Molec. gen. Genet. 156, 5 5 - 6 0 (1977) © by Springer-Verlag 1977
Genetic Studies with a Phosphoglucose Isomerase Mutant of Saccharomyces cerevisiae P. K. Maitra and Zita Lobo Tata Institute of Fundamental Research, Bombay 400005, India
Summary. A mutation pgil in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic
Materials and Methods
enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2 + :2 in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: t s m 1 3 4 - l y s 2 - p g i l - t y r l approximately 15 map units from tyrl. The mutation pgil defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgil locus.
Strains The wild type haploid strain of S. cerevisiae and glucose 6-phosphate isomerase-deficient mutant 9520b (called henceforth as pgil) have been described (Maitra, 1971). The following strains were obtained from the Yeast Genetic Stock Center at Berkeley, California: X2928-3D-1C, c~adel gall leul his2 ura3 trpl metl4; 2101-4C, a gall sue malhis5-2 trp5 2 leu2-1 lysl-1 tyr6-1 ilvl-1 arg; A364A, a adel ade2 ural his7 lys2 tyrl gall tsm134; 212-244-A1, gIcl. The diploid D649, a M A L 2 trpl pet6 ade2 lys2 mal his4 leu2 ade l thr4 was obtained from the Yeast Genetics Workshop at the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Media
Introduction Mutations affecting glucose 6-phosphate isomerase have been studied both biochemically and genetically in Escherichia coli (see Vinopal et al., 1974). A single locus controls the synthesis of this enzyme. The locus codes for a protein that could be either a regulatory or a structural element. A mutation causing the loss of phosphoglucose isomerase in Saccharomyces cerevisiae was reported from this laboratory several years ago (Maitra, 1971). The mutation was characterised biochemically and examination of the thermolability of the enzyme from the mutant and revertants had led to the suggestion that a structural determinant of phosphoglucose isomerease was affected. This paper reports the results of genetic experiments with this mutant. For offprints contact." P.K. Maitra
YEP Medium contained in 100 ml 1 g peptone, 0.3 g yeast extract and 1.5 g agar for solid media. 50 mM Sugars or 150 mM ethyl alcohol were added as the carbon source. The minimal medium used has been described by Winge and Roberts (1958). Supplements for auxotrophic markers were as described by Sherman et al. (i974). The mutant pgil was grown in YEP medium or in the minimal medium supplemented with fructose. Doubling periods were computed during exponential growth in shaken cultures at 30 °.
Genetic Methods Diploids were obtained by mass mating of two haploid parents of opposite mating types on YEP fructose plates and connterselecting the parents on minimal glucose media. Occasionally they were obtained by micromanipulating zygotes. A sliding micromanipulator was used in conjunction with an Ergaval microscope from Carl Zeiss Jena. Sporulation was achieved in agar plates containing potassium acetate and yeast extract, glucose being omitted. Dissection of tetrads was done by standard procedures (Sherman et al., 1974); spores were germinated in YEP fructose plates. Unless otherwise mentioned~ all tetrad analyses refer to asci in which all the four spores germinated, leaving out an occasional tetrad showing deviation from 2 + :2- segregation. Map distances were calculated using the formula cited by Sherman et al. (1974).
56
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase
Enzyme and Metabolite Assays
Table 1. Enzyme levels in haploid and diploid strains of yeast
These have been described (Maitra, 1971). Segregation of the pgi marker was followed in tetrads both by growth on YEP glucose plates and by assay of phosphoglucose isomerase in toluene lysates. For this, 107-108 cells from a colony or a streak were collected with a platinum loop in 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4 and containing 2 mM 2-mercaptoethanol and 2 mM EDTA. The procedure of toluenization was described earlier. Cell extracts made by a French pressure cell were used for more quantitative measurement of enzyme activities. Protein was determined by ultraviolet absorption (Layne, 1957).
