures and facilities for light microscopy, Valerie Knowlton, who performed the ultramicrotomy work, and Dr David Chitwood, who provided sitosterol for these experiments. We also appreciate Dr Donna G. Cookmeyer's assistance in the preparation of this manuscript.
1023
REFERENCES Bailey, R. B. and Parks, L. W. (1975). Yeast sterol esters and their relationship to the growth of yeast. J. Bacteriol. 124,606-612. Cannon, J. F. and Tatchell, K. (1987). Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase. Mol. Cell. Biol. 7,2653-2663. Casey, W. M., Burgess, J. P. and Parks, L. W. (1991). Effect of sterol side-chain structure on the feed-back control of sterol biosynthesisin yeast. Biochim. Biophys. Acta 1081,279-284. Cross, F., Hartwell L. H., Jackson, C. and Konopka, J. B. (1988). Conjugation in Saccharomyces cerevisiae. Ann. Rev. Cell Biol. 4,429-457. Dahl, C., Biemann, H. P. and Dahl, J. (1987). A protein kinase antigenically related to pp60'-"" possibly involved in the yeast cell cycle control: Positive in vivo regulation by sterol. Proc. Natl. Acad. Sci. USA 84, 4012-4016. Dahl, J. S. and Dahl, C. E. (1985). Stimulation of cell proliferation and polyphosphinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol. Biochem. Biophys. Res. Comm. 133,844-850. Elion, E. A., Grisafi, P. L. and Fink, G. R. (1990). FUS3 encodes a cdc2 +/CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60, 649-664. Gollub, E. G., Liu, K., Dayan, J., Adlersberg, M. and Sprinson D. S. (1977). Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3 oxidosqualene. J . Biol. Chem. 252, 2846-2854. Gonzales, R. A. and Parks, L. W. (1977).Acid-labilization of sterols for extraction from yeast. Biochim. Biophys. Acta 489,507-509. Hartwell, L. H. (1980). Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J. Cell Biol. 85,811-822. Hartwell, L. H. (1973). Synchronization of haploid yeast cell cycles, a prelude to conjugation. Exper. Cell Res. 76,111-1 12. Kawasaki, S., Rampogal M., Chin J. and Bloch, K. (1985). Sterol control of the phosphatidylethanolaminephosphatidylcholine conversion in the yeast mutant GL7. Proc. Natl. Acad. Sci. USA 82,5715-5719. Lewis, T. A., Taylor, F. R. and Parks, L. W. (1985). Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae. J . Bacteriol. 163, 199-207. Lorenz, R. T. and Parks, L. W. (1990). Effects of lovastatin (mevinolin)on sterol levels and on activity of azoles in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 34,1660-1665. Lorenz, R. T., Casey, and Parks, L. W. (1989). Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae. J. Bacteriol. 17, 6169-61 73.
1024 Lorenz, R. T. and Parks, L. W. (1987). Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast. J. Bact. 169,3707-37 11. Low, C., Rodriguez, R. J. and Parks, L. W. (1985). Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol. Arch. Biochem. Biophys. 240,530-538. McCaffrey, G.,Clay, F. J., Kelsay, K. and Sprague, G . F. Jr. (1987). Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell. Biol. 7,268&2690. Osumi, M. (1974). Mating reaction in Saccharomyces cerevisiae. Arch. Microbiol. 97,27-38. Papahadjopoulos, D., Poste, G., Schaeffer, B. E. and Vail, W. J. (1974). Membrane fusion and molecular segregation in phospholipid vesicles. Biochim. Biophys. Acta 352,lO-28. Parks, L. W. (1978). Metabolism of sterols in yeast. CRC Crit. Rev. Microbiol. 6,301-341. Pinto, W. J. and Nes, W. D. (1983). Stereochemical specificity for sterols in Saccharomyces cerevisiae. J . Biol. Chem. 258,4472-4476. Rampogal, M., Zundel, M . and Bloch, K. (1990). Sterol effects on phospholipid biosynthesis in the yeast strain GL7. J. Lipid Res. 31,653-658. Rampogal, M. and Bloch, K. (1983). Sterol synergism in yeast. Proc. Natl. Acad. Sci. USA 80,712-715.
