Current Genetics
Current Genetics (1983) 7 : 3 9 3 - 397
© Springer-Verlag 1983
Trehalose: Its Role in Germination of Saccharomyces cerevisiae Anita D. Panek and Edilson J. Bernardes Departamento de Bioqu~mica, Instituto de Qulmica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, 21941, Rio de Janeiro, RJ, Brasil
Summary. Mutants with specific lesions were used to differenciate between the functions of glycogen and trehalose in S. cerevisiae. Diploids which harbor the glcl/glcl mutation depend upon the phosphorylated, less active form of glycogen synthase and show a more active, phosphorylated form, of the enzyme trehalase. These conditions are due to a lesion in the regulating subunit of the cAMP-dependent protein kinase. Such cells are unable to sporulate. Diploids which contain the sstl/sstl mutation have normal glycogen metabolism but their trehalose-6-phosphate synthase is not active. Such strains sporulate but germination is poor and only one-spore tetrads are formed. These results confirm that glycogen is needed to trigger sporulation while trehalose plays a role in the germination process. Different systems, I and II, of trehalose accumulation were proposed. System I would require the UDPG-linked trehalose synthase, whereas system II would constitute an alternative pathway, specifically induced or activated by the expression of a MAL gene. The presence of system II in its constitutive form in the constructed diploids would favour trehalose synthesis during growth on glucose, however, it did not overcome the glycogen deficiency during sporulation nor the lack of trehalose for germination. It seems that only system I, namely trehalose 6-P-synthase, plays a role in the germination process. Key
words:
Germination
Trehalose
-
Glycogen
-
- Saccharomyces cerevisiae
Offprint requests to: A. D. Panek
Sporulation
-
Introduction
The significance of two storage carbohydrates in yeast cells has intrigued investigators over the years. It is clear that in view of the many aspects of a cell cycle maintenance, differentiation and survival - no exclusive role should be assigned to any energy reserve. However, nature must have had some reason for providing the yeast cell with both glycogen and trehalose. The accumulation of trehalose under conditions leading to sporulation was reported by Roth in 1970. In a following paper Kane and Roth (l 974) demonstrated that trehalose and glycogen are synthesized to the same extent in both sporulating and non-sporulating strains. However, only in sporulating cells is glycogen synthesis followed by a period of breakdown coincident with the appearance of mature spores. Corroborating these results Colonna and Magee (1978) and Clancy et al. (1982) demonstrated that under these conditions a sporulation specific amyloglucosidase activity is responsible f o r glycogenolysis. It should be emphasized that in all these investigations different diploid strains of Saceharomyces cerevisiae are employed and no specific role for trehalose was assigned. Recently, Thevelein et al. (1982) demonstrated by NMR spectroscopy that trehalose in Pichia pastoris is rapidly mobilized upon induction of germination. The use of mutants harboring well defined deficiencies will certainly bring forth unequivocal evidence and permit better insight into this problem. This paper intends to demonstrate that trehalose does not seem to be required for sporulation but plays a definite role during germination of S. cerevisiae while glycogen is indeed a pre-requisite for spore formation. The significance of the alternative pathway for trehalose synthesis (Operti et al. 1982) is also discussed in relation to spore formation and germination.