Strain
PGI1 Haploid pgil Haploid PGII/PGI1 Diploid PGI1/pgil Diploid
Milliunits per mg protein Glucose 6-phosphate isomerase
Pyruvate kinase
1,940 15 1,775 550
3,880 13,300 3,500 4,600
Extracts were made from cells grown for 30 h on YEP fructose medium
Chemicals Sugars (Sigma Chemical Co.) used as carbon sources for growth of the pgi muta~nt were freed from contaminating glucose by incubating solutions with glucose oxidase (Worthington Biochemicals Corporation) and occasionally neutralising the acid produced with dilute alkali. These were sterilised by filtration. For digestion of asci glusulase (Endo Laboratories) and the snail enzyme (/~-glucuronidase and aryl sulphatase) from Boehringer were found equally effective. Most other enzymes and substrates were from Boehringer.
Results
Segregation of pgil The mutant 9520b carrying pgil was crossed with the strain X2928-3D-1C and the purified diploid was found to be glucose-positive unlike the haploid strain bearing the mutant phenotype. However, the activity of phosphoglucose isomerase in a diploid heterozygous for this locus (see below) was only a third of that of a homozygous diploid. This showed that the mutation was semidominant. In spore tetrads the mutation segregated 2-- :2- on the basis of growth on YEP glucose or minimal glucose media and by assay of phosphoglucose isomerase in spore clones. Further backcrosses of spores bearing pgil with appropriate strains wild type for this enzyme confirmed these results. At least 100 tetrads were tested for growth on glucose media and were found to segregate 2 + :2 ; approximately 20 of these tetrads were examined for phosphoglucose isomerase activity in toluene lysates. These results showed that the negativity of phosphoglucose isomerase was due to a single gene, pgil. The mutant 9520b was found to produce petite clones, designated 9520s, at a frequency of 90% (Maitra, 1971); however, this property was found to be recessive in heterozygous diploids. To see whether this trait was a property of the mutation pgil, the progeny spores were examined for growth on YEP alcohol plates and also streaked for single colonies on YEP fructose plates to look for segregation of small colonies. The property of petite generation and the marker pgi were found to be independent. However, unlike the character pgil which segregated as
2+:2 , the property of generating petite colonies did not segregate as a nuclear gene. In a particular cross of 9520b x wild type, 17 spores from 18 tetrads grew poorly on YEP alcohol. One spore bearing pgil that grew well on alcohol and did not segregate any petite progeny during vegetative propagation, was recrossed with another wild type; progeny spores from this cross gave the expected 2 + :2- segregation for the pgi character, while 6 spores from 20 tetrads were found to be segregating into big and small colony types; 2 of these 6 spores carried the mutated allele of the pgil gene. Attempts to sporulate diploids of 9520s x wild type were unsuccessful.
Nature of the pgil Mutation The mutation pgil was recovered from two successive backcrosses to strains wild type for this character and was hybridised with another wild type strain. Results in Table 1 compare the glucose 6-phosphate isomerase activities of haploid and diploid strains homozygous and heterozygous for this genotype. The heterozygous diploid PGI1/pgil has a much reduced activity of phosphoglucose isomerase. Their pyruvate kinase activities are also shown as controls. The results indicate that the activities of pyruvate kinase are similar in all the strains except in the phosphoglucose isomerase mutant. For the mutant the activity of pyruvate kinase and those of a number of glycolytic enzymes (not shown here) is much higher: This possibly reflects a higher steady state level o f glucose 6-phosphate that accumulates in the isomerase mutant under these conditions (Maitra and Lobo, 1971). Results in Figure 1 for the thermal inactivation of this activity indicate a biphasic kinetics (1.4 and 3.2 min half-lives) as against a monophasic kinetics (3.1 min half-life) for the homozygous wild type diploid. Further, the stabler of the two components in the heterozygous diploid decays nearly as fast as the activity from the homozygous wild type diploid.
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase
5/
This indicates a heterogeneity in the species of phosphoglucose isomerase synthesized by the former. Its reduced activity is thus consistent with the engagement of active subunits made by the wild type parent with the structurally faulty subunits made by the mutant parent. It had been shown earlier that yeast phosphoglucose isomerase is a dimer of identical subunits (Noltmann, 1972). Figure l also shows the pronounced heat sensitivity of the enzyme from the spontaneous glucose revertant T1CR3 confirming the earlier conclusion that the mutation pgil determines the structure of the enzyme (Maitra, 1971). However, the enzyme from the wild type loses activity much faster than was shown earlier. Whether this is due to the higher concentration of protein (12 mg per ml, versus 0.5 mg per ml) in the enzyme solutions during heating has not been determined.