M. E. TOME0 ET AL.
Rodriguez, R. J., Low,. C., Bottema, C. D. K. and Parks, L. W. (1985). Multiple functions for sterols in Saccharomyces cerevisiae. Biochim. Biophys. Acta 837, 336-343. Rodriguez, R.J. and Parks, L. W. (1983). Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking requirements in yeast sterol auxotrophs. Arch. Biochem. Biophys. 225, 861-871. Rodriguez, R. J., Taylor, F. R. and Parks, L. W. (1982). A requirement for ergosterol to permit growth of yeast sterol auxotrophs on cholestanol. Biochem. Biophys. Res. Comm. 106,435-441. Rodriguez, R. J. and Parks, L. W. (1982). Application of high-performance liquid chromatographic separation of free sterols to the screening of yeast sterol mutants. Anal. Biochem. 119,200-204. Shinabarger, D. L., Keesler, G. A. and Parks, L. W. (1989). Regulation by heme of sterol uptake in Saccharomyces cerevisiae. Steroids 53,607-623. Snyder, M. and Davis, R. W. (1988). S P A ] : a gene important for chromosome segregation and other mitotic functions in S. cerevisiae. Cell 54,743-754. Trueheart, J., Boeke, J. D. and Fink, G. R. (1987). Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced protein. MoZ. Cell. Biol. 7,23162328.
VOL. 8
1015-1 024 ( 1992)
Effect of Sterol Alterations on Conjugation in Saccharomyces cerevisiae MICHELE E. TOMEO, GREGEORY FENNER, SHIRLEY R. TOVE AND LEO W. PARKS*
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, U.S.A. Received 23 March 1992; accepted 28 April 1992
Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols. The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols. After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding. KEYWORDS - Saccharomyces
cerevisiae; mating; conjugation; sterols.
INTRODUCTION Conjugation in Saccharomyces cerevisiae normally requires recognition between two haploid cells of opposite mating types and proceeds through a series of reactions culminating in the formation of a zygote. Specifically, both cells respond to the opposite mating pheromone by producing surface agglutinins, arresting in G1 cell-division phase, and elongating toward the pheromonal signal, forming a structure known as a schmoo. Once the cells have made contact, they undergo cytoplasmic and nuclear fusion. The resulting zygote divides mitotically resultingin the production ofnewdiploidcells(Cross et al., 1988). Our laboratory became interested in a possible function for sterols in the yeast mating process when we observed that sterol auxotrophic strains exhibited poor mating and aberrant budding behavior during mating experiments. Under aerobic conditions, wild-type yeast do not take up exogenously supplied sterol and produce ergosterol endogenously for fulfillment of their cellular requirements (Parks, 1978). The exclusion is precise and without regard to the suitability of the sterol for the various functions in the cell. Sterol auxotrophs, which do take up sterol aerobically, are viable only when they contain a concomitant *To whom correspondence should be addressed. 0749-503X/92/12l015-10 $10.00 0 1992 by John Wiley & Sons Ltd
mutational defect in heme biosynthesis (Gollub et al., 1977; Lewis et al., 1985). In fact, further investigations in our laboratory have found that hemes play a negative role in the regulation of sterol uptake for these mutants (Lorenz and Parks, 1987; Shinabarger et al., 1989).Previously, our laboratory has described multiple functions for sterols in the vegetative growth of S. cerevisiae (Lorenz et al., 1989; Rodriguez and Parks, 1983). Variability in the mating response that we observed appeared to be dependent on the sterol that was available. This was surprising since we had not seen substantial variations in the vegetative growth response to the different sterols that were being supplied. We therefore initiated a study of the effect of substituting aberrant sterols for the natural yeast sterol, ergosterol, on mating in S. cerevisiae. MATERIALS AND METHODS Yeast strains
All strains used in this study are presented in Table 1. Media and growth conditions
The YNB medium used for growth contained 2% glucose, 0.67% yeast nitrogen base w/o amino acids,
1016
M. E. TOME0 ETAL.
Table 1. Strains
FY14 FY24 FY34 Y93 JC482 JCS30-4B
Reference or Source
Genotype
Strain
M A T a . heml,ergl::URA3,ade2,leu2 M A T a , heml, ergl::URA3, ade2, his3-200, trp 1-90 M A T a I M A T a . hemllheml, erg]::URA31ergl:: URA3, ade2/ade2 M A T a , ade2-101, his 3-200,lys2-801,trpl-901, ura3 M A T a . hisl, leu2, ura3-52 MATa, hisl, leu2, ura3-52
Lorenz and Parks (1990) This study This study Snyder and Davis (1988) Cannon and Tatchell(l987) K. Tatchell (North Carolina State University, Raleigh, NC)