394
A.D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination ofSaccharomyees cerevisiae
Table 1. Yeast strains employed: genotypes and sources Diploids
No
a parent genotype
No
a parent genotype
Source of haploids
DE4 DE6 AP5 AP7 AP8 AP9 AP10 AP11 NT1 NT2
PG1-2C DE4-3A 20Q1-3B DE6-5D BR15-14B DE6-5D DE6-5D NT2-5 B 7Q-2A 7Q-2A
ade 2-40 real GLC SST ade 2-40 MAL2 c GLC SST glcl-2 mal gal2 SST lys2 MAL2 c sstl GLC glcl-1 MAL4 c his1 SST lys2 MAL2 c sstl GLC lys2 MAL2 c sstl GLC glc4-1 real gal2 SST glc4-1 mal gal2 S S T glc4-1 mal gal2 S S T
PS-8C Q6R2 DE6-4A DE6-4D BR15-15D Q6R2 DE6-4A NT2-4A GS1-36 DE6-4A
ade 2-40 MAL2-47 c GLC SST lys2 MAL6 sstl GLC lys2 maI sstl GLC lys2 G L C M A L 2 c s s t l . glcl-1 MAL4 e ura3 trpl S S T lys2 MAL6 sstl GLC lys2 mal sstl GLC glc4-1 real gal2 S S T glcl-1 mal gal2 S S T lys2 mal sstl GLC
(1) (2, 3) (4, 2) (2) (2) (2, 3) (2) (2) (5, 6) (2)
Source key: (1) F. Zimmermann, Technische Hochschule, Darmstadt, GFR (2) prepared at the Universidade Federal do Rio de Janeiro, Brazil (3) spontaneous revertant of strain Q6 from K. van de Poll, Rejks Universiteit, Utrecht, Netherlands (4) H. Ruis, Institut fur Algemeinen Biochimie, Wien, Austria (5) J. Pringle, University of Michigan, USA (6) E. Cabib, National Institutes of Health, Bethesda, USA
Material and Methods Yeast Cultures. The genotypes of the various strains used in this study are listed in Table 1. Strain Q6R2 is derived from Saccharomyces carlsbergensis and the remaining strains from S. eerevisiae. All diploids were obtained in our laboratory. Isolation, induction of sporulation and dissections were performed as described by Hawthorn and Mortimer (1960) and by Sherman (1963). Glycogen was detected by staining the colonies directly on plates with a 0.2% 12 in 0.4% KI solution (Chester 1967). Trehalose 6-phosphate synthase activity was determined by a modification of the classical method as described by Operti et al. (1982). The presence of trehalose was detected by incubating the cells in buffered glucose, extracting with 50 mM TCA and determining trehalose by the anthrone method as described by Oliveira et al. (1981).
Results and Discussion Strains of S a c c h a r o m y c e s harboring two different mutant alleles were used to construct the diploids. The s s t l mutation is characterized by the absence o f trehalose 6-phosphate synthase activity or o f any other isozyme which would catalyze the synthesis of trehalose from ADPG or GDPG instead o f UDPG (Operti et al. 1982). The g l c l mutation is a regulatory pleiotropic mutation which reduces glycogen and trehalose accumulation (Rothman-Denes and Cabib 1970; Padr~o et al. 1982). With respect to glycogen, the mutation was described as affecting the conversion o f glycogen synthase from its less active (D) form to the more active (I) form. The absence of trehalose accumulation is due to a defect in protein kinase regulation which causes the hydrolytic enzyme, trehalase, to remain always in its phosphorylated,
active form (Ortiz et al. 1983). The glc4 mutation has a very similar phenotype. All diploids containing double glc mutations sporulated very poorly (Table 2). A similar result was reported for a diploid homozygous for the g l c l - 1 gene (Pringle et al. 1974). Diploid AP8 contains, moreover, the dominant, constitutive M A L 4 gene. As previously reported (Operti et al. 1982), the constitutivity of a M A L gene induces or activates an alternative system o f trehalose synthesis (system II). The introduction Of a MAL gene into a g l c l - 1 mutant overcomes the deficiency in trehalase regulation by allowing for trehalose accumulation during growth through a pathway not directly controlled by the UDPG linked trehalose-6phosphate synthase (Panek et al. 1978). The idea was to verify whether a diploid