100
50
20
o 10 -6 2~
I I 21-
Chromosomal Location 1
I 0It
T1CR 3 I 2
I 4
I I 6 8 Minutes
F [ 10 12 mt 60 C
I 14
I 16
_
Fig. 1. Thermoiability of glucose d-phosphate isomerase from strains of S. cerevisiae: a wild type diploid strain homozygous for the PGII character, PGI1/PGI1 ; a diploid strain heterozygous for this gene, pgil/PGI1; and a haploid glucose revertant, T1CR3. A fraction precipitating between 55% and 75% saturation of (NH4)2SO~ from extracts of cells grown on YEP glucose was used as the source of the enzyme. Heating for the periods indicated was done in a 25 m M potassium phosphate (pH 7.4) buffer containing 1 mM 2-mercaptoethanol, 1 m M E D T A a n d 0.5 mg bovine serum albumin per ml. Samples were chilled and assayed immediately. Results are expressed as percentages of the activity prior to heating
Table 2. Location o f p g i l on c h r o m o s o m e IIR of S. cerevisiae
Cross
Ascus type PD
NPD
T
Map distance cM
[
pgil-lys2 pgil-tsm 134 lys2-tsm134
39 11 27
1 5 1
49 40 20
31 62 27
II
pgit-tyrl
33
0
14
15
The parental configuration of markers in the diploids used in crosses I and II were respectively a
pgil
tsm134
lys2
adel
+
+
c~
+
+
+
+
metl
ural ade2
+
tyrl
+
trp4
+
adel
+
The segregation of pgil from crosses in which the centromere marker trpl (Mortimer and Hawthorne, 1975) was segregating indicated that it was not linked to any of the ccntromeres of S. cerevisiae. The second division segregation frequency was 62% (52 in 84 tetrads). The marker pgil was found to be linked to a number of landmarks on the right arm of chromosome II, viz., tsm134, lys2 and tyrl. However, germination of spores from diploids heterozygous for these four markers was very poor. Amongst nearly 200 tetrads examined in whichpgil was either in conjunction or in repulsion with any of tyrl, lys2 and tsm134 in various combinations, no more than 50 spores germinated. We have, however, been able to follow the segregation of pgil from tsm134 and lys2 in one diploid, and of pgil from tyrl in another. The results are shown in Table 2. The calculated map distances as also inspection of the exchange pattern of gene pairs in individual asci suggest the order: tsm134- lys2-pgil - tyrl, pgil being located closer to tyrl than to lys2 (Fig. 2). The marker glcl could not be tried since it did not produce any distinctive iodine coloration (Chester, 1967) in clones grown on fructose medium, a condition permissive for the marker pgil. However, we have found that strains bearing glcl have wild type levels of phosphoglucose isomerase activity.
+
Suppression
and a
c~ pgil
Earlier work had suggested (Maitra, 1971) that the mutant 9520b carrying pgil harboured a defect in the structural gene coding for phosphoglucose isomer-
58
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase I
©
cyh 1
r
i
gal I
tsm 134
i
i
lys2
pgil
I
tyr 1
Fig. 2. Location ofpgil on chromosome II of S. cerevisiae (Mortimer and Hawthorne, 1975)
Table 3. Segregation of suppressors ofpgil in crosses with wild type Revertant
Ascus types showing segregation of growth on glucose media 4 + :0
T1CR3 pgiR/3 T1CR1
9 4 9
3 + :10 0 23
2 + :20 0 9
ase. A cross was made between a suitable segregant carrying pgit and the strain 2101-4C carrying mutant alleles suppressible by nonsense suppressors; haploid segregants carrying some of these alleles together with pgil were reverted spontaneously for growth on glucose and 38 such revertants were examined for a coincident suppression of the nutritional markers. No simultaneous suppression of pgil with that of any of the nonsense alleles was seen. Two of the revertants (T1CR3 andj pgiR/3) were crossed to a wild type strain, the resultant diploids sporulated and tetrads were dissected tQ examine the segregation of the mutant and the suppressor loci. The germination of spores from such crosses was very poor; however, no glucose-negative spore could b e found amongst nearly 200 spores from each cross suggesting that the reversion mutations are very close to the pgil gene. This is consistent with intragenic suppression. Results in Table 3 show the analysis of the few 4-spore tetrads that germinated fully. When the phosphoglucose isomerase activity was examined in toluene lysates from these spore clones by heating them for 30 s at 60 °, two spores from each tetrad lost their enzyme activity by nearly 95% while those from the other two spores remained essentially unaltered or decreased up to 10%. This confirmed the 2 + :2 segregation of the wild type and the thermolabile revertant enzymes.