Table 2. Description of sterols Sterol Ergosterol Cholesterol 7-Dehydrocholesterol Sitosterol Stigmasterol
IUPAC Name
Features
Ergosta-S,7,22-trien-3Pol Cholesta-5-en-3pol Cholesta-5,7-dien-3Pol 24-a-Ethyl-cholesta-5-en-3~ol 24-a-Ethyl-cholesta-5,22-dien-3pol
AS, 7,22, C-24 P-methyl AS AS, 7 AS, C-24 a-ethyl AS, 22, (2-24 a-ethyl
and 1% casamino acids (Difco Laboratories) and was supplemented with 20 pg/ml each of adenine, methionine, tryptophan, histidine and 30 pg/ml of leucine. The medium was buffered with 0.05 Msuccinic acid and adjusted to pH 5.5. Selective YNB medium contained no casamino acids and only the appropriate amino acids. The rich medium, YPD, contained 0.5% yeast extract, 1% peptone and 2% glucose. Cholesterol and ergosterol were recrystallized and sitosterol, 7-dehydrocholesterol and stigmasterol were purified by high-performance liquid chromatography (HPLC; Rodriguez and Parks, 1982) before being added at a concentration of 5 pg/ml in Tergitol Nonidet P-4&95% ethanol (1 :1 [vol/vol]). Structural descriptions of the different sterols are given in Table 2. Unsaturated fatty acid (UFA) supplementation consisted of a mixture of oleic and palmitoleic acids (4: 1 [vol/vol])at a concentration of 0.01YOin Tergitol Nonidet P-4&95% ethanol (1 :1 [vol/vol]). 6-Aminolevulinic acid (ALA) was dissolved in distilled water and filter sterilized (0.25 pm pore size, type HA; Millipore Corp., Bedford, MA) for use at a concentration of
10 pg/ml. Cultures were grown aerobically at 30°C with constant shaking. Growth was monitored by a Klett-Summerson photoelectric colorimeter equipped with a green filter. HPLC purification
Sterols were purified using a Beckman 110A HPLC equipped with a Beckman Ultrasphere ODS 5 pm semi-preparative C18 reverse-phase 10.0 mm i.d. x 25 cm column. The solvent system was an isocratic mixture of methanol/ethanol/water (1710:200:90) run at 5 ml/min. A single peak was collected from each of the sterols being purified, with absorbance being measured at 210 nm. Sterol analysis
Strains FY14, FY24 and FY34, grown to stationary phase in 10 ml of medium containing the sterols to be tested, were centrifuged and washed twice with distilled water. Cell pellets were acid labilized (Gonzales and Parks, 1977) and alkaline saponified
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SACCHAROMYCES CEREVISIAE
(Bailey and Parks, 1975). Sterols were extracted with n-hexane, evaporated to dryness under N,, resuspended in iso-octane, and analysed by gas chromatography (5890A; Hewlett Packard) equipped with a flame ionization detection system. Sterol separation was achieved using an SPB-1 fused silica column, 30 m x 0.32 mm i.d., 0-25 pm film thickness (Supelco Inc., Bellefonte, PA). An oven temperature of 235°C was maintained and the injector and detector were operated at 280°C. Helium was the carrier gas maintained at 32 psi, and the make-up gas was N,. Data were analysed by a Waters Maxima 820 data acquisition processor (Milford, MA) on an IBM/AT work station. Quantitative mating procedures
Matings were performed using the method of Hartwell (1980) with modifications. The haploids were grown overnight at 30°C and harvested for the mating experiments during exponential growth. Cells (1 x lo7)of each mating type were collected on a nitrocellulose filter (type HA; Millipore Corp., Bedford, MA) and the filter was transferred to a YNB medium plate with the designated sterol, UFA, ALA and amino acid supplementation. After 2, 4, 8 and 12 h, the cells were washed from the filters with 10ml of succinic acid buffer, diluted appropriately, and plated on YPD to determine total cell number and on selective YNB to recover prototrophs. Mating efficiency was determined as the ratio of diploid cell number over total cell number multiplied by 100. Results are averages from triplicate experiments. Rescue of the low mating ejiciency
The cells were grown overnight in liquid media containing UFAs, ALA and amino acids. Sterol supplementation was with stigmasterol alone (at 5 pg/ml), ergosterol alone (5 pg/ml), or with one of the following sterol combinations: ergosterol 0.1 pg/ ml stigmasterol 4.9 pg/ml; ergosterol 0.5 pg/ml+ stigmasterol 4.5 pg/ml; ergosterol 2.5 pg/ml+ stigmasterol 2-5 pg/ml. The YNB plates used to conduct the mating experiments were of the same composition as the liquid growth media. Once these cells were removed from the nitrocellulose filter following the mating interval, all the cells, except those mated solely on ergosterol, were plated onto medium containing only stigmasterol as the sterol supplement.