containing this alternative pathway o f trehalose synthesis could overcome the deficient carbohydrate accumulation caused by the g l c l - 1 mutation and provide energy for sporulation. The extremely low sporulation observed with diploid AP8 (less than 3%) shows that this is not the case. The presence of glycogen is indeed a pre-requisite for the formation o f spores in S a c c h a r o m y c e s . All other diploid strains analyzed sporulated and gave a positive test for glycogen (Table 2). These diploids were allowed to germinate. An analysis of the data in Table 3 shows a significant influence of trehalose 6-phosphate synthase activity (system I) upon the germination capacity o f the spores. Heterozygous diploids for the s s t l gene which controls trehalose synthase activity originated spores which germinated with lower efficiency (strains DE6, AP5 and
395
A. D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination of Saccharomyces cerevisiae Table 2. Sporulation and glycogen accumulation Strains
Genotype
I2-KI test
Sporulation
DE4
SST GLC
++
normal
++
normal
+
normal
+++
normal
SST GLC
DE6
SST GLC sstl GLC
AP5
SST GLC sstl glcl
AP7
sstl GLC sstl GLC
AP8
none
SST glcl.1 SST glcl.1
AP9
sstl GLC
++
low
++
low
-
none
+
none
+
normal
sstl GLC
AP10
sstl GLC sstl GLC
AP11
SST glc4-1 SST glc4-1
NT1
SST glc4-1 SST glel-1
NT2
SST GLC sstl glc4-1
NT2). Introduction of a g l c l deficient allele (diploid AP5) had no effect upon the total percentage of germination when compared to strain DE6. The same can be said about the g l c 4 allele in diploid NT2. There was, however, a low percentage of 4 spore tetrads in both cases which suggests that the glc sst condition might be lethal (Table 3). Some g l c l - 2 segregants were assayed and showed normal trehalose-6-phosphate activity "in vitro" suggesting that the parental types predominate in the segregation (Table 4). In glycogen negative segregants with normal trehalose synthase activity, no accumulation of trehalose was observed due to the activation of trehalase by the glc mutation. When we looked at diploids homozygous for s s t l , no 4 spore tetrads were seen at all (strains AP7, AP9 and AP10). Of the 46 asci dissected from these diploids which are characterized by a homozygous lesion in trehalose-6-phosphate synthase (system I) but normal glycogen accumulation capacity, the total germination obtained (Table 3) corresponded mostly to one-spore tetrads. The presence of a M A L constitutive gene in these diploids did not improve the pattern of germination. Here again, the existence of the alternative pathway of trehalose synthesis which would operate during growth, did not overcome the trehalose-6-P-synthase deficiency. This result strengthens our working hypothesis that cells of S a c c h a r o m y c e s are able to form a second independent pool of trehalose, through system I1, which might have a specific function in regulating the efficient uptake of
Table 3. Role of storage carbohydrates during germination Diploids
Genotype
Dissected Asci
Germination (%)
4 spore tetrads (%)
DE4
SST GLC
22
87.0
60.0
22
52.0
27.0
20
50.0
15.0
32
17.0
0.0
7
10.0
0.0
7
21.0
0.0
10
42.5
0.0
SST GLC
DE6
SST GLC sstl GLC
AP5
SST GLC sstl glcl-2
AP7
sstl GLC sstl GLC
AP9
sstl GLC sstl GLC
AP10
sstl GLC sstl GLC
NT2
S S T GLC sstl glc4-1
396
A.D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination of S a c c h a r o m y c e s
eerevisiae
Table 4. Segregation o f g l c and s s t mutations Strain
AP5-1A 1B
Glycogen synthesis
Trehalose synthesis
Genotype
Trehalose-6-Psynthase activity
-
-
glc S S T
+
-
-
glc S S T
+
1C
+
-
GLC sst
1D
+
-
GLC sst
n.d. n.d.
-
glc S S T
+
-
-
glc S S T
+
+
-
GLC sst
+
-
GLC sst
n.d. n.d.
AP5-2A 2B 2C 2D AP5-3A 3B 3C 3D
-
-
glc S S T
+
+
-
GLC sst
+
-
GLC sst
+
-
GLC sst
n.d. n.d. n.d.