Characterisation of Enzyme-negative Revertant T1CR1 All but one of the glucose-positive revertants of the mutation pgil regained the enzyme activity. The sole exception was a revertant T1CR1 which grew on glucose but had no visible enzyme activity in excess of the mutant level. When crossed with the wild type,
T1CR1 segregated the suppressor gene (Table 3). The equality of the number of 4 + ' 0 - and 2 + :2- asci showed that the gene was unlinked to the pgil locus. The gene pgil coding for phosphoglucose isomerase segregated 2 + : 2 - in all the three tetrad classes, seen by examining toluenized cell suspensions for the enzyme activity. In order to rule out the possibility that the revertant T1CR1 had regained an enzyme activity too unstable to detect in cell extracts prepared either at 0 ° or in toluene lysates p r e p a r e d at 23 ° or at 37 °, the residual enzyme activity in the revertant was compared to that present in mutant extracts. The specific activities of phosphoglucose isomerase in the mutant and revertant extracts were 10 and 8 milliunits per mg protein respectively ; further, the enzyme activity in the mutant extract decayed at 60 ° with a halflife of 25 s, while that in the revertant had a half-life of 30 s. Both the extracts were able to catalyse the isomerisation of glucose 6-phosphate to fructose 6phosphate measured by coupling the latter with ATP and fructose 6-phophate kinase in an aldolase assay system (Maitra and Lobo, 1971). The revertant T1CR1 was found to be unable to make colonies on YEP glucose plates at 37 ° although at 30 ° it did; on YEP fructose and YEP mannose, however, it grew at both the temperatures. When further revertants of T1CR1 were collected on YEP glucose plates at 37 ° , there was no visible alteration seen in the specific activity or thermolability of phosphoglucose isomerase. We consider it very unlikely therefore that the revertant T1CR1 had regained by extragenic suppression a particularly unstable variant of glucose 6-phosphate isomerase. The suppressor mutation allowing growth of the revertant T1CR1 on glucose media is dominant over its wild type allele. The diploid obtained by crossing this revertant with a strain bearing pgil is able to grow on glucose. The inhibition of growth of the isomerase mutant by sugars was shown earlier to be related to the intracellular pool of glucose 6-phosphate (Maitra, 1971). A partial block in the enzymes catalysing glucose phosphorylation might then allow the mutant to grow on glucose. The revertant T 1CR1 was therefore examined for glucokinase and hexokinase activities. However, these enzyme levels in the revertant were comparable to those in the wild type. Further, double mutants lacking the isomerase and the hexokinases (obtained from crosses between the former and a hexokinaseless mutant) were glucosenegative although the rate of glucose phosphorylation was considerably reduced. A suppressor of pyruvate kinase mutation conferring growth on glucose media and causing decreased sugar phosphorylation (Maitra and L o b o , 1977) was also unable to restore growth of the pgil strains on glucose.
59
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase Table 4. Equilibrium of phosphoglucose isomerase reaction in wild type, revertants Strain
PGI a mu/mg protein
Wild type
1500
pgil
I2
T12A
Doubling time in YEP glu ~ h 1.8
> 30
Sugar added
pgil
strain and glucose
Rate of sugar utilisation /amoles m i n x g wet wt
laMoles/g wet cells
F6P G6P
F6P"
G6P a
Fru a Glu
4.7 8.8
0.3 0.6
1.2 2.5
0.25 0.24
Fru Glu
11.2 2.7
0.8
Genetic Studies with a Phosphoglucose Isomerase Mutant of Saccharomyces cerevisiae P. K. Maitra and Zita Lobo Tata Institute of Fundamental Research, Bombay 400005, India
Summary. A mutation pgil in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic
Materials and Methods
enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2 + :2 in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: t s m 1 3 4 - l y s 2 - p g i l - t y r l approximately 15 map units from tyrl. The mutation pgil defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgil locus.
