Isolation and Characterization of Saccharomyces cerevisiae Mutants ...

Vol. 129, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1977, p. 1335-1342 Copyright © 1977 American Society for Microbiology

Printed in U.S.A.

Isolation and Characterization of Saccharomyces cerevisiae Mutants Defective in Glycerol Catabolism GEORGE F. SPRAGUE, JR.,1 AND JOHN E. CRONAN, JR.* Departments of Biology and of Molecular Biophysics and Biochemistry,* Yale University, New Haven, Connecticut 06510

Received for publication 16 August 1976

Mutants of the yeast Saccharomyces cerevisiae that are defective in the catabolism of glycerol were isolated, and two types of mutants were obtained. One type was deficient in glycerol kinase activity, whereas the other type was deficient in sn-glycerol 3-phosphate dehydrogenase activity. Genetic analysis indicated that each mutant strain owed its phenotype to a single nuclear mutation, and that the two mutations were complementary. The mutations were not linked to each other or to any of 10 loci tested. In addition, neither mutation was centromere linked. Possible mechanisms for the regulation of these enzymes were tested by growing the parental strain in the presence of various carbon sources.

Glycerol catabolism has been examined extensively in bacteria, particularly Escherichia coli. The studies of Lin and his co-workers have led to a genetic and biochemical description of the components of the glycerol catabolic pathway of this organism. These components include a glycerol kinase (EC 2.7.1.30) (9) and a membrane-bound sn-glycerol 3-phosphate (G3P) dehydrogenase (EC 1.1.99.5) (4, 11, 12), as well as proteins that are involved in the facilitated diffusion of glycerol (20), the transport of G3P (8), and the anaerobic oxidation of G3P (11, 12). The synthesis of these proteins is repressed by the gene product of a single regulatory gene, and specific repression is overcome by an inducing metabolite, G3P (3). In addition, the components of the pathway are under general catabolite control (5). Other species of bacteria possess comparable pathways for the dissimilation of glycerol. Generally, however, glycerol utilization has not been studied as thoroughly in other bacteria as it has been in E. coli. For example, Bacillus subtilis contains a glycerol kinase, a G3P dehydrogenase, a G3P permease, and a glycerol facilitator protein (16), and there is evidence that G3P is the inducer of the system. Strain differences in the inducibility of glycerol kinase have been reported (19). Pleiotropic mutants that cannot be induced for the components of the glycerol catabolic pathway have been isolated. However, constitutive revertants of these mutants have not been obtained, an observation that suggests a regulatory mechanism different I Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

from that found in E. coli (16). The diversity of glycerol catabolic pathways and their regulation among bacteria has been reviewed recently

(14).

Studies of glycerol catabolism in the fungi are still in their infancy. Gancedo et al. (6) have reported inducible glycerol kinase and G3P dehydrogenase activities in the yeast Candida utilis. Recently, Courtright (1, 2) described inducible glycerol kinase and G3P dehydrogenase activities in Neurospora crassa. The N. crassa glycerol kinase is located in the cytosol, whereas the G3P dehydrogenase is located in the mitochondria. A mutant deficient in the inducible glycerol kinase activity has been isolated (10). Glycerol can be used as the sole source of carbon and energy by the yeast Saccharomyces cerevisiae. The glycerol catabolic pathway of this yeast also includes a glycerol kinase (26) and a G3P dehydrogenase (21). Mutants defective in either of these enzyme activities have not been isolated previously. The isolation of such mutants as glycerol nonutilizers will provide good evidence that these enzymes constitute the major pathway of glycerol catabolism in S. cerevisiae. In this study we report the isolation and the biochemical and genetic characterization of such mutants. In addition, we have examined the effect of differing growth conditions on the levels of these enzymes in wild-type cells. (This work was part of a dissertation presented by G.F.S. to the faculty of Yale University in partial fulfillment of the requirements for the Ph.D. degree.)

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SPRAGUE AND CRONAN

MATERIALS AND METHODS Yeast strains. The strains of S. cerevisiae used in this study are listed in Table 1. Media and growth conditions. The minimal medium of Wickerham (25) was used (S medium). S-N medium lacked (NH4)2SO4. L-Amino acids and nucleotide bases were supplemented as needed at 20 ,ug/ml (adenine, uracil, histidine), 30 ug/ml (tyrosine, lysine, leucine, isoleucine), or 150 ,ug/ml (valine). SC medium, in addition to the nutrients listed above, contained: methionine, arginine, tryptophan (all at 20 4g/ml); phenylalanine (50 j,g/ml); glutamic acid (100 ,ug/ml); and serine (375 ,ug/ml). Scaa medium contained 0.1% vitamin-free Casamino Acids. YEP medium (1% yeast extract and 2% peptone [Difco], supplemented with adenine and uracil as above) was used for genetic manipulations. The sporulation regimen utilized presporulation medium (0.8% yeast extract, 0.3% peptone [Difco], and 10% dextrose) and sporulation medium (1% potassium acetate, 0.1% yeast extract, and 0.05% dextrose). Carbon sources (dextrose, glycerol, acetate, ethanol, or lactate) were added to 2%. Solid media contained 2% agar. Growth was followed in a Klett-Summerson colorimeter at 540 nm (green filter) (1 KU _ 2 x 105 cells/ ml). All experiments were performed while the cells were in the logarithmic phase of growth. The growth temperature was 30°C. Mutagenesis. A haploid strain of yeast was mutagenized withN-methyl-N'-nitro-N-nitrosoguanidine (NTG) as described by Hartwell (7). The mutagenized cells were washed by centrifugation, suspended in S lactate medium, and allowed to grow overnight. The culture was then passed through a nystatin enrichment procedure, as follows (22). The over-