+
1017
Light microscopy
The mating cells were photographed using a Zeiss Axioskop microscope equipped with differential interference contrast optics. Cell preparation for electron microscopy
Cells were prepared and mated as above except that liquid media were used instead of plates. At the designated time points, cells were harvested by centrifugation and washed in 0.1 M-sodium cacodylate buffer, pH 6.8. The cells were fixed in 3% glutaraldehyde with added lOmM-Cacl, for 16h at 4°C. Glutaraldehyde was removed by three washes in sodium cacodylate buffer. The cells were treated with a solution containing 0.1 M-P-mercaptoethanol, 0.02 M-EDTA and 0.2 M-Tris-HC1 for 10 min at room temperature. Three more washes, in sodium cacodylate buffer, were followed by postfixation with 2% osmium tetroxide for 2 h at 4°C. Once again, the cells were washed in sodium cacodylate buffer three times. For ease in further handling, the cells were embedded in 2% agarose and 1 mm cubes were cut from this mixture. The tissue was then dehydrated through three changes in each of a graded series of ethanols (30,50,70,95 and 100%) at 4”C, where they remained for at least 8 h. The tissue was then brought to room temperature in 100% ethanol. The cells were embedded in Spurr’s resin (firm recipe) in a series of steps (ethanol/Spurr’s [2:1, 1:1, 1:2, 100% Spurr’s, three changes]) with the full resin infiltration steps being performed under gentle vacuum. Hardening took place in a 70°C oven. Thin sections were prepared on a LKB Nova microtome and placed on 75-mesh grids. After staining with Reynold’s lead citrate for 4 min, the grids were examined on a Jeol 100s electron microscope. Chemicals
Ergosterol, cholesterol, 7-dehydrocholesterol, Tergitol Nonidet P-40, ALA, EDTA, Tris and amino acids were purchased from Sigma Chemical Co. (St Louis, MO). Stigmasterol was from Research Plus (Bayonne, NJ). Sitosterol was a generous gift from Dr David Chitwood (USDA, Beltsville, MD). Glutaraldehyde, sodium cacodylate, osmium tetroxide and Spurr’s resin were purchased from Ladd Research (Burlington, VT). Sodium citrate was received from Mallinckrodt Inc. (Paris, KY). Lead nitrate was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ).
1018 P-Mercaptoethanol and all the solvents used were obtained from Fisher Scientific (Pittsburgh, PA) and were redistilled before use.
M.E. TOME0 ETAL.
.---
1
A
RESULTS
FYI4 and FY24 exhibit similar growth responses to various sterol supplements Despite differences in the lag time for these cells grown on the tested sterols, haploids FY14, FY24 and their diploid, FY34, showed comparable doubling times and final densities in stationary phase (Figure 1, Table 3). The stigmasterol-grown cells of FY34, for example, which appeared to be growing more slowly (Figure lC), possessed a doubling time that was comparable to that of these same cells grown on ergosterol (Table 3). These data demonstrate that during exponential growth the cells are metabolizing at a similar rate regardless of their specific sterol supplement. Also, the sterols were extracted from the same cells used in the growth experiments and analysed for sterols by gas chromatography; it was found that the proffered sterols were recovered unmodified at the end of growth (data not shown). Sterol auxotrophic cells mate more eficiently on ergosterol Consistent with our growth experiments, the mating cells were fed exogenous sterol at a concentration of 5 pg/ml. This amount has been shown previously to satisfy fully the membrane sterol requirement of these cells (Rodriguez et al., 1985). ALA, which was found to increase the mating efficiency of these cells, was also added at a concentration of 10 pg/ml along with the methionine and UFA supplements. Furthermore, we chose to mate cells that were harvested in the exponential phase of growth to ensure the population of yeast tested was fully viable at the start of these experiments. The results of mating the sterol auxotrophs grown on the different sterols are shown in Table 4. Mating in these strains occurs relatively rapidly and can be demonstrated 2 h after the opposite mating types are mixed. In fact, the largest increase in mating efficiency for the sterol auxotrophs (FY 14 and FY24) mated to each other can be seen in the period from 2 to 4 h after mixing. Beyond this time, some outgrowth of the diploids should have occurred, as shown previously in a study using wildtype yeast (Hartwell, 1973). The mating percentages shown do not distinguish between the diploids and their progeny; instead, the numbers reflect all the
0
10
20
30
40
Time (hrs) 1000
E
0
10
20
30
40
50
40
50
Time (hrs) 1000
C
0
10
20
30
60
Time (hrs)
Figure 1. Determination of growth patterns of FY14, FY24 and their diploid under various sterol conditions. Cells were grown on 5 pg/ml of ergosterol supplemented with unsaturated fatty acids at 100 pg/ml and ALA at 10 pg/ml. The exponentially growing cells were harvested by centrifugation and any excess exogenous sterol was removed from the cells using a 0.5% tergito1 wash. After resuspendingthe cells in succinate buffer (pH 5 . 9 , a 10 pl inoculum was placed in 10 ml of media containing either one of the test sterols or no sterol as a control. The growth curves shown are the results from the third transfer onto these test sterols. Data presented are from triplicate experiments. (A) FY14, (B) FY24 and (C) FY34 grown on the followingsterols: 0, ergosterol; 0 , cholesterol; A, sitosterol; A , 7-dehydrocholesterol; 0, stigmasterol; I, no sterol.