Strains The wild type haploid strain of S. cerevisiae and glucose 6-phosphate isomerase-deficient mutant 9520b (called henceforth as pgil) have been described (Maitra, 1971). The following strains were obtained from the Yeast Genetic Stock Center at Berkeley, California: X2928-3D-1C, c~adel gall leul his2 ura3 trpl metl4; 2101-4C, a gall sue malhis5-2 trp5 2 leu2-1 lysl-1 tyr6-1 ilvl-1 arg; A364A, a adel ade2 ural his7 lys2 tyrl gall tsm134; 212-244-A1, gIcl. The diploid D649, a M A L 2 trpl pet6 ade2 lys2 mal his4 leu2 ade l thr4 was obtained from the Yeast Genetics Workshop at the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Media
Introduction Mutations affecting glucose 6-phosphate isomerase have been studied both biochemically and genetically in Escherichia coli (see Vinopal et al., 1974). A single locus controls the synthesis of this enzyme. The locus codes for a protein that could be either a regulatory or a structural element. A mutation causing the loss of phosphoglucose isomerase in Saccharomyces cerevisiae was reported from this laboratory several years ago (Maitra, 1971). The mutation was characterised biochemically and examination of the thermolability of the enzyme from the mutant and revertants had led to the suggestion that a structural determinant of phosphoglucose isomerease was affected. This paper reports the results of genetic experiments with this mutant. For offprints contact." P.K. Maitra
YEP Medium contained in 100 ml 1 g peptone, 0.3 g yeast extract and 1.5 g agar for solid media. 50 mM Sugars or 150 mM ethyl alcohol were added as the carbon source. The minimal medium used has been described by Winge and Roberts (1958). Supplements for auxotrophic markers were as described by Sherman et al. (i974). The mutant pgil was grown in YEP medium or in the minimal medium supplemented with fructose. Doubling periods were computed during exponential growth in shaken cultures at 30 °.
Genetic Methods Diploids were obtained by mass mating of two haploid parents of opposite mating types on YEP fructose plates and connterselecting the parents on minimal glucose media. Occasionally they were obtained by micromanipulating zygotes. A sliding micromanipulator was used in conjunction with an Ergaval microscope from Carl Zeiss Jena. Sporulation was achieved in agar plates containing potassium acetate and yeast extract, glucose being omitted. Dissection of tetrads was done by standard procedures (Sherman et al., 1974); spores were germinated in YEP fructose plates. Unless otherwise mentioned~ all tetrad analyses refer to asci in which all the four spores germinated, leaving out an occasional tetrad showing deviation from 2 + :2- segregation. Map distances were calculated using the formula cited by Sherman et al. (1974).
56
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase
Enzyme and Metabolite Assays
Table 1. Enzyme levels in haploid and diploid strains of yeast
These have been described (Maitra, 1971). Segregation of the pgi marker was followed in tetrads both by growth on YEP glucose plates and by assay of phosphoglucose isomerase in toluene lysates. For this, 107-108 cells from a colony or a streak were collected with a platinum loop in 0.5 ml of 50 mM potassium phosphate buffer, pH 7.4 and containing 2 mM 2-mercaptoethanol and 2 mM EDTA. The procedure of toluenization was described earlier. Cell extracts made by a French pressure cell were used for more quantitative measurement of enzyme activities. Protein was determined by ultraviolet absorption (Layne, 1957).