Parental phenotypes were determined by the I2-KI test and trehalose synthesis in non proliferating conditions. In segregants negative for glycogen, trehalose-6-phosphate synthase was measured (see Methods); n.d. = not determined
maltose. Only system I would contribute for the accumulation required during germination. Diploids which harbor the g l c l / g l c l mutation do not convert glycogen synthase into the more active, independent form; moreover, trehalase activity is always high. Such strains do, probably, not synthesize glycogen and they rapidly breakdown any trehalose that might be formed: they do not sporulate. Diploids which harbour the s s t l / s s t l mutation have normal glycogen metabolism but they do not synthesize trehalose. Such strains sporulate confirming that glycogen accumulation is required to trigger this process. If we assume that the spores formed by the three sstl/sstl diploids are not defective themselves and would be able to germinate should they possess normal trehalose 6-phosphate synthase activity, than the results here presented point towards a specific function for trehalose in the process of germination o f S a c c h a r o m y c e s , dependent upon the activity of the classical trehalose synthase described by Cabib and Leloir (1958). For the fission yeast S c h i z o s a c c h a r o r n y c e s pombe there is good evidence that both accumulation o f trehalose and induction of trehalase activity during sporulation are the preparatory steps for germination and not necessarily a pre-requisite for sporulation itself (Inoue and Shimoda 1981). In ascospores of P i c h i a p a s t o r i s trehalose accumulates in dormant spores because of the low activity o f trehalase and its strong inhibition by ATP. During early germination, energy is supplied by a rapid breakdown o f trehalose due to a highly active trehalase (Thevelein et al. 1982). The trehalase activation process could play an essential role in the induction of germination but
spores would have to contain the disaccharide which is not the case with the s s t l / s s t l mutants. Cause and effect relations are difficult to establish but it is stimulating to believe that studies with well characterized mutants will allow more definite conclusions to be drawn. Clearly, trehalose does not play a unique role in the regulation of energy metabolism in yeast. Its function during starvation or adaptation to new culture conditions (Panek 1975; Lillie and Pringle 1980; Panek, 1963) or as osmoregulator of the cytosol (Keller et al. 1982) should not be overlooked. It is rewarding to speculate why nature provided yeast cells with two different storage carbohydrates and assigned a specific role in sporulation for the polysaccharide, whereas, the disaccharide, trehalose, was chosen for the germination step. This work was supported by grants from CNPq, CEPG, FINEP and FUJB; our thanks to Mauro S. Operti for the enzyme assays and to Vaine R. Reis for assistance in preparing the manuscript.
Acknowledgements.
References Cabib E, Leloir LF (1958) J Biol Chem 231:259-275 Chester, VE (1967) Nature 214:1237-1238 Clancy MJ, Smith LM, Magee PT (1982) Mol Cel Biol 2:171178 Colonna WJ, Magee PT (1978) J Bacteriol 134:844-853 Hawthorne DC, Mortimer RK (1960) Genetics 45:1085-1110 Inoue H, Shimoda C (1981) Mol Gen Genet 183:32-36 Kane SM, Roth R (1974) J Bacteriol 118:8-14 Keller F, Schellenberg M, Wiemken A (1982) Arch Microbiol 131:298-301
A. D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination of Saccharomyces cerevisiae Lillie SH, Pringle JR (1980) J Bacteriol 143:1384-1394 Oliveira DE, Rodrigues EGC, Mattoon JR, Panek AD (1981) Curr Genet 3 : 2 3 5 - 2 4 2 Operti MS, Oliveira DE, Freitas-Valle AB, Oestreicher EG, Mattoon JR, Panek AD (1982) Curt Genet 5 : 6 9 - 7 6 Ortiz CH, Maia JCC, Tenan MN, Braz-Padr~o GR, Mattoon JR, Panek AD (1983) J Bacteriol 153:644-651 Padrgo GRB, Malamud DR, Panek AD, Mattoon JR (1982) Mol Gen Genet 185:255-261 Panek AD (1963) Arch Biochem Biophys 100:422-425 Panek AD (1975) Eur J Appl Microbiol 2 : 3 9 - 4 6 Panek AD, Sampaio AL, Braz GRC, Mattoon JR (1978) In: Bicila ~ M, Horecker BL, Stoppani AOM (eds) Biochemistry and Genetics of Yeasts. Pure and Applied Aspects. Academic Press, NY, pp 145-160