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SACCHAROMYCES CEREVISIAE
Table 3. Growth rates of sterol auxotrophs fed exogenous sterols Doubling time* (h) Sterol
FYI4
FY24
FY34
Ergosterol Cholesterol Sitosterol 7-Deh ydrocholesterol Stigmasterol
2.13 2.37 2.14 2.35 2.66
4.27 3.88 4.04 3.24 3.69
2.18 1.17 1.89 1.89 1.89
*Values represent the doubling times as measured by turbidity using the following formula: 0.3 (time 2-time I)/(log klett 2-log klett 1).
diploids that are present at the designated time points. However, the numbers reported here demonstrate that, especially in the case of stigmasterolmated cells, the mating efficiency does not increase dramatically simply by keeping the cells exposed to each other for longer periods of time. Ergosterol was the best sterol for mating of those tested, followed by cholesterol, 7-dehydrocholesterol, sitosterol and finally stigmasterol. In fact, ergosterol possessed a 30-fold better mating efficiency than stigmasterol at 4 h and continued to demonstrate a 22-fold better mating efficiency than stigmasterol even after 12 h. Sterol prototrophs Y93 and JC482 mated at approximately 70% efficiency after 6 h of exposure (data not shown). In these experiments, crossing either FY14 or FY24 to the wild types JC530-4B and JC482 increased the mating efficiencies for all the sterols tested. Ergosterol, however, remained the best sterol for mating, even under these conditions, where only one partner was a sterol auxotroph. In addition, we recognize that the FY 14 x JC5304B cross generally resulted in more prototrophs than the FY24 x JC482 cross. We are unable to explain this difference. Furthermore, the mating defect seen with stigmasterol-grown cells cannot be completely compensated simply by mating them with a wild-type partner because these crosses show less than half the efficiency of comparable matings on ergosterol. Cells mated on stigmasterol demonstrate a conjugation defect late in the mating process Because stigmasterol elicited the lowest recovery of zygotes, we examined the morphology of the mated
1019
pairs. Cells mating on stigmasterol that appear to be fixed in a prezygotic state contain a prominent septum between the two mating cells. Figure 2 demonstrates the typical appearance of these cells during mating. Unlike the wild type (Figure 2A) and ergosterol-grown auxotrophic cells (Figure 2B) which produced many zygotes within 5 h of mating, the majority of the stigmasterol-grown cells were caught at a precytoplasmic fusion step (Figure 2D). No differencesin the earlier steps ofconjugation such as agglutin production and shmoo formation could be detected; however, many of the stigmasterolmated cells continued to exhibit this cytoplasmic fusion defect even after 12 h of exposure to their opposite mating type. In fact, some of the cells which appeared to be unable to fuse had begun to resume mitotic growth, forming a new haploid bud (Figures 2D and 3C). The apical appearance of this bud is in contrast to the usual emergence of the diploid bud at the isthmus between successfully mated pairs (Figure 2A, B). Electron microscopy shows the septum between the cells mated on stigmasterol
The stigmasterol-mated cells which had arrested in the mating process were better characterized by electron microscopy. Using this technique, we were able to identify that not only the cell membrane but also the cell wall was left undissolved between these non-fusing cells (Figure 3B). Figure 3A shows a normal zygote and its primary bud, which has formed after 4 h of incubation on ergosterol medium. Comparatively, Figure 3C demonstrates the appearance of the majority of the stigmasterol-cultured cells after 12 h ofincubation. Apparently, once these cells are desensitized to mating pheromone, and the mating reaction has been abandoned, these haploid cells begin another round of replication including bud formation. Although some stigmasterol-mated cells examined had successfully fused after this extended period of time, many of these cells showed only partial dissolution of the septum between them, indicating that cytoplasmic fusion had occurred only very recently (Osumi, 1974). The addition of ergosterol to stigmasterol-mated cells successfully rescued mating
Based on the finding that the normal yeast sterol ergosterol allowed for so much greater mating efficiency than stigmasterol, it was suggested that the addition of small amounts of ergosterol to
