Current Genetics (1982) 5:69-76

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© Springer-Verlag 1982

Relationships between Trehalose Metabolism and Maltose Utilization in Saccharomyces cerevisiae III. Evidence for Alternative Pathways of Trehalose Synthesis

M. S. Operti I , D. E. Oliveira I , A. B. Freitas-Valle 1, E. G. Oestreicher I , J. R. Mattoon 2, and A. D. Panek 1 1 Departamento de Bioqulmica, Instituto de Quimica, Universidade Federal do Rio de Janeiro, Caixa Postal 1573, 21941 Rio de Janeiro, Brasil 2 Department of Biology, College of Letters, Arts and Sciences, University of Colorado, Colorado Springs, Colorado, USA

Summary. A specific deficiency in UDPG-linked trehalose-6-phosphate synthase in the yeast, Saceharomyces eerevisiae has been associated with a single nuclear gene, sstl. Strains bearing this abnormal allele lacked the capacity to accumulate trehalose during growth on glucose or galactose medium or when incubated with glucose in nonproliferating conditions. However, sstl strains still exhibited trehalose accumulation during growth on maltose medium, provided they contained a gene for maltose fermentation (MAL gene). Introduction of a constitutive MAL e gene into an sstI strain rendered the strain capable of accumulating trehalose during growth on glucose medium, but did not restore the normal capacity to convert glucose to trehalose in nonproliferating conditions. Different systems, I and II, of trehalose accumulation are proposed. System I would require the UPDGlinked synthase, whereas system II, which is normally specific for maltose, would utilize a different enzyme. It is unlikely that system II produces trehalose by transglucosylation, since it converted glucose to trehalose in MAL e sstl strains. The results indicate that maltose specifically induces the production of the MAL geneproduct, which, in turn, would stimulate the formation (or activation) of system II. Key words: Saccharomyces - Trehalose - Constitutive MAL genes - Mutants defective in trehalose-6-phosphate synthase

Introduction The glucose disaccharide, trehalose, is a storage carbohydrate in Saccharomyces cerevisiae and other yeasts with, Offprint requests to." A. D. Panek

as yet, no clearly defined function during the different phases of the cell cycle. Recent investigations have revealed the existence of at least two types of nuclear genes which alter the regulation of trehalose biosynthesis and hydrolysis. The mutation glcl, which causes defective regulation of glycogen synthesis (Rothman-Denes and Cabib 1970) has two effects on trehalose metabolism: 1) Cells lose the capacity to accumulate trehalose during growth on galactose (Panek et al. 1979). 2) Strains do not accumulate trehalose when incubated with glucose in nonproliferating conditions. The second type of nuclear gene which alters trehalose metabolism also affects maltose fermentation. It has recently been demonstrated (Oliveira et al. 1981) that the polygenic set of MAL genes which controls maltose fermentation also regulates trehalose accumulation. When a strain contains a given MAL gene in the constitutive condition (MALe), the cells will accumulate large amounts of trehalose during growth on glucose medium. This property, designated TAC(+) (active trehalose accumulation) phenotype, is absent when cells contain normal inducible MAL genes, and is lost when a MAL e gene mutates to a nonfermenting real allele. In other words, strains lacking the constitutive allele accumulate much less trehalose during growth on glucose, that is, they display a tac(-) phenotype. A special relationship between maltose metabolism and trehalose has also been shown by comparing trehalose accumulation in maltose growth medium with that obtained in media containing glucose or galactose as primary carbon source. All strains tested which are capable of maltose fermentation (inducible or constitutive) accumulate trehalose during growth on maltose. That this effect is specifically related to maltose and not to the derepression of cytochromes which occurs in maltose medium is shown using a glcl strain. Galactose, which allows for even more cytochrome derepression 0172-8083/82/0005/0069/$ 01.60

70

A.D. Panek et al.: Alternative Pathways for Trehalose Synthesis in Yeast

Table 1. Yeast strains employed: genotypes and sources I. Haploids Strain

Genotype

Source

Q6R2 Q6 BR16-7D API-5C

~ lys2 MAL6 sstl c~lys2 fdp MAL6 sstl a trp MAL6 a ura3 trp l MAL4c

(2) (1) (3) (3)