Strain
PGI1 Haploid pgil Haploid PGII/PGI1 Diploid PGI1/pgil Diploid
Milliunits per mg protein Glucose 6-phosphate isomerase
Pyruvate kinase
1,940 15 1,775 550
3,880 13,300 3,500 4,600
Extracts were made from cells grown for 30 h on YEP fructose medium
Chemicals Sugars (Sigma Chemical Co.) used as carbon sources for growth of the pgi muta~nt were freed from contaminating glucose by incubating solutions with glucose oxidase (Worthington Biochemicals Corporation) and occasionally neutralising the acid produced with dilute alkali. These were sterilised by filtration. For digestion of asci glusulase (Endo Laboratories) and the snail enzyme (/~-glucuronidase and aryl sulphatase) from Boehringer were found equally effective. Most other enzymes and substrates were from Boehringer.
Results
Segregation of pgil The mutant 9520b carrying pgil was crossed with the strain X2928-3D-1C and the purified diploid was found to be glucose-positive unlike the haploid strain bearing the mutant phenotype. However, the activity of phosphoglucose isomerase in a diploid heterozygous for this locus (see below) was only a third of that of a homozygous diploid. This showed that the mutation was semidominant. In spore tetrads the mutation segregated 2-- :2- on the basis of growth on YEP glucose or minimal glucose media and by assay of phosphoglucose isomerase in spore clones. Further backcrosses of spores bearing pgil with appropriate strains wild type for this enzyme confirmed these results. At least 100 tetrads were tested for growth on glucose media and were found to segregate 2 + :2 ; approximately 20 of these tetrads were examined for phosphoglucose isomerase activity in toluene lysates. These results showed that the negativity of phosphoglucose isomerase was due to a single gene, pgil. The mutant 9520b was found to produce petite clones, designated 9520s, at a frequency of 90% (Maitra, 1971); however, this property was found to be recessive in heterozygous diploids. To see whether this trait was a property of the mutation pgil, the progeny spores were examined for growth on YEP alcohol plates and also streaked for single colonies on YEP fructose plates to look for segregation of small colonies. The property of petite generation and the marker pgi were found to be independent. However, unlike the character pgil which segregated as
2+:2 , the property of generating petite colonies did not segregate as a nuclear gene. In a particular cross of 9520b x wild type, 17 spores from 18 tetrads grew poorly on YEP alcohol. One spore bearing pgil that grew well on alcohol and did not segregate any petite progeny during vegetative propagation, was recrossed with another wild type; progeny spores from this cross gave the expected 2 + :2- segregation for the pgi character, while 6 spores from 20 tetrads were found to be segregating into big and small colony types; 2 of these 6 spores carried the mutated allele of the pgil gene. Attempts to sporulate diploids of 9520s x wild type were unsuccessful.
Nature of the pgil Mutation The mutation pgil was recovered from two successive backcrosses to strains wild type for this character and was hybridised with another wild type strain. Results in Table 1 compare the glucose 6-phosphate isomerase activities of haploid and diploid strains homozygous and heterozygous for this genotype. The heterozygous diploid PGI1/pgil has a much reduced activity of phosphoglucose isomerase. Their pyruvate kinase activities are also shown as controls. The results indicate that the activities of pyruvate kinase are similar in all the strains except in the phosphoglucose isomerase mutant. For the mutant the activity of pyruvate kinase and those of a number of glycolytic enzymes (not shown here) is much higher: This possibly reflects a higher steady state level o f glucose 6-phosphate that accumulates in the isomerase mutant under these conditions (Maitra and Lobo, 1971). Results in Figure 1 for the thermal inactivation of this activity indicate a biphasic kinetics (1.4 and 3.2 min half-lives) as against a monophasic kinetics (3.1 min half-life) for the homozygous wild type diploid. Further, the stabler of the two components in the heterozygous diploid decays nearly as fast as the activity from the homozygous wild type diploid.
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase
5/
This indicates a heterogeneity in the species of phosphoglucose isomerase synthesized by the former. Its reduced activity is thus consistent with the engagement of active subunits made by the wild type parent with the structurally faulty subunits made by the mutant parent. It had been shown earlier that yeast phosphoglucose isomerase is a dimer of identical subunits (Noltmann, 1972). Figure l also shows the pronounced heat sensitivity of the enzyme from the spontaneous glucose revertant T1CR3 confirming the earlier conclusion that the mutation pgil determines the structure of the enzyme (Maitra, 1971). However, the enzyme from the wild type loses activity much faster than was shown earlier. Whether this is due to the higher concentration of protein (12 mg per ml, versus 0.5 mg per ml) in the enzyme solutions during heating has not been determined.