1020
M. E. TOME0 ET AL.
Table 4. Mating efficiencies in sterol auxotrophic strains fed exogenous sterols. Sterol*? Time Cross
(h)
Ergosterol
Cholesterol
7-Dehydrocholesterol
Sitosterol
Stigmasterol
JC530-4B x FY 14
2 4 8 12
20.0 f 1.58 35.0 f6.70 67.8 f5.40 79.6 f3.32
19.3 f3.91 32.7f 1.91 54.4 f5-68 66.4f4.39
3.7 f0.8 1 25.0 f3.47 38.0f 1.91 67.2 f8.20
2.8 f0.29 10.2 f2.64 26.0 3.87 60.9 f5.12
2.0 f0.60 3.2f0.55 11.0f0.47 30-2f5.19
JC482 x FY24
2 4 8 12
12.0f 1.91 29-9f4.27 55.9 f4.50 72.1 f7.09
4.9 & 0.92 26.4 f 1.40 48.0 f5.57 45.9 f9.27
2.0 0.55 8.4f0.98 18.9f 1.07 42.2f 5.87
1.2& 0.30 11.7f2.12 33.7 f5.79
1.7f 1.14 4.2 f 1-79 10.8f 1.10 13.9 f2.30
2 4 8 12
13.3 f5.25 19.5 f7.1 3 21.6 f2.70 28.7 f 1.50
1.3f0.21 6.7 2.26 11.2f3.50 15.0 & 3.29
0.54 f0.08 3.1 f 1.65 7.3 f2.11 9.0 f0.9 1
0.50 f0.50 2.6 f0.50 6.4f 1.51 8.8f0.91
0.25 f0.14 0.62 f0.29 0.77f0.17 1.30f0.19
FY14 x FY24
*
*
5.5 f2.15
*Sterol was supplied at 5 pg/ml, and the cells used for these mating experiments were taken at exponential phase. tValues represent k the standard deviation for the experiments performed in triplicate expressed as YOmating events based on the following formula: (diploid cells/total cell no.) x 100% =mating efficiency.
stigmasterol-mated cells would increase their mating efficiency. Therefore, cells that had been previously grown on stigmasterol were used to inoculate medium containing either 0.1, 0.5 or 2-5pg/ml ergosterol, along with enough stigmasterol to maintain the total sterol concentration at 5 pg/ml. The mating experiment was conducted using the same procedure as before (see Methods), and the cells were allowed to incubate for 4 h before they were plated to selective medium. It was discovered from this experiment that as little as 0.5pg/ml of ergosterol produced 1.4 0.43% mating, as opposed to approximately 0.70% mating for cells grown only on stigmasterol. Even more convincingly, the addition of equivalent amounts of ergosterol and stigmasterol (2.5 pg/ml) allowed for a mating efficiency that was equal to that of ergosterol alone, 12.2f 1.42% and 16.4f3.43%, respectively. DISCUSSION Several vegetative functions have been proposed for sterol in the yeast S. cerevisiae, with some of these requirements being best fulfilled by the features of the native yeast sterol, ergosterol (Dahl et al., 1987; Dahl and Dahl, 1985; Kawasaki et al., 1985; Pinto and Nes, 1983; Rampogal et al., 1990; Rampogal
and Bloch, 1983; Rodriguez et al., 1982, 1985; Rodriguez and Parks, 1983). We have now shown that sterol also plays a role in the yeast conjugation process. The sterol auxotrophs, FY14 and FY24, which require sterol supplementation in order to grow aerobically, mate more efficiently when they are grown on ergosterol than when they have been grown on cholesterol, 7-dehydrocholesterol, sitosterol or stigmasterol. Interestingly, while ergosterol fulfills this role in mating the most efficiently, all of the other sterols tested do allow for some mating in our experiments. However, the cells mated only very poorly on stigmasterol, and it would appear, based on our observations using light and electron microscopy, that many of these cells were unable to mate due to their failure to undergo cytoplasmic fusion. This phenomenon raises the question of how non-ergosterol sterols substituted into the yeast membranes for ergosterol affect the process of membrane fusion during mating. We have demonstrated in previous research that yeast strain RD5R has the ability to alter its fatty acid and phospholipid composition as an accommodation to different exogenous sterols. These changes have been proposed to modulate the fluid state of the plasma membrane (Low et al., 1985). Papahadjopoulos et al. (1974), have shown
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SA CCHAROMYCES CEREVISIAE
1021
Figure 2. Light micrographs showing the typical appearance of the wild type and the sterol auxotrophs after 4 h of mating on various sterols. Bar indicates 0.3 p. (A) JC482 x JC530-4B mated on stigmasterol; (B) FY 14 x FY24 mated on ergosterol; (C) FY 14 x FY24 mated on 7-dehydrocholesterol; (D) FY 14 x FY24 mated on stigmasterol.
that the presence of negatively charged phospholipids in vesicles increases their ability to fuse. Perhaps alterations in either the phospholipids or fatty acids or both of these sterol auxotrophs grown on stigmasterol have deleterious effects on the normal fusigenic properties of their cellular membrane, leading to subsequent difficulties in mating. Alternatively, a second possible explanation for the lowered mating efficiency of the sterol auxotrophs
grown on different sterols is that the sterol molecule interacts with some protein which enables cells to fuse during mating. Several researchers have characterized the FUS genes, FUSI, FUS2 (McCaffrey et al., 1987; Trueheart et ul., 1987) and FUS3 (Elion et al., 1990), whose gene products function specifically in the mating events of cytoplasmic fusion and G1 arrest, respectively, in S. cerevisiue. It has been concluded that FUSl and probably FUS2 act at the