II. Diploids Strain a parent No SB1 DE6

c~parent Genotype

No

BRI6-7D a trpMAL6 Q6R2 DE4-3A a ade2-4OMAL2 c Q6R2

Genotype a lys2MAL6sst] alys2MAL6 sstl

Source key: (1) K. van de Poll, Rejks Universiteit, Utrecht, Netherlands. The strain code Q6 was assigned by the present authors. (2) this strain is a spontaneous revertant of strain Q6 obtained by incubating a large population of cells in glucose medium (YED) and isolating a colony produced from a single cell. (3) obtained in our laboratory

than maltose, does not support trehalose accumulation during growth. In contrast, substantial trehalose is produced when the same strain is grown on maltose medium (Panek et al. 1979). The accumulated evidence, then, supports the hypothesis that S. cerevisiae would contain two trehalose accumulation systems (I and II). System I would be required for trehalose accumulation during growth when galactose is the carbon source. The same system would be required for trehalose accumulation under nonproliferating conditions when either glucose or galactose is the carbon source. System II is normally maltose-specific; it would operate when cells are grown on maltose medium. It appears very likely that this same system would be responsible for the accumulation of trehalose during growth on glucose (TAC(+) phenotype) as long as the maltose gene is constitutively expressed (MALe). The glcl mutation would specifically abolish system I, leaving system II unchanged. Further evidence for the existence of the two systems is provided by the discovery of another type of mutant which exhibits substrate-specific trehalose accumulation, sst phenotype (Panek et al. 1979). Like the glcl mutation, sst strains do not produce trehalose from glucose in nonproliferating conditions nor do they accumulate more than small amounts of the disaccharide during growth on either galactose or glycerol. Nevertheless, sst strains accumulate substantial amounts of trehalose during growth on maltose. Therefore, an sst

strain would also seem to be defective in system 1 but would retain system II. Although the phenotype o f sst strains resembles that of glcl the two defects are under separate genetic control, since they exhibit genetic complementation (Panek et al. 1979). The present results indicate that the sst phenotype is due to a single nuclear gene which will be designated sstl. Strains bearing this mutation were used to demonstrate that trehalose accumulation during growth is maltose-specific. Evidence is presented that the sstl mutation results in an extremely low UDPG-dependent trehalose-6-phosphate synthase activity. It is also shown that the TAC(+) phenotype segregates independently from the sstl gene. The results support the hypothesis that the UDPG-linked enzyme is associated only with system I and that a second enzymatic pathway must function, system II.

Materials and Methods Yeast Cultures. The genotypes of the various strains used irl this study are listed in Table 1. Strain Q6R2 is derived from Saccharornyces carlsbergensis and the remaining strains from S. cerevisiae. Growth Conditions. Cells were grown in media containing 1% yeast extract and 1% carbon source, Cultures were grown in 100 ml of medium contained in 500 ml flasks which were incubated at 28 °C on rotary shaker operated at 150 rpm. Harvested cells were washed twice with distilled water. Nonproliferating Conditions. Washed cells were incubated at 28 °C in 0.05 M phosphate buffer, pH 6.0 in the presence of 20 mM carbon source at a proportion of 3 mg dry wt/ml. Suspensions were shaken at 150 oscillations per rain. Analytical Methods. Growth of cultures was followed by turbidity measurements at 570 nm. Glucose was determined either enzymatically by the glucose oxidase-peroxidase method (Raabo and Terkildsen 1960), or, when nonproliferating conditions were used, according to Nelson 1944. Under these conditions maltose and galaetose were also determined by the same procedure. Trehalose was extracted with 0.5 M trichloroacetic acid (Trevelyan and Harrison 1952) and determined by anthrone (Brin 1966). Glycogen was extracted with 0.25 M Na2CO 3 and determined as glucose after a 2 h hydrolysis at 37 ~C with Rhizopus amyloglucosidase (Becker 1978). Proteins were estimated according to Lowry et al. (1951), using bovine serum albumin as standard. For permeabilized cells, protein determinations were carried out with 1 mg samples after digestion at 100 °C for 15 min in 1 ml of 1 N NaOH. After cooling, the digested sample was neutralized with 1 N HC1 and used for pro~ein determination. It was found that i mg dry wt of yeast contains approximately 0.4 mg of protein which is in good agreement with values reported in the literature (Steward 1975). Cell-Free Extracts. Washed cells (0.75-1.2 g dry wt) were resuspended in 6 ml of 0.1 M imidazole buffer, pH 7.0 and disrupted in a Braun Shaker with 8 g of glass beads (0.45 rnm diameter). The glass beads were allowed to settle and the supernatant fluid was decanted. The beads were washed with 4 rnl of the

