Vol. 45, No. 4, July 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages663-671
DE NOVO PROTEIN SYNTHESIS IS ESSENTIAL FOR T H E R M O T O L E R A N C E ACQUISITION IN A Saccharomyces cerevisiae TREHALOSE SYNTHASE MUTANT
Claudia Gross and Kenneth Watson* Division of Molecular and Cellular Biology, School of Biological Sciences, University of New England, Armidale, NSW 2351, Australia Received March 30, 1998 Received after revision, April 1, 1998
SUMMARY
Heat shock (25~ to 37~ for 30 rain) acquisition of thennotolerance (at 50~ was observed in a yeast trehalose synthase mutant and the corresponding control strain. The acquisition of thennotolerance in the control strain was maintained for a significantly longer time than in the trehalose synthase mutant. The heat shock was associated with the synthesis of specific heat shock proteins and, in the case of the control strain, also trehalose accumulation. Inhibition of protein synthesis during the heat shock totally abolished acquisition of thermotolerance in both strains but not trehalose accumulation in the control. It was concluded that trehalose may only be required for prolonged stress protection while heat shock proteins are required for heat shock acquisition of thermotolerance. Key words: heat shock proteins, trehalose, thennotolerance, yeast. INTRODUCTION The heat shock response refers to the synthesis of a specific set of proteins, the heat shock proteins (hsps), in cells that are exposed to a non-lethal heat shock.
These proteins, highly
conserved from bacteria to higher eukaryotes, have been widely implicated in thermotolerance (1,2). A characteristic feature of the heat shock response is the acquisition of thennotolerance, whereby cells subjected to a mild temperature increase acquire resistance to a subsequent exposure to an otherwise lethal heat stress. In yeast, the intracellular content of the disaccharide trehalose has also been shown to increase in response to various stresses and has been associated with conferral of thermotolerance (3,4,5). Despite the wealth of data on thermotolerance, there exists an apparent contention in the literature concerning the relative significance between hsps and trehalose as thennoprotectants. Few reports, with notable exceptions (5,6,7), have considered their action as complementary.
* To whom correspondence should be addressed, Tel: +61 2 6773 3125. Fax: +61 2 6773 3267. E-mail:
[email protected] 1039-9712/98/100663-09505.00/0 663
Copyright 9 1998 by Academic Press Australia, All rights of reproduction in any fiwm reserved,
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Most evidence implicating the involvement o f either hsps or trehalose in thermotolerance is fundamentally by association, however, many reports concentrate on one, ignoring the possible validity of the other. For example, the high stress resistance of an h s r l mutant was recently attributed largely to the affects of trehalose (8), without investigating the influence of high levels of constitutive hsp expression exhibited by this strain (9), Furthermore, in work related to the role of hsp 104 in yeast thermotolerance, trehalose is not measured in the strains under examination (10-13). Also, evidence has been presented to support the greater importance of hsps over trehalose (10,11,14) and vice versa (5,15,16). Reconciliation of conflicting factors necessitates measurement of both these stress-associated metabolites under the same conditions, in the same strain. The availability of trehalose deficient mutants allowed the design of experiments to test relative contributions of hsps and trehalose to thermotolerance. However, here again, published data on tolerance in such mutants has been equivocal as researchers have generally only measured trehalose levels. Moreover, measurement of the kinetics ofthermotolerance have been confined to a single time point for a given temperature. For example, 50.5~ for 8 rain (17) and 52~ for 8 mix (16). In the present studies, we have examined in detail the thermotolerance of a mutant deficient in trehalose accumulation and the corresponding trehalose proficient control strain. Parameters measured included levels of hsps and trehalose as a function of intrinsic and heat shock induced thermotolerance.
The data supported the conclusion that hsps rather than
trehatose are key factors in the heat shock acquisition ofthermotolerance. MATERIALS A N D M E T H O D S Yeast strains and culture conditions. Strains Klg 102 (trehalose-6-phosphate synthase deficient) and VFP1-8C (18) (gifts from A. Panek) were grown at 25~ on a rotary shaker (180 rpm) in " ~ P meditan (0.5% yeast extract, 0.5% bacteriological peptone, 0.3% (NH4)2SO4 , 0.3% KH2PO4 and 2% glucose). Experimental cultures were grown to mid,logarithmic phase, approximately 3 x 106 to 6 x 106 cells mt -~, corresponding to an optical density at 600ran of 0,15 -:0.2. All experiments were repeated a minimum of three times and data presented are representative of consistent results. Heat treatment and thermntolerance assays. Intrinsic thermotolerance was determined following a heat stress. The temperature of exponentially growing cells was rapidly raised from 25~ to 45~ in a 70~ water-bath, with subsequent incubation at 50~ in an oscillating water bath. Samples were taken at various times during a 60 mix time course. Induced thermotolerance was measured by subjecting cultures to a heat shock at 37~ for 30 rain, prior to a heat stress as described above.
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In all cases, 0.5 ml samples were taken from treated cultures, transferred to microfuge tubes and cooled on ice to 25~ The samples were diluted appropriately in YEP medium, plated in duplicate onto YEP agar and incubated at 28~ for 48 h. Thennotolerance was expressed as the percentage of viability with reference to a control sample, taken from the same culture prior to heat treatment. Trehalose determination. Trehalose was extracted from 80 ml of washed cells (5-10 mg dry weight) at 4~ with 0.5M trichloroacetic acid and estimated according to a modified anthrone procedure (19). Protein analysis. Protein synthesis of control and heat shocked cells was examined by incorporation of [35S]-methionine into protein. Cultures (60 ml) were pelleted and resuspended in 2 ml of supernatant. [35S]-methionine (100 gCi; specific activity t150 Ci mmo1-1) was added to both 25~ and 37~ test cultures, which were subsequently incubated for 30 rain. Incorporation of label was terminated by transferring 1.5 ml samples to microfuge tubes containing 150 /al of 100 mg m1-1 unlabelled methionine. Cells were pelleted and proteins extracted as previously described (1). The protein concentration of extracts was determined using a Coomassie blue protein microassay procedure (Pierce) based on the Bradford method (20). Where required, protein synthesis was inhibited by the addition of 20 gg ml -~ cycloheximide, 20-30 rain prior to heat treatment. The efficacy of this procedure was confirmed by the absence of protein profiles following polyacrylamide gel electropboresis (see below). SDS-polyacrylamide gel electrophoresis was employed to separate 10 ~tg protein samples and low range molecular mass standards (Bio-Rad, Sydney). Stacking and resolving gels were 4% and 10% polyacrylamide respectively. Gels were stained, dried and exposed to Hyperfilm MP (Amersham, Sydney) at -70~ for 2-5 days prior to developing. Western transfer and immuno-deteetion. Unlabelled protein extracts from control and heat shocked cultures, subjected to SDS-PAGE in the manner described above, were transferred to Hybond-C super nitrocellulose (Amersham) using the Novablot system (Pharmacia LKB) according to manufacturer's instructions. Detection of bound anti-hsp antibodies was carried out using an ECL detection kit (Amersham). Final washes of membranes, prior to detection, were modified to 3 x 5 rain in PBS / 0.3% Tween 20 followed by 3 x 5 min in PBS / 0.1% Tween 20. Primary antibodies comprising hsps 104, 70 (Affinity BioReagents), 60 (StressGen) and 90 (gift from Dr. P. Piper) were used at dilutions of 1:1000, 1:10 000, 1:1000 and 1:750 respectively. Membranