FEMS Microbiology Letters 193 (2000) 117^121
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Role for trehalase during germination of spores in the ¢ssion yeast Schizosaccharomyces pombe F.F. Beltran, R. Castillo, J. Vicente-Soler, J. Cansado, M. Gacto * Department of Genetics and Microbiology, Faculty of Biology, University of Murcia, 30071 Murcia, Spain Received 26 July 2000; accepted 4 October 2000
Abstract Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1 , which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulationspecific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Trehalose ; Trehalase ; Spore germination; Fission yeast
1. Introduction Yeasts accumulate trehalose as a reserve carbohydrate in resting stages of the growth cycle, such as spores and stationary-phase cells [1]. In addition, growing cells show high levels of trehalose as protectant in response to environmental stresses [2]. The synthesis and mobilisation of this non-reducing disaccharide involves the participation of various enzymes, including trehalose-6P synthase, trehalose-6P phosphatase and trehalase [3]. Two distinct trehalose-hydrolysing enzymes (trehalases) have been reported in yeasts [1]. Neutral trehalases are cytosolic enzymes regulated by cAMP-dependent phosphorylation which are involved in the degradation of endogenous trehalose [4]. Acid trehalases are mainly vacuolar or cell surface enzymes. Localisation of acid trehalases in either vacuoles or cell walls supports the concept that they belong to the exoplasmic compartment of the cell and function as scavenger hydrolases responsible for the utilisation of extracellular trehalose [4^6].
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Trehalose and glycogen are the main storage carbohydrates in spores of the ¢ssion yeast Schizosaccharomyces pombe [7,8]. Trehalose is mobilised at the onset of spore germination while the glycogen content remains constant throughout germination and outgrowth [8]. Hence, trehalose, rather than glycogen, can be considered as preferential energy and carbon source during germination in the ¢ssion yeast, which implies a critical role for the enzyme activities involved in its degradation. In S. pombe two di¡erent trehalases are present [9,10]. Neutral trehalase is post-translationally activated upon increases in the level of cAMP, and its corresponding gene ntp1 has been cloned and sequenced [11]. Disruption mutants in this gene show better survival than wild-type cells after multiple stresses, including dehydration, osmostress and heat shock [12]. Another trehalase, which appears to be sporulation-specific, shows optimal activity at acidic pH and it is found in walls of asci and spores of the ¢ssion yeast [8,9]. The absence of this enzyme from vegetative cells suggests some function in the germination process but its physiological role in the life cycle of S. pombe remains to be established. In this work we have analysed the e¡ect of disrupting ntp1 , which encodes neutral trehalase, on spore germination. We have also used ntp1-disrupted spores to outline the possible function of acid trehalase in this process.
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 7 1 - 7
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2. Materials and methods 2.1. Strains and culture conditions The strains of S. pombe used in this work are listed in Table 1. To obtain spores, cells were pre-grown in YES liquid medium, containing 2% glucose plus 0.6% yeast extract, until the mid-exponential phase. These cells were washed and further incubated for 5^6 days in liquid MEL sporulation medium, which contained 3% malt extract in 50 mM sodium phosphate bu¡er pH 5.9 [7]. Spores were puri¢ed and isolated from sporulated cultures as described in [13]. Germination was performed in YES liquid medium and followed by phase-contrast microscopy analysis and cell counting. The development from dormant spores to actively growing vegetative cells was divided into three distinct stages [14]: (i) breaking of dormancy, that was accompanied by darkening or the loss of refractility of the spores under phase-contrast microscopy, (ii) swelling and initial emergence of the germ-tube, with the formation of pear-shaped spores, and (iii) subsequent outgrowth leading to the appearance of a cell plate. For growth of particular strains the culture media were supplemented with the required nutritional markers (75 mg l31 ). Induction of di¡erential germination was performed in EMM minimal medium without uracil to allow only growth of prototrophic cells [15]. 2.2. Enzyme assays and trehalose determination Neutral trehalase was assayed as described previously [12]. Acid trehalase was assayed at pH 4.2 as indicated in [9]. For inhibition experiments 5 mM phloridzin (Sigma) was used and the spore concentration kept constant at 107 spores ml31 . Trehalose was extracted from spores by treatment with boiling water for 20 min [16] and determined by the anthrone method [17]. These water soluble extracts were con¢rmed to be composed solely of trehalose as sugar by thin layer chromatography on silica gel 60 plates (Merck) using butanol^ethanol^water (5:3:2, v/v) as solvent. Sugar spots were detected by charring at 95³C after spraying with 25% H2 SO4 .
Fig. 1. Germination of spores from strains P968 (wild-type, left panel) and its isogenic derivative MMT48 (ntp1-disrupted, right panel). A representative result is shown. 2.4^2.6U107 spores were inoculated in YES medium and incubated for 24 h at 28³C. Symbols: dormant spores (a), darkened spores (b), pear-shaped spores (O), spores with a cell plate (F).