I022
M. E. TOME0 ET AL.
Figure 3. Electron micrographs demonstrating the appearance of the sterol auxotrophic strains mated on ergosterol or stigmasterol. (A) Zygote on ergosterol after 4 h of mating. Bar indicates 1 p. (B) The intraparentaljunction of a prezygote mated on stigmasterol. Bar indicates 0.5 p. (C) Cells mating on stigmasterol for 12 h; the interparental junction is still visible. Bar indicates
point of contact between mating cells and somehow allow for the dissolution and reorganization of the cellular membrane, which leads to zygote formation (Trueheart et al., 1987). Successful mating may require sterol to work in conjunction with these fusion proteins to permit proper membrane reorganization. We have shown that the haploid strain mutant atfusl produces normal yeast sterols (unpublished experiments). Little can be concluded from the results concerning which sterol structural feature is most important in facilitating mating. Cholesterol was found to be the second most efficient sterol for mating, and this sterol lacks the unsaturations at C-7, C-22 and the alkyl group at C-24, indicating that the absence of these features does not preclude efficient mating in
S. cerevisiue. The greatest structural difference between ergosterol and the poorest mating sterol, stigmasterol, is the presence of an ethyl group at C24 with an a orientation. The fact that sitosterol also contains a C-24 a-ethyl group but still allows for four-fold better mating than stigmasterol further complicates these findings. The occurrence of unsaturation at C-5 has been found to be a necessary structural feature for fulfillment of the sparking function in yeast (Rodriguez et ul., 1982, 1985; Rodriguez and Parks, 1983). A sterol lacking the C5 has not been tested for its ability to allow efficient mating. Clearly, a lot more sterols will have to be tested before we can fully understand how sterol structure is affecting this process. Even then, the results may not be unambiguous. We have shown
EFFECT OF STEROL ALTERATIONS ON CONJUGATION IN SA(XHAROMYCES CEREVISIAE
that the side-chain features of ergosterol appear to contribute cumulatively to feed-back inhibition of sterol synthesis (Casey et al., 1991). A similar situation may exist in the mating reactions. Regardless of which specific feature(s) of the sterol molecule facilitates mating in S. cerevisiae, we have shown that ergosterol plays a role in mating by demonstrating that cells previously growing on stigmasterol can be rescued from their mating deficiency by the introduction of ergosterol to these cells. While addition of only 0.5 pg/ml of ergosterol partially relieved the mating defect of the stigmasterol mating cells, 2.5 pg/ml of ergosterol allowed for complete restoration of mating ability. Working with the yeast strain RDSR, Rodriguez et ul. (1985) have shown that this amount of sterol is enough to fulfill the bulk role of sterol in S. cerevisiae. Probably no other eukaryotic cellular component has as many functional natural analogs as do the sterols. Indeed, attempting to show a precisely defined requirement for a specific feature of the ergosterol molecule can be exacerbated by imposed physiological adjustments in the cell. Yeast have a very efficient mechanism for excluding exogenously supplied sterols aerobically, presumably by allowing for a selective advantage for the use of ergosterol. It is paradoxical, therefore, that a myriad of sterols seem to be accommodated efficiently in the growth of the cells (Figure l), providing suitable mutations are present which permit the uptake of sterols from the media. We have shown here that at least one critical step in the cytoplasmic fusion even in conjugation has a marked preference for ergosterol. Our continuing challenge is to dissect that process and discern the sterol-dependent step. ACKNOWLEDGEMENTS This research was supported by the National Science Foundation (DCB881437), the National Institutes of Health of the U.S. Public Health Service (DK37222), U.S. Army Research Office staff research award to S.R.T. (DAAL03-89-D-003D7), and the North Carolina Agricultural Research Service. The authors gratefully acknowledge the assistance of Dr Kelly Tatchell, who provided cultures and facilities for light microscopy, Valerie Knowlton, who performed the ultramicrotomy work, and Dr David Chitwood, who provided sitosterol for these experiments. We also appreciate Dr Donna G. Cookmeyer's assistance in the preparation of this manuscript.