J. BACTERIOL.

night culture was diluted and growth was permitted for one or two generations. The culture was then washed three times with S-N medium by centrifugation and resuspended in S-N medium. After 8 to 24 h of nitrogen starvation, the culture was pelleted by centrifugation and resuspended in S glycerol medium. When growth resumed, the antibiotic nystatin was added to 10 ,ug/ml. After 1 h of nystatin treatment, the culture was plated onto solid S lactate medium at a sufficient dilution to yield 100 to 200 colonies per petri plate. The colonies were transferred by replica-plating to S glycerol medium. Colonies that grew on the former but not the latter medium were considered presumptive glycerol nonutilizers. They were then purified by single-colony isolation on S lactate medium, and their phenotype was retested. They were characterized further by growth tests and finally by enzymatic assay. Preparation of crude enzyme extracts. Cultures (100 ml) of strains to be assayed were grown in S medium supplemented with the appropriate carbon source. While still in the logarithmic phase of growth, the cells were harvested by centrifugation, washed twice with 0.1 M (hydroxymethyl)aminomethane-hydrochloride, pH 7.4, and resuspended in 4.0 ml of this buffer. The cells were ruptured by passage twice through a French pressure cell at 18,000 lb/in2 (American Instrument Co., Silver Spring, Md.). The homogenate was centrifuged (14,000 x g for 20 min), and the supernatant fluid was used as the enzyme source. Enzyme assays. Glycerol kinase was assayed by two different procedures. Assay 1 was essentially the procedure of Lin et al. (15). The assay mixture contained 20 mM glycerol, 20 mM adenosine-5'-triphosphoric acid (ATP), 20 mM MgCl2, 1.5 mM nicotinamide adenine dinucleotide, oxidized form (NAD+), cell extract (0.1 to 0.5 mg of protein/ml), and 15 U of rabbit muscle G3P dehydrogenase per ml TABLE 1. Yeast strains in 0.4 M hydrazine sulfate-1 M glycine, pH 9.5. The Strain Source Genotype a production of reduced NAD was monitored spectroA364A a adel ade2 ural his7 L. H. Hart- photometrically at 340 nm using a Gilford recording spectrophotometer (model 240). One unit of enzyme tyrl Iys7 gall well reduced 1 ,umol of NAD+ per min. D286-2A a adel hisl F. Sherman Glycerol kinase was also assayed by measuring D273-11A a, adel hisl G. R. Fink the amount of [14C]G3P produced when [14C]glycerol D517-4B F. Sherman a ade2 lys9 was incubated with a crude enzyme preparation (asD603-1A a ade2 his7 G. R. Fink JB143 a ade2 leu2 P. T. Magee say 2), in a modification of the procedure of NewsM2 a trp ilv2 P. T. Magee holme et al. (18). The assay mixture contained 20 a hisl ilv2 M15 P. T. Magee mM MgCl2, 20 mM ATP, 25 mM NaF, 1 mM ethylXC38-74B a leu2 ura3 ade2 his4 P. T. Magee enediaminetetraacetic acid, 20 mM glycerol (1.6 IA.Ci/ ,tmol), cell extract (0.01 to 0.06 mg of protein), and gal2 80 mM tris(hydroxymethyl)aminomethane-hydroS5 gutl; other markers This paper chloride at an appropriate pH, in a final volume of as in strain A364A 0.1 ml. The reaction mixture was incubated at 30°C, S22 gut2; other markers This paper and the reaction was terminated at desired times by as in strain A364A the addition of 2 volumes of 95% ethanol. The samG1-16A,B,C,D Single-ascus segre- This paper ples were kept in ice for 30 min. They were then gants of S5 x JB143 applied to squares of diethylaminoethyl-cellulose G2-18A,B,C,D Single-ascus segre- This paper (Whatman DE81) paper, and the papers were gants of S22 x washed in bulk with the following series of soluJB143 tions: 4% (wt/vol) glycerol, water (three times), and a Genetic symbols are as described by Mortimer finally 95% ethanol. The papers were dried, and the and Hawthorne (17). For explanation of gutl and radioactivity was determined by liquid scintillation gut2, see text. counting using Aquasol I liquid scintillation cock-