3. Results 3.1. Germination in control and ntp1-disrupted spores In a previous work we described that ntp1-disrupted strains of S. pombe, which are devoid of neutral trehalase activity, were able to sporulate [12]. In the present study we analysed the germination of these spores in liquid medium and found marked di¡erences when compared to wild-type spores. Fig. 1 summarises a representative follow-up of the germination process in spores from either the homothallic h90 wild-type strain P968 or the ntp1-disrupted MMT48 strain of S. pombe. As shown, normal spores followed the typical germination stages of S. pombe in a rather synchronous manner (see Section 2), whereas ntp1-disrupted spores showed a comparatively sluggish germination pattern. Moreover, germination and subsequent outgrowth was less e¤cient in the population of disruption mutant spores. These results indicate that under germination conditions the absence of ntp1 signi¢cantly reduces the conversion of spores to vegetative cells in S. pombe and therefore that neutral trehalase may be required for normal germination.
Table 1 List of strains Strain P968 MM1 MM2 MMT4 MMT38 MMT48 MMTD MMD
Relevant genotype
Source
90
h ura4-D18 h ade6-M216 leu1^32 ura4-D18 h3 ade6-M210 leu1^32 ura4-D18 h ade6-M216 leu1^32 h3 ade6-M210 leu1^32 ura4-D18 vntp1: :ura4 h90 ura4-D18 vntp1: :ura4 h ade6-M216 leu1^32./h3 ade6-M210 leu1^32 ura4-D18 h ade6-M216 leu1^32 ura4-D18/h3 ade6-M210 leu1^32 ura4-D18 vntp1: :ura4
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P. Perez M. Yamamoto A. Duran [12] this work this work this work this work
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We considered the possibility that the above results were artifactually due to non-matching ura4-auxotrophies of mutant (ura4 ) and control reference strain (ura4-D18). Hence, we repeated the same type of experiments by using spores of the diploid strains MMD (ntp1-disrupted) and MMTD (control), which were obtained upon mating the corresponding heterothallic strains (Table 1) and selection for intragenic complementation of the ade marker in EMM medium without adenine. Germination was performed in these cases in EMM medium without uracil to allow only outgrowth of spores carrying the ura4 cassette. The results obtained (data not shown) were entirely compatible with those described above for the haploid strain P968 and its counterpart MMT48. 3.2. Degradation of trehalose during germination of wild-type and ntp1-disrupted spores Trehalose decreases steeply after the onset of germination and then increases gradually in the cultures as spores outgrow and vegetative cells accumulate [8]. Consistent with this, we found that trehalose began to decrease in isolated wild-type spores during germination without a distinct lag period and that the initial level of the sugar was reduced to about one third after 6^8 h of incubation (Fig. 2). This decrease was almost coincidental with a predominance of spore darkening and pear-like morphology. We then determined the trehalose content in isolated dormant spores of ntp1-disrupted strains, which showed a
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Fig. 3. E¡ect of phloridzin (5 mM) on germination of spores from strains P968 (wild-type) and its isogenic derivative MMT48 (ntp1-disrupted). In each case, 107 spores were incubated at 28³C in YES medium in the absence or presence of the inhibitor and germination analysed by phase-contrast microscopy. A representative result is shown.
two-fold increase relative to the amount of trehalose present in normal spores (Fig. 2). A comparison of the trehalose pool in normal and ntp1-disruption mutant spores during germination revealed that the change in trehalose content was slower in ntp1-disrupted spores. However, the decrease in trehalose in such spores was comparable to that shown by their normal counterparts (i.e. about 60 nmol trehalose mobilised per 107 germinating spores) in spite of the fact that the ¢nal e¤ciency of germination was lower in ntp1-disrupted spores. The degradation of trehalose in ntp1-disrupted spores was unexpected and indicated that another trehalase present in spores of the ¢ssion yeast, likely the sporulation-speci¢c acid trehalase [9], may also be involved in trehalose degradation during germination. 3.3. Inhibition of acid trehalase in ntp1-disrupted spores Phloridzin (phloretin-2P-L-glucoside) is an inhibitor of some microbial trehalases [18]. This glycoside behaves as a non-competitive inhibitor of the sporulation-speci¢c acid trehalase of S. pombe (apparent Ki = 2 mM) whereas it is without signi¢cant e¡ect in assays of neutral trehalase in vitro. When phloridzin was added to ntp1-disrupted spores in germination medium such a compound induced a further arrest in germination, which was thus completely blocked (Fig. 3). Also no changes in the trehalose pool were observed in ntp1-disrupted spores under these conditions (not shown). In contrast, no noticeable abnormality was found in the germination pattern of wild-type spores in germination medium containing phloridzin (Fig. 3). 4. Discussion
Fig. 2. Trehalose content in cultures of strains P968 (wild-type; open symbols) and its isogenic derivative MMT48 (ntp1-disrupted ; closed symbols) during germination. Conditions were as for Fig. 1. Symbols: trehalose (a, b), appearance of vegetative cells (E, F).