100
50
20
o 10 -6 2~
I I 21-
Chromosomal Location 1
I 0It
T1CR 3 I 2
I 4
I I 6 8 Minutes
F [ 10 12 mt 60 C
I 14
I 16
_
Fig. 1. Thermoiability of glucose d-phosphate isomerase from strains of S. cerevisiae: a wild type diploid strain homozygous for the PGII character, PGI1/PGI1 ; a diploid strain heterozygous for this gene, pgil/PGI1; and a haploid glucose revertant, T1CR3. A fraction precipitating between 55% and 75% saturation of (NH4)2SO~ from extracts of cells grown on YEP glucose was used as the source of the enzyme. Heating for the periods indicated was done in a 25 m M potassium phosphate (pH 7.4) buffer containing 1 mM 2-mercaptoethanol, 1 m M E D T A a n d 0.5 mg bovine serum albumin per ml. Samples were chilled and assayed immediately. Results are expressed as percentages of the activity prior to heating
Table 2. Location o f p g i l on c h r o m o s o m e IIR of S. cerevisiae
Cross
Ascus type PD
NPD
T
Map distance cM
[
pgil-lys2 pgil-tsm 134 lys2-tsm134
39 11 27
1 5 1
49 40 20
31 62 27
II
pgit-tyrl
33
0
14
15
The parental configuration of markers in the diploids used in crosses I and II were respectively a
pgil
tsm134
lys2
adel
+
+
c~
+
+
+
+
metl
ural ade2
+
tyrl
+
trp4
+
adel
+
The segregation of pgil from crosses in which the centromere marker trpl (Mortimer and Hawthorne, 1975) was segregating indicated that it was not linked to any of the ccntromeres of S. cerevisiae. The second division segregation frequency was 62% (52 in 84 tetrads). The marker pgil was found to be linked to a number of landmarks on the right arm of chromosome II, viz., tsm134, lys2 and tyrl. However, germination of spores from diploids heterozygous for these four markers was very poor. Amongst nearly 200 tetrads examined in whichpgil was either in conjunction or in repulsion with any of tyrl, lys2 and tsm134 in various combinations, no more than 50 spores germinated. We have, however, been able to follow the segregation of pgil from tsm134 and lys2 in one diploid, and of pgil from tyrl in another. The results are shown in Table 2. The calculated map distances as also inspection of the exchange pattern of gene pairs in individual asci suggest the order: tsm134- lys2-pgil - tyrl, pgil being located closer to tyrl than to lys2 (Fig. 2). The marker glcl could not be tried since it did not produce any distinctive iodine coloration (Chester, 1967) in clones grown on fructose medium, a condition permissive for the marker pgil. However, we have found that strains bearing glcl have wild type levels of phosphoglucose isomerase activity.
+
Suppression
and a
c~ pgil
Earlier work had suggested (Maitra, 1971) that the mutant 9520b carrying pgil harboured a defect in the structural gene coding for phosphoglucose isomer-
58
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase I
©
cyh 1
r
i
gal I
tsm 134
i
i
lys2
pgil
I
tyr 1
Fig. 2. Location ofpgil on chromosome II of S. cerevisiae (Mortimer and Hawthorne, 1975)
Table 3. Segregation of suppressors ofpgil in crosses with wild type Revertant
Ascus types showing segregation of growth on glucose media 4 + :0
T1CR3 pgiR/3 T1CR1
9 4 9
3 + :10 0 23
2 + :20 0 9
ase. A cross was made between a suitable segregant carrying pgit and the strain 2101-4C carrying mutant alleles suppressible by nonsense suppressors; haploid segregants carrying some of these alleles together with pgil were reverted spontaneously for growth on glucose and 38 such revertants were examined for a coincident suppression of the nutritional markers. No simultaneous suppression of pgil with that of any of the nonsense alleles was seen. Two of the revertants (T1CR3 andj pgiR/3) were crossed to a wild type strain, the resultant diploids sporulated and tetrads were dissected tQ examine the segregation of the mutant and the suppressor loci. The germination of spores from such crosses was very poor; however, no glucose-negative spore could b e found amongst nearly 200 spores from each cross suggesting that the reversion mutations are very close to the pgil gene. This is consistent with intragenic suppression. Results in Table 3 show the analysis of the few 4-spore tetrads that germinated fully. When the phosphoglucose isomerase activity was examined in toluene lysates from these spore clones by heating them for 30 s at 60 °, two spores from each tetrad lost their enzyme activity by nearly 95% while those from the other two spores remained essentially unaltered or decreased up to 10%. This confirmed the 2 + :2 segregation of the wild type and the thermolabile revertant enzymes.