397
Pringle JR, Friedman M, Fiechter A (1974) Proceedings of the Fourth International Symposium on Yeasts: Part I p 37 Roth R (1970) J Bacteriol 101:53-57 Rothman-Denes LB, Cabib E (1970) Proc Natl Acad Sci 66: 967 974 Sherman F (1963) Genet 4 8 : 3 7 5 - 3 8 5 Thevelein JM, Hollander JA, Den Shulman RG (1982) Proc Natl Acad Sci USA 79:3503-3507 Van de Poll KW, Schamhart DHJ (1977) Mol Gen Genet 154: 61-66
C o m m u n i c a t e d b y F. K a u d e w i t z Received May 10, 1983
Current Genetics (1983) 7 : 3 9 3 - 397
© Springer-Verlag 1983
Trehalose: Its Role in Germination of Saccharomyces cerevisiae Anita D. Panek and Edilson J. Bernardes Departamento de Bioqu~mica, Instituto de Qulmica, Universidade Federal do Rio de Janeiro, Centro de Tecnologia, 21941, Rio de Janeiro, RJ, Brasil
Summary. Mutants with specific lesions were used to differenciate between the functions of glycogen and trehalose in S. cerevisiae. Diploids which harbor the glcl/glcl mutation depend upon the phosphorylated, less active form of glycogen synthase and show a more active, phosphorylated form, of the enzyme trehalase. These conditions are due to a lesion in the regulating subunit of the cAMP-dependent protein kinase. Such cells are unable to sporulate. Diploids which contain the sstl/sstl mutation have normal glycogen metabolism but their trehalose-6-phosphate synthase is not active. Such strains sporulate but germination is poor and only one-spore tetrads are formed. These results confirm that glycogen is needed to trigger sporulation while trehalose plays a role in the germination process. Different systems, I and II, of trehalose accumulation were proposed. System I would require the UDPG-linked trehalose synthase, whereas system II would constitute an alternative pathway, specifically induced or activated by the expression of a MAL gene. The presence of system II in its constitutive form in the constructed diploids would favour trehalose synthesis during growth on glucose, however, it did not overcome the glycogen deficiency during sporulation nor the lack of trehalose for germination. It seems that only system I, namely trehalose 6-P-synthase, plays a role in the germination process. Key
words:
Germination
Trehalose
-
Glycogen
-
- Saccharomyces cerevisiae
Offprint requests to: A. D. Panek
Sporulation
-
Introduction
The significance of two storage carbohydrates in yeast cells has intrigued investigators over the years. It is clear that in view of the many aspects of a cell cycle maintenance, differentiation and survival - no exclusive role should be assigned to any energy reserve. However, nature must have had some reason for providing the yeast cell with both glycogen and trehalose. The accumulation of trehalose under conditions leading to sporulation was reported by Roth in 1970. In a following paper Kane and Roth (l 974) demonstrated that trehalose and glycogen are synthesized to the same extent in both sporulating and non-sporulating strains. However, only in sporulating cells is glycogen synthesis followed by a period of breakdown coincident with the appearance of mature spores. Corroborating these results Colonna and Magee (1978) and Clancy et al. (1982) demonstrated that under these conditions a sporulation specific amyloglucosidase activity is responsible f o r glycogenolysis. It should be emphasized that in all these investigations different diploid strains of Saceharomyces cerevisiae are employed and no specific role for trehalose was assigned. Recently, Thevelein et al. (1982) demonstrated by NMR spectroscopy that trehalose in Pichia pastoris is rapidly mobilized upon induction of germination. The use of mutants harboring well defined deficiencies will certainly bring forth unequivocal evidence and permit better insight into this problem. This paper intends to demonstrate that trehalose does not seem to be required for sporulation but plays a definite role during germination of S. cerevisiae while glycogen is indeed a pre-requisite for spore formation. The significance of the alternative pathway for trehalose synthesis (Operti et al. 1982) is also discussed in relation to spore formation and germination.