YEAST GLYCEROL CATABOLISM MUTANTS

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not be capable of growth when glycerol is supplied as the sole source of carbon and energy. However, such mutants should be capable of growth when other nonfermentable substrates such as lactate or ethanol are supplied as carbon and energy sources. The mutant isolation procedure described above was based on these assumptions. When the isolation procedure was performed, several presumptive glycerol utilization mutants were obtained. After purification, the growth characteristics of the strains were tested. Those strains capable of growth when supplied with dextrose, lactate, ethanol, or acetate, but not glycerol, as carbon source were selected for biochemical analysis. Biochemical characterization of glycerol utilization mutants. Two presumptive mutant strains were initially selected for biochemical analysis. Extracts of these two strains and of the parental strain, A364A, were prepared. Each extract was assayed for glycerol kinase and G3P dehydrogenase activities. Strain A364A contained significant levels of each enzyme (Table 2). One of the two mutant strains, strain S5, lacked glycerol kinase activity, even when the concentration of extract protein exceeded that used in the assay of strain A364A by 20-fold. However, strain S5 possessed normal levels of G3P dehydrogenase activity. The second mutant strain, strain S22, lacked G3P dehydrogenase activity, whereas the level of glycerol kinase activity was normal (Table 2). To test for the presence of an inhibitor, extracts of the two mutant strains were mixed, and micromanipulator. Complementation tests were performed by glycerol kinase and G3P dehydrogenase activistreaking strains to be tested in a grid pattern on a ties were determined. In each case, the exYEPD agar plate. After a period of time to allow pected amount of enzyme activity was obmating at the intersections, the cells were trans- tained, indicating that the lack of detectable ferred by replicating to two minimal medium agar activity was not due to the production of an plates (one containing glycerol and the other con- inhibitor by either of the mutant strains (Table taining lactate as the carbon source). Growth at an 2). intersection on the S glycerol plate indicated comThe spectrophotometric assay for glycerol kiplementation. Chemicals and other supplies. Glusulase was nase activity must be performed at pH 9.5 for from Endo Laboratories, Garden City, N.Y. NTG efficient trapping of dihydroxyacetone phos-

tail. One unit of enzyme catalyzed the production of 1 jumol of G3P per min. G3P dehydrogenase was assayed according to the procedure of Lin et al. (15). The assay measures the production of the reduced derivative (formazan) of an artificial electron acceptor, [3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyl]tetrazolium bromide (MTT). Phenazine methosulfate was included in the assay as an intermediate electron acceptor. A molar extinction coefficient (E550) of 8.1 x 103 M-1 cm-' for the formazan derivative of MTT was used to calculate enzyme activity. One unit of enzyme caused the reduction of 1 ,umol of MTT per min. Protein concentrations were determined by the biuret method (13). Glycerol metabolism in vivo. The incorporation of glycerol into macromolecules was determined by adding [U-_4C]glycerol (specific activity, 5 ,uCi/200 mg) to a culture of cells growing logarithmically in Scaa medium with lactate as the carbon source. At the indicated time, an aliquot was removed and added to an equal volume of 10% trichloroacetic acid. Incorporation into the acid-insoluble fraction was determined by filtering the aliquots on glass fiber filters, washing three times with 5% acid, drying, and determining the radioactivity by liquid scintillation counting. Genetic analyses. Standard yeast genetic techniques were used for genetic analysis of mutant strains. Mating were done by mixing strains on YEP agar plates containing dextrose as carbon source (YEPD). Diploids were sporulated by allowing growth on presporulation agar plates for 1 day followed by incubation on sporulation agar plates for 3 to 5 days. The sporulated culture was suspended in sterile water and treated with glusulase. Tetrads were dissected on an agar slab using a de Fonbrune

from Aldrich Chemical Co., Milwaukee, Wis. MTT, phenazine methosulfate, rabbit muscle G3P dehydrogenase, NAD+, nystatin, and ATP (type II) were all from Sigma Chemical Co., St. Louis, Mo. [U-_4C]glycerol (250 ,Ci/0.17 mg) and Aquasol I liquid scintillation cocktail were from New England Nuclear Corp., Boston, Mass. Millipore filters (type HA, 0.45 am) were from Millipore Filter Corp., Bedford, Mass. DE81 paper and glass fiber filters (GF/ A) were from Whatman. The agar was Special AgarNoble from Difco Laboratories, Detroit, Mich. All other chemicals were reagent grade. was

RESULTS

Isolation of mutants. Mutants defective in glycerol kinase or G3P dehydrogenase should

TABLz 2. Glycerol kinase and G3P dehydrogenase activities in wild-type and mutant strains Sp act ([U/mg of protein] x 103)a Source of extract

Glycerol kinase

G3P dehydrogenase

38.6 16.0 A364A