A. D. Panek et al. : Alternative Pathways for Trehalose Synthesis in Yeast

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A

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GALACTOSE

= -" I

~. I

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= I

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GALACTOSE GLUCOSE I HOURS

,=. 2C I0

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GLUCOSE

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0

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10

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Fig. 1. Trehalose accumulation during growth of strain Q6R2 on different carbon sources Fig. 2. Trehalose accumulation by strain Q6R2 in nonproliferating conditions in buffered carbon sources. For accumulation studies in buffered glucose or galactose, cells were pre-grown up to 2nd log in 1% galactose. For accumulation of trehalose in the presence of maltose, growth was carried out in 1% maltose medium. In all cases uptake of sugar during the non-proliferating incubation was determined Fig. 3. Glycogen accumulation by strain Q6R2 in nonproliferating conditions in buffered glucose. Cells were pre-grnwn in 1% glucose, 1% yeast extract medium and harvested during stationary phase

same buffer, and the washings were combined with the first homogenate fluid. The resulting cell-free extract was then centrifuged at 3,000 x g for 20 min. The residue was discarded and the supernatant fluid centrifuged again for 60 rain at 130,000 x g to remove mitochondria and ribosomal fractions. All operations were performed at 4 °C. The final supernatant fluid could be frozen at - 2 0 °C for several days without loss of trehalose6-phosphate synthase activity.

Trehalose-6-Phosphate Synthase Determination. The synthase activity was measured using the assay described by Cabib and Leloft (1958) modified as follows: the reaction mixture contained 12 ~moles of imidazole buffer, pH 7.0, 1 #mole of UDPG, 5 /~moles of glucose-6-phosphate, 5 gmoles of MgSO 4 and the enzyme preparation ( 5 0 - 1 0 0 #g protein) in a final volume of 0.2 ml. After 3 min incubation at 37 °C the reaction was stopped by heating at 100 °C for 3 min and the tubes were cooled on ice. The UDP formed was then determined by adding 25 gl of a solution containing 5 ~moles of imidazole buffer, pH 7.0, 1 gmole of phosphoenol pyruvate and 10 ~moles of KC1. The reaction was started by adding 4 gg of pyruvate kinase in 25 #1 of 0.1 M MgSO 4. Incubations were carried out for 30 min at 37 °C. The resulting pyruvate was determined as its 2,4-dinitro phenylhydrazone derivate according to Friedmann and Haugen (1943). Alpha-Glucosidase Activity. A convenient method for the determination of a-gtucosidase activity was provided by the hydrolysis of p-nitrnphenol-a-D-glucoside (PNPG) by permeabilized yeast cells. Samples of 20 mg (dry wt.) were harvested from the growth medium, washed in a round bottom glass tube with cold water and a second time with 0.05 M phosphate buffer. The cell pellet was subjected to five cycles of freezing in liquid nitrogen and thawing at 37 °C (30 s for each step), and the permeabilized cells were then resuspended in 0.05 M phosphate buffer, pH 6.8, containing 10 - 3 M mercaptoethanol. For the final dilution, total activity of c~-glucosidase in each strain had to be taken into consideration, and volumes adjusted accordingly. A 50 #1 aliquot of the diluted cell cuspension was added to 1 ml

of 5 mM PNPG in the same buffer and incubated at 30 °C. The reaction was stopped by addition of 1 ml of a 0.1 M sodium carbonate solution. The release of p-nitrophenol was followed by measuring absorbance at 410 rim. F~r each strain, proportionality between time of incubation and enzymatic release p-nitrophenol was determined (Oliveira et al. 1981).