394
A.D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination ofSaccharomyees cerevisiae
Table 1. Yeast strains employed: genotypes and sources Diploids
No
a parent genotype
No
a parent genotype
Source of haploids
DE4 DE6 AP5 AP7 AP8 AP9 AP10 AP11 NT1 NT2
PG1-2C DE4-3A 20Q1-3B DE6-5D BR15-14B DE6-5D DE6-5D NT2-5 B 7Q-2A 7Q-2A
ade 2-40 real GLC SST ade 2-40 MAL2 c GLC SST glcl-2 mal gal2 SST lys2 MAL2 c sstl GLC glcl-1 MAL4 c his1 SST lys2 MAL2 c sstl GLC lys2 MAL2 c sstl GLC glc4-1 real gal2 SST glc4-1 mal gal2 S S T glc4-1 mal gal2 S S T
PS-8C Q6R2 DE6-4A DE6-4D BR15-15D Q6R2 DE6-4A NT2-4A GS1-36 DE6-4A
ade 2-40 MAL2-47 c GLC SST lys2 MAL6 sstl GLC lys2 maI sstl GLC lys2 G L C M A L 2 c s s t l . glcl-1 MAL4 e ura3 trpl S S T lys2 MAL6 sstl GLC lys2 mal sstl GLC glc4-1 real gal2 S S T glcl-1 mal gal2 S S T lys2 mal sstl GLC
(1) (2, 3) (4, 2) (2) (2) (2, 3) (2) (2) (5, 6) (2)
Source key: (1) F. Zimmermann, Technische Hochschule, Darmstadt, GFR (2) prepared at the Universidade Federal do Rio de Janeiro, Brazil (3) spontaneous revertant of strain Q6 from K. van de Poll, Rejks Universiteit, Utrecht, Netherlands (4) H. Ruis, Institut fur Algemeinen Biochimie, Wien, Austria (5) J. Pringle, University of Michigan, USA (6) E. Cabib, National Institutes of Health, Bethesda, USA
Material and Methods Yeast Cultures. The genotypes of the various strains used in this study are listed in Table 1. Strain Q6R2 is derived from Saccharomyces carlsbergensis and the remaining strains from S. eerevisiae. All diploids were obtained in our laboratory. Isolation, induction of sporulation and dissections were performed as described by Hawthorn and Mortimer (1960) and by Sherman (1963). Glycogen was detected by staining the colonies directly on plates with a 0.2% 12 in 0.4% KI solution (Chester 1967). Trehalose 6-phosphate synthase activity was determined by a modification of the classical method as described by Operti et al. (1982). The presence of trehalose was detected by incubating the cells in buffered glucose, extracting with 50 mM TCA and determining trehalose by the anthrone method as described by Oliveira et al. (1981).