1023
REFERENCES Bailey, R. B. and Parks, L. W. (1975). Yeast sterol esters and their relationship to the growth of yeast. J. Bacteriol. 124,606-612. Cannon, J. F. and Tatchell, K. (1987). Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase. Mol. Cell. Biol. 7,2653-2663. Casey, W. M., Burgess, J. P. and Parks, L. W. (1991). Effect of sterol side-chain structure on the feed-back control of sterol biosynthesisin yeast. Biochim. Biophys. Acta 1081,279-284. Cross, F., Hartwell L. H., Jackson, C. and Konopka, J. B. (1988). Conjugation in Saccharomyces cerevisiae. Ann. Rev. Cell Biol. 4,429-457. Dahl, C., Biemann, H. P. and Dahl, J. (1987). A protein kinase antigenically related to pp60'-"" possibly involved in the yeast cell cycle control: Positive in vivo regulation by sterol. Proc. Natl. Acad. Sci. USA 84, 4012-4016. Dahl, J. S. and Dahl, C. E. (1985). Stimulation of cell proliferation and polyphosphinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol. Biochem. Biophys. Res. Comm. 133,844-850. Elion, E. A., Grisafi, P. L. and Fink, G. R. (1990). FUS3 encodes a cdc2 +/CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60, 649-664. Gollub, E. G., Liu, K., Dayan, J., Adlersberg, M. and Sprinson D. S. (1977). Yeast mutants deficient in heme biosynthesis and a heme mutant additionally blocked in cyclization of 2,3 oxidosqualene. J . Biol. Chem. 252, 2846-2854. Gonzales, R. A. and Parks, L. W. (1977).Acid-labilization of sterols for extraction from yeast. Biochim. Biophys. Acta 489,507-509. Hartwell, L. H. (1980). Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J. Cell Biol. 85,811-822. Hartwell, L. H. (1973). Synchronization of haploid yeast cell cycles, a prelude to conjugation. Exper. Cell Res. 76,111-1 12. Kawasaki, S., Rampogal M., Chin J. and Bloch, K. (1985). Sterol control of the phosphatidylethanolaminephosphatidylcholine conversion in the yeast mutant GL7. Proc. Natl. Acad. Sci. USA 82,5715-5719. Lewis, T. A., Taylor, F. R. and Parks, L. W. (1985). Involvement of heme biosynthesis in control of sterol uptake by Saccharomyces cerevisiae. J . Bacteriol. 163, 199-207. Lorenz, R. T. and Parks, L. W. (1990). Effects of lovastatin (mevinolin)on sterol levels and on activity of azoles in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 34,1660-1665. Lorenz, R. T., Casey, and Parks, L. W. (1989). Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae. J. Bacteriol. 17, 6169-61 73.
1024 Lorenz, R. T. and Parks, L. W. (1987). Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast. J. Bact. 169,3707-37 11. Low, C., Rodriguez, R. J. and Parks, L. W. (1985). Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol. Arch. Biochem. Biophys. 240,530-538. McCaffrey, G.,Clay, F. J., Kelsay, K. and Sprague, G . F. Jr. (1987). Identification and regulation of a gene required for cell fusion during mating of the yeast Saccharomyces cerevisiae. Mol Cell. Biol. 7,268&2690. Osumi, M. (1974). Mating reaction in Saccharomyces cerevisiae. Arch. Microbiol. 97,27-38. Papahadjopoulos, D., Poste, G., Schaeffer, B. E. and Vail, W. J. (1974). Membrane fusion and molecular segregation in phospholipid vesicles. Biochim. Biophys. Acta 352,lO-28. Parks, L. W. (1978). Metabolism of sterols in yeast. CRC Crit. Rev. Microbiol. 6,301-341. Pinto, W. J. and Nes, W. D. (1983). Stereochemical specificity for sterols in Saccharomyces cerevisiae. J . Biol. Chem. 258,4472-4476. Rampogal, M., Zundel, M . and Bloch, K. (1990). Sterol effects on phospholipid biosynthesis in the yeast strain GL7. J. Lipid Res. 31,653-658. Rampogal, M. and Bloch, K. (1983). Sterol synergism in yeast. Proc. Natl. Acad. Sci. USA 80,712-715.
M. E. TOME0 ET AL.
Rodriguez, R. J., Low,. C., Bottema, C. D. K. and Parks, L. W. (1985). Multiple functions for sterols in Saccharomyces cerevisiae. Biochim. Biophys. Acta 837, 336-343. Rodriguez, R.J. and Parks, L. W. (1983). Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking requirements in yeast sterol auxotrophs. Arch. Biochem. Biophys. 225, 861-871. Rodriguez, R. J., Taylor, F. R. and Parks, L. W. (1982). A requirement for ergosterol to permit growth of yeast sterol auxotrophs on cholestanol. Biochem. Biophys. Res. Comm. 106,435-441. Rodriguez, R. J. and Parks, L. W. (1982). Application of high-performance liquid chromatographic separation of free sterols to the screening of yeast sterol mutants. Anal. Biochem. 119,200-204. Shinabarger, D. L., Keesler, G. A. and Parks, L. W. (1989). Regulation by heme of sterol uptake in Saccharomyces cerevisiae. Steroids 53,607-623. Snyder, M. and Davis, R. W. (1988). S P A ] : a gene important for chromosome segregation and other mitotic functions in S. cerevisiae. Cell 54,743-754. Trueheart, J., Boeke, J. D. and Fink, G. R. (1987). Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced protein. MoZ. Cell. Biol. 7,23162328.