Genetic Analysis. Isolation of diploids, induction of sporulation, dissections and tetrad analysis were performed as described by Hawthorn and Mortimer (1960), and by Sherman (1963). Maltose fermentation was followed by indicator color change in agar medium containing 0.33% bromocresolpurple. Special Chemicals. All enzymes and special reagents were purchased from Sigma Chemical, Co, St. Louis, MO, USA.

Results

Substrate-Specific Trehalose Accumulation in Strain Q6R2 It was previously s h o w n t h a t thefdp m u t a n t , Q6, c a n n o t a c c u m u l a t e trehalose d u r i n g g r o w t h o n galactose or glycerol, b u t t h a t s u b s t a n t i a l a c c u m u l a t i o n o f the disaccharide o c c u r s d u r i n g g r o w t h o n m a l t o s e m e d i u m ( P a n e k et al. 1979). Since t h e fclp m u t a t i o n p r e v e n t s g r o w t h o n glucose ( V a n de Poll a n d S c h a m h a r t 1977), a s p o n t a n e o u s r e v e r t a n t , strain Q 6 R 2 , w h i c h grew o n glucose m e d i u m , was isolated. As s h o w n in Fig. 1, strain Q6R2 a c c u m u l a t e d large a m o u n t s o f t r e h a l o s e d u r i n g g r o w t h on maltose, but not during growth on media containing glucose or galactose. T h e r e f o r e , t h e substrate-specific t r e h a l o s e a c c u m u l a t i o n (sst p h e n o t y p e ) was n o t a f f e c t e d b y reversion ( o r s u p p r e s s i o n ) o f t h e fdp m u t a t i o n .

72

A.D. Panek et al. : Alternative Pathways for Trehalose Synthesis in Yeast

Table 2. Trehalose accumulation by normal and sstl strains in nonproliferating conditions. Glucose was the substrate used

60

5C

Strain

Genotype

.T

Trehalose content (mg/g dry wt) 0 min

--~4t

20 min

o J a:

Q6R2

8.0

sstl

8.0

BR16-7D

SST

21.3

48.0

SBI-IA

7.1

48.8

9.5

51.8

5.1

5.1

1D

SST SST sstl sstl

8.9

8.5

SB1-2A 2B 2C 2D

SST SST sstl SST

9.2 14.5 2.1 14.5

42.1 32.9 4.9 39.6

SB1-3A 3B 3C 3D

SST sstl sstl

7.9 4.0 7.8

-

-

56.6 7.9 11.2 -

SB1-4A 4B 4C 4D

SST SST sst l sstl

8.3 17.7

26.6 24.2

5.6

6.2

3.1

7.0

SB1-5A

SST sstl sstl SST

37.0 13.4 14.6 31.3

58.0 13.3 15.8 59.0

1B

1C

5A (SS._.T) 5D (SIT)

g z0

•~

a 5C (ssj} 5B (sstl

i ~ HOURS

A(SS_T) 5B(ssl)

70 g

50

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30

HOURS

5B

5C 5D

Likewise, strain Q6R2 was deficient in the ability to accumulate trehalose in nonproliferating conditions, as shown in Fig. 2. No detectable accumulation ocurred when glucose was used as substrate, and only a weak accumulation was observed with galactose. Normal, nonproliferating cells have previously been shown to accumulate extremely high levels of trehalose when galactose is used as substrate (Costa-Carvalho et al. 1978). Similarly, although maltose supports trehalose accumulation during growth, no detectable increase in trehalose occurred when this carbohydrate wasused in nonproliferating conditions. Thus, the conditions for trehalose accumulation in strains Q6R 2 are highly restricted: substantial accumulation occurs only during growth on maltose medium.

Differences between sst and glcl

This restricted trehalose accumualtion phenotype closely resembles that exhibited by strains carrying the nuclear mutation glcl (Panek et al. 1979) However, two experiments indicate that the two defects are different.