Results and Discussion Strains of S a c c h a r o m y c e s harboring two different mutant alleles were used to construct the diploids. The s s t l mutation is characterized by the absence o f trehalose 6-phosphate synthase activity or o f any other isozyme which would catalyze the synthesis of trehalose from ADPG or GDPG instead o f UDPG (Operti et al. 1982). The g l c l mutation is a regulatory pleiotropic mutation which reduces glycogen and trehalose accumulation (Rothman-Denes and Cabib 1970; Padr~o et al. 1982). With respect to glycogen, the mutation was described as affecting the conversion o f glycogen synthase from its less active (D) form to the more active (I) form. The absence of trehalose accumulation is due to a defect in protein kinase regulation which causes the hydrolytic enzyme, trehalase, to remain always in its phosphorylated,
active form (Ortiz et al. 1983). The glc4 mutation has a very similar phenotype. All diploids containing double glc mutations sporulated very poorly (Table 2). A similar result was reported for a diploid homozygous for the g l c l - 1 gene (Pringle et al. 1974). Diploid AP8 contains, moreover, the dominant, constitutive M A L 4 gene. As previously reported (Operti et al. 1982), the constitutivity of a M A L gene induces or activates an alternative system o f trehalose synthesis (system II). The introduction Of a MAL gene into a g l c l - 1 mutant overcomes the deficiency in trehalase regulation by allowing for trehalose accumulation during growth through a pathway not directly controlled by the UDPG linked trehalose-6phosphate synthase (Panek et al. 1978). The idea was to verify whether a diploid containing this alternative pathway o f trehalose synthesis could overcome the deficient carbohydrate accumulation caused by the g l c l - 1 mutation and provide energy for sporulation. The extremely low sporulation observed with diploid AP8 (less than 3%) shows that this is not the case. The presence of glycogen is indeed a pre-requisite for the formation o f spores in S a c c h a r o m y c e s . All other diploid strains analyzed sporulated and gave a positive test for glycogen (Table 2). These diploids were allowed to germinate. An analysis of the data in Table 3 shows a significant influence of trehalose 6-phosphate synthase activity (system I) upon the germination capacity o f the spores. Heterozygous diploids for the s s t l gene which controls trehalose synthase activity originated spores which germinated with lower efficiency (strains DE6, AP5 and
395
A. D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination of Saccharomyces cerevisiae Table 2. Sporulation and glycogen accumulation Strains
Genotype
I2-KI test
Sporulation
DE4
SST GLC
++
normal
++
normal
+
normal
+++
normal
SST GLC
DE6
SST GLC sstl GLC
AP5
SST GLC sstl glcl
AP7
sstl GLC sstl GLC
AP8
none
SST glcl.1 SST glcl.1
AP9
sstl GLC
++
low
++
low
-
none
+
none
+
normal
sstl GLC
AP10
sstl GLC sstl GLC
AP11
SST glc4-1 SST glc4-1
NT1
SST glc4-1 SST glel-1
NT2
SST GLC sstl glc4-1
NT2). Introduction of a g l c l deficient allele (diploid AP5) had no effect upon the total percentage of germination when compared to strain DE6. The same can be said about the g l c 4 allele in diploid NT2. There was, however, a low percentage of 4 spore tetrads in both cases which suggests that the glc sst condition might be lethal (Table 3). Some g l c l - 2 segregants were assayed and showed normal trehalose-6-phosphate activity "in vitro" suggesting that the parental types predominate in the segregation (Table 4). In glycogen negative segregants with normal trehalose synthase activity, no accumulation of trehalose was observed due to the activation of trehalase by the glc mutation. When we looked at diploids homozygous for s s t l , no 4 spore tetrads were seen at all (strains AP7, AP9 and AP10). Of the 46 asci dissected from these diploids which are characterized by a homozygous lesion in trehalose-6-phosphate synthase (system I) but normal glycogen accumulation capacity, the total germination obtained (Table 3) corresponded mostly to one-spore tetrads. The presence of a M A L constitutive gene in these diploids did not improve the pattern of germination. Here again, the existence of the alternative pathway of trehalose synthesis which would operate during growth, did not overcome the trehalose-6-P-synthase deficiency. This result strengthens our working hypothesis that cells of S a c c h a r o m y c e s are able to form a second independent pool of trehalose, through system I1, which might have a specific function in regulating the efficient uptake of
Table 3. Role of storage carbohydrates during germination Diploids
Genotype
Dissected Asci
Germination (%)
4 spore tetrads (%)
DE4
SST GLC
22
87.0
60.0
22
52.0
27.0
20
50.0
15.0
32
17.0
0.0
7
10.0
0.0
7
21.0
0.0
10
42.5
0.0
SST GLC
DE6
SST GLC sstl GLC
AP5
SST GLC sstl glcl-2
AP7
sstl GLC sstl GLC
AP9
sstl GLC sstl GLC
AP10
sstl GLC sstl GLC
NT2
S S T GLC sstl glc4-1
396
A.D. Panek and E. J. Bernardes: Trehalose: Its Role in Germination of S a c c h a r o m y c e s
eerevisiae
Table 4. Segregation o f g l c and s s t mutations Strain
AP5-1A 1B
Glycogen synthesis
Trehalose synthesis
Genotype
Trehalose-6-Psynthase activity
-
-
glc S S T
+
-
-
glc S S T
+
1C
+
-
GLC sst
1D
+
-
GLC sst
n.d. n.d.