Disruption of tps1 , which encodes trehalose-6P synthase in S. pombe, halts spore germination [16]. This ¢nding is congruent with the interpretation that trehalose is an important carbon and energy source during germination. In this context, one would anticipate that spores showing disruption in ntp1 would mimic the behaviour of those
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disrupted in tps1 since in either of these mutants the ¢nal e¡ect should be an ine¡ective mobilisation of trehalose. Nevertheless, our results indicate that this is not the case because, although at a slower rate, trehalose hydrolysis takes place in ntp1-disrupted spores to an extent which is able to support in part spore germination. Obviously, this can be explained by the occurrence in spores of other trehalase activity. The existence of additional neutral trehalase activity is di¤cult to rationalise since it escapes biochemical detection in ntp1-disrupted cells and no signi¢cant homology was detected with other S. pombe genes during cloning of ntp1 as to suggest the existence of related enzymes [12]. Instead, a candidate enzyme to account for these results would be the sporulation-speci¢c acid trehalase described in spores and asci of the ¢ssion yeast [8,9]. However, this enzyme appears to be mostly located at the cell surface and thus the mechanism by which cytosolic trehalose becomes hydrolysed is uncertain. In order to render soluble the bulk of acid trehalase, a treatment of the cell wall fraction of spores with Novozym (Sigma) is required, suggesting that this trehalase is either covalently bound to the cell wall matrix or entrapped by integral components of the wall framework ([9] and our unpublished results). Whatever the case, it should be mentioned that in our case a minor, although still signi¢cant, amount of acid trehalase is found to be soluble rather than sedimentable in cell extracts (9^17% depending on the particular strains). Hence, the possibility remains that this enzyme fraction, presumably intracellular, might be responsible for the decrease in trehalose content in ntp1disrupted spores. Should acid trehalase reach intracellular trehalose in a direct way, it would be then unnecessary to assume a temporal alteration of the spatial separation between external enzyme and internal substrate as suggested by the compartmentation hypothesis [8]. The data presented support the two trehalase enzymes being somehow involved in the in vivo mobilisation of trehalose but that only neutral trehalase a¡ords a normal developmental pattern of germination. When ntp1-disrupted spores were incubated in germination medium supplemented with phloridzin the simultaneous absence of both trehalase activities blocked germination, whereas wild-type spores still showed unaltered germination. This indicates that the activity of neutral trehalase alone is su¤cient to mobilise trehalose during the normal process. It could be argued that the in vivo e¡ect of phloridzin is due to side e¡ects that might be independent on the inhibitory action of this compound on the acid trehalase. However, since germination proceeds normally in wildtype spores in the presence of phloridzin (Fig. 3), the target of the inhibitor in disruption mutant spores appears rather speci¢c. Related to this, it is worth mentioning that pka1sck1 double mutant spores of S. pombe, which lack two protein kinases presumably involved in neutral trehalase activation [19], are also impaired in germination [20]. Moreover, independent observations indicate that pka1-
disrupted spores are very slow to germinate [21]. Taken together, these evidences support our conclusion that neutral trehalase activity normally plays a signi¢cant role in this process. An interesting question arises as to why the conversion of spores to vegetative cells in ntp1-disrupted cells is comparatively low although the amount of trehalose hydrolysed during germination is equivalent to that degraded in wild-type spores. We do not know the reason for this but the existence of additional functions for neutral trehalase, other than trehalose hydrolysis, may deserve further research. An alternative interpretation might be that, since the absolute level of trehalose is higher in the germinating mutant than in the corresponding wild-type strain, the di¡erence in the germination e¤ciency might be due to a potentially inhibitory e¡ect of trehalose on germination. It should be mentioned in this respect that trehalose counteracts the renaturation of partially denatured proteins by chaperone proteins [22]. If the chaperone function is operative during germination, the high trehalose level of the ntp1 mutant spores might inhibit their function. Another intriguing aspect to be explained concerns the markedly heterogeneous behaviour of mutant spores during germination. The rate of entry into the initial steps of germination (darkening) is dependent upon a relatively high external glucose concentration [23]. The appearance in the germination medium of actively growing vegetative cells brings about a signi¢cant drop in the level of this sugar. Such glucose limitation might help to explain the absence of germination in ntp1-disrupted spores that were initially delayed. Likely, this would act as a secondary blocking factor, superimposed to the early delay due to ntp1-disruption, producing the sluggish behaviour of part of the mutant spore population in spite of its uniform genetic background. In summary, our approach reveals for the ¢rst time that the lack of neutral trehalase severely reduces spore germination in ¢ssion yeast and that the sporulation-speci¢c acid trehalase somehow participates in the degradation of endogenous trehalose in the absence of neutral trehalase, thus playing an ancillary role during germination. Acknowledgements We thank P. Perez, A. Duran (University of Salamanca) and M. Yamamoto (University of Tokyo) for supplying yeast strains. We also thank F. Garro for excellent technical assistance. This work was supported in part by grant PB97-1049 from DGES, Spain.
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