Characterisation of Enzyme-negative Revertant T1CR1 All but one of the glucose-positive revertants of the mutation pgil regained the enzyme activity. The sole exception was a revertant T1CR1 which grew on glucose but had no visible enzyme activity in excess of the mutant level. When crossed with the wild type,
T1CR1 segregated the suppressor gene (Table 3). The equality of the number of 4 + ' 0 - and 2 + :2- asci showed that the gene was unlinked to the pgil locus. The gene pgil coding for phosphoglucose isomerase segregated 2 + : 2 - in all the three tetrad classes, seen by examining toluenized cell suspensions for the enzyme activity. In order to rule out the possibility that the revertant T1CR1 had regained an enzyme activity too unstable to detect in cell extracts prepared either at 0 ° or in toluene lysates p r e p a r e d at 23 ° or at 37 °, the residual enzyme activity in the revertant was compared to that present in mutant extracts. The specific activities of phosphoglucose isomerase in the mutant and revertant extracts were 10 and 8 milliunits per mg protein respectively ; further, the enzyme activity in the mutant extract decayed at 60 ° with a halflife of 25 s, while that in the revertant had a half-life of 30 s. Both the extracts were able to catalyse the isomerisation of glucose 6-phosphate to fructose 6phosphate measured by coupling the latter with ATP and fructose 6-phophate kinase in an aldolase assay system (Maitra and Lobo, 1971). The revertant T1CR1 was found to be unable to make colonies on YEP glucose plates at 37 ° although at 30 ° it did; on YEP fructose and YEP mannose, however, it grew at both the temperatures. When further revertants of T1CR1 were collected on YEP glucose plates at 37 ° , there was no visible alteration seen in the specific activity or thermolability of phosphoglucose isomerase. We consider it very unlikely therefore that the revertant T1CR1 had regained by extragenic suppression a particularly unstable variant of glucose 6-phosphate isomerase. The suppressor mutation allowing growth of the revertant T1CR1 on glucose media is dominant over its wild type allele. The diploid obtained by crossing this revertant with a strain bearing pgil is able to grow on glucose. The inhibition of growth of the isomerase mutant by sugars was shown earlier to be related to the intracellular pool of glucose 6-phosphate (Maitra, 1971). A partial block in the enzymes catalysing glucose phosphorylation might then allow the mutant to grow on glucose. The revertant T 1CR1 was therefore examined for glucokinase and hexokinase activities. However, these enzyme levels in the revertant were comparable to those in the wild type. Further, double mutants lacking the isomerase and the hexokinases (obtained from crosses between the former and a hexokinaseless mutant) were glucosenegative although the rate of glucose phosphorylation was considerably reduced. A suppressor of pyruvate kinase mutation conferring growth on glucose media and causing decreased sugar phosphorylation (Maitra and L o b o , 1977) was also unable to restore growth of the pgil strains on glucose.
59
P.K. Maitra and Z. Lobo: Genetics of Yeast G6P Isomerase Table 4. Equilibrium of phosphoglucose isomerase reaction in wild type, revertants Strain
PGI a mu/mg protein
Wild type
1500
pgil
I2
T12A
Doubling time in YEP glu ~ h 1.8
> 30
Sugar added
pgil
strain and glucose
Rate of sugar utilisation /amoles m i n x g wet wt
laMoles/g wet cells
F6P G6P
F6P"
G6P a
Fru a Glu
4.7 8.8
0.3 0.6
1.2 2.5
0.25 0.24
Fru Glu
11.2 2.7
0.8