-
glc S S T
+
-
-
glc S S T
+
+
-
GLC sst
+
-
GLC sst
n.d. n.d.
AP5-2A 2B 2C 2D AP5-3A 3B 3C 3D
-
-
glc S S T
+
+
-
GLC sst
+
-
GLC sst
+
-
GLC sst
n.d. n.d. n.d.
Parental phenotypes were determined by the I2-KI test and trehalose synthesis in non proliferating conditions. In segregants negative for glycogen, trehalose-6-phosphate synthase was measured (see Methods); n.d. = not determined
maltose. Only system I would contribute for the accumulation required during germination. Diploids which harbor the g l c l / g l c l mutation do not convert glycogen synthase into the more active, independent form; moreover, trehalase activity is always high. Such strains do, probably, not synthesize glycogen and they rapidly breakdown any trehalose that might be formed: they do not sporulate. Diploids which harbour the s s t l / s s t l mutation have normal glycogen metabolism but they do not synthesize trehalose. Such strains sporulate confirming that glycogen accumulation is required to trigger this process. If we assume that the spores formed by the three sstl/sstl diploids are not defective themselves and would be able to germinate should they possess normal trehalose 6-phosphate synthase activity, than the results here presented point towards a specific function for trehalose in the process of germination o f S a c c h a r o m y c e s , dependent upon the activity of the classical trehalose synthase described by Cabib and Leloir (1958). For the fission yeast S c h i z o s a c c h a r o r n y c e s pombe there is good evidence that both accumulation o f trehalose and induction of trehalase activity during sporulation are the preparatory steps for germination and not necessarily a pre-requisite for sporulation itself (Inoue and Shimoda 1981). In ascospores of P i c h i a p a s t o r i s trehalose accumulates in dormant spores because of the low activity o f trehalase and its strong inhibition by ATP. During early germination, energy is supplied by a rapid breakdown o f trehalose due to a highly active trehalase (Thevelein et al. 1982). The trehalase activation process could play an essential role in the induction of germination but
spores would have to contain the disaccharide which is not the case with the s s t l / s s t l mutants. Cause and effect relations are difficult to establish but it is stimulating to believe that studies with well characterized mutants will allow more definite conclusions to be drawn. Clearly, trehalose does not play a unique role in the regulation of energy metabolism in yeast. Its function during starvation or adaptation to new culture conditions (Panek 1975; Lillie and Pringle 1980; Panek, 1963) or as osmoregulator of the cytosol (Keller et al. 1982) should not be overlooked. It is rewarding to speculate why nature provided yeast cells with two different storage carbohydrates and assigned a specific role in sporulation for the polysaccharide, whereas, the disaccharide, trehalose, was chosen for the germination step. This work was supported by grants from CNPq, CEPG, FINEP and FUJB; our thanks to Mauro S. Operti for the enzyme assays and to Vaine R. Reis for assistance in preparing the manuscript.
Acknowledgements.
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C o m m u n i c a t e d b y F. K a u d e w i t z Received May 10, 1983