Fig. 4 A and B. Trehalose accumulation during growth of segregants from tetrad SB1-5, on different carbon sources. (A) 1% galactose, (B) 1% maltose

First, as reported in the above publication, when an sst haploid is crossed with a glcl haploid, the resulting diploid exhibits normal accumulation o f trehalose (80 mg/g dry wt in 45 rain) using glucose in nonproliferating conditions. Second, unlike g l c l , the sst defect in Q6R2 does not result in defective glycogen metabolism. As shown in Fig. 3, strain Q6R2 accumulated glycogen readily when cells were incubated in buffered glucose. It appears, therefore, that the sst defect is limited to trehalose metabolism, whereas the g l c l mutation is pleiotropic.

Inheritance o f sst

Genetic experiments indicate that the substratespecific trehalose accumulation phenotype is controlled b y a single nuclear gene. Strain QaR2 was crossed with a norreal tester strain, BR16-7D, which exhibited normal accumulation of trehalose from glucose in nonproliferating conditions. Both strains contained the inducible maltose fermentation gene, M A L 6 . Five complete tetrads were analyzed for trehalose production in non-

A. D. Panek et al. : Alternative Pathways for Trehalose Synthesis in Yeast Table 3. Trehatose-6-phosphate synthase activity in normal and mutant strains. Cells were grown on 1% maltose, 1% yeast extract medium and harvested in stationary phase Strain

Genotype

Trehalose phosphate synthase ~moles UDP x min -1 x (rag prot.) -1

AP1-5C Q6R2 SB1-5A SB1-5B

SST sstl SST sstl

0.28 0.01 0.20 0.01

proliferating conditions as shown in Table 2. In tetrad 3 segregant D did not grow enough to permit analysis. Although the data are limited, it appears likely that the sst phenotype is controlled by a single gene which will be designated sstl henceforth. All tetrads except tetrad 2 exhibited two strains that showed low trehalose accumulation capacity. As discussed below, 2:2 segregation of the defect was found in 7 additional tetrads derived from a second cross (DE6). The sstl gene is recessive (Panek et al. 1979). Besides the loss of trehalose accumulation capacity in nonproliferating cells, strain Q6R2 exhibited defective accumulation during growth on galactose but not on maltose medium (Fig. 1). As shown in Fig. 4, both traits were inherited together; only the normal SB1 segregants, 5A and 5D, converted galactose to trehalose (panel A). However, maltose was converted to trehalose in either type of segregant, as shown in panel B.

Deficiency in UDPG - Linked Trehalose Phosphate Synthase in sstl Strains When the activity of UDPG-linked trehalose-6-phosphate synthase in cell-free homogenates of sstl strain Q6R2 was determined, it was found to be almost undetectable (Table 3). A value of 0.01 pmoles UDP formed x rain -1 x (mg protein)- 1 was obtained compared to 0.28/Jmoles x min -1 x (mg protein) - I for normal strain AP1-5C. Since both strains were grown on maltose and, therefore, produced trehalose during growth, one would have anticipated that the mutant strain should have exhibited a substantial enzyme activity. Since this was not the case and since incubations of cell-free extracts from strain Q6R2 with UDP did not lead to any change in the concentration of the added nucleotide, it appears very likely that an enzyme different from the normal UDPG-linked synthase, and not detected by the assay (UDP formation), must function during growth on maltose.

73

Inheritance o f the Enzyme Deficiency To determine whether the deficiency in trehalose-6phosphate synthase was inherited together with the sstl gene, enzymatic activities of SB1 segregants were determined. As shown in Table 3, normal segregant 5A exhibited substantial activity of the synthase, whereas the sstl mutant, segregant 5B, showed only a trace of activity.

Test for Activators and Inhibitors of Trehalose Phosphate Synthase To test for the absence of possible activators or cofactors in the mutant and for the presence of inhibitors of the synthase, cell-free extracts form normal and mutant cells grown on maltose were mixed and incubated for 20 rain at 37 °C. No changes in synthase activity were observed. However, the possibility that a cryptic form of the enzyme exists and might not be activated under our experimental conditions has to be taken into consideration.

Independence o f MAL - Directed Trehalose Accumulation from sstl-Dependent Trehalose Formation Since the distinguishing characteristic of the sstl phenotype is its specificity for maltose, the possibility might be considered that UDPG-linked trehalose-6-phosphate synthase would be utilized whenever glucose is the carbon source, and that a second pathway of trehalose synthesis would utilize maltose directly, presumably through a transglucosylation reaction. However, as noted earlier (Oliveira et al. 1981), when'a yeast strain contains a constitutive MAL gene, active accumulation of trehalose occurs in the presence of glucose in the growth medium. This effect suggests that trehalose produced when maltose is the substrate, is in fact, derived from glucose produced by the hydrolytic action of intracellular aglucosidase, and not by a direct transglucosylation. To determine whether the UDPG-linked trehalose phosphate synthase is invariably involved when glucose serves as substrate, a diploid strain DE6, was constructed by crossing sstl strain, Q6 R2, with strain DE4-3A which contains a constitutive MAL2 e gene and consequently exhibits the TAC(+) phenotype. The meiotic progeny obtained by sporulation of this diploid was then tested for trehalose accumulation under two conditions: 1) during growth on glucose medium, TAC(+), and 2) in nonproliferating conditions utilizing glucose as carbon source (sstl). As shown in Table 4, both traits segregated 2 : 2 and their segregation was independent. For example, in tetrad DE6-1, segregant 1C converted large amounts

74

A. D. Panek et al.: Alternative Pathways for Trehalose Synthesis in Yeast

Table 4. Independence of TA C(+) from sstl Strain

TAC(+)a mg/g cells dry wt

Table 5. Independence of system I from system IIa. (Cells were harvested 1 h after depletion of glucose in growth medium)

Trehalosein N.P.C. mg/g cells dry wt

Strain

0 min

30 min

DE6-1A 1B 1C 1D

70.6 5.0 116.6 3.3

33.3 22.0 38.0 11.7

55.8 45.8 29.7 4.7

DE6-2A 2B 2C 2D

7.9 58.5 39.0 1.9

20.0 24.6 4.7 8.4

35.0 22.7 23.7 7.2

DE6-4A 4B 4C 4D

2.4 4.1 95.0 67.6

13.5 20.8 104.1 13.2

8.6 51.4 145.8 8.4

DE6-5A 5B 5C 5D

69.0 2.1 4.2 5.0

32.8 2.0 3.4 4.8

78.9 28.3 1.7 3.8

DE6-6A 6B 6C 613

5.9 83.1 101.5 11.8

20.5 35.1 80.7 22.2

23.1 32.9 111.1 66.7

DE6-7A 7B 7C 7D

8.3 6.0 59.5 7.8

13.8 10.4 40.4 16.2

38.7 10.4 39.8 56.9

DE6-8A 8B 8C 8D

3.8 3.4 35.6 111.0

18.6 11.8 12.5 38.3

44.3 8.4 12.5 27.7

Cells were harvested one hour after exhaustion of glucose from growth medium. For nonproliferating conditions (N.P.C.) in the presence of 20 mM glucose cells were taken from 2nd log culture

of glucose to trehalose during growth, but when cells partially loaded with trehalose were incubated with glucose in nonproliferating conditions, trehalose content actually declined. A similar effect was seen in segregants 2B, 4D, 6B and 7C. All of these segregants contain the M A L 2 c gene (seen by the presence of c~-glucosidase), clearly indicating that TAC(+) phenotype and the sstl phenotype are determined by mutations in separate genes. When DE6 segregants were tested for UDPGdependent trehalose phosphate synthase activity it was again found that sstl segregants were deficient in this activity even in TAC(+) strains, which accumulated large amounts of trehalose during growth on glucose medium, e.g. segregant DE6-1C (Table 5). Clearly then, t h e M A L 2 cdependent system, which permits active trehalose accu-

Trehalose-6-P synthase tzmoles UDP x min-1 x (rag prot) -1

c~glucosidase nmoles PNPG x min- 1 x (mg prot) -~1

DE6-1A 1B 1C 1D

0.34 0.35 0.00 0.00

1.27 0.02 0.81 0.03

DE6-2A 2B 2C 2D

0.35 0.05 0.27 0.09

0.01 2.73 3.03 0.03

DE6-4A 4B 4C 4D

0.01 0.13 0.18 0.03

0.05 0.01 1.17 3.26

DE6-5A 5B 5C 5D

0.44 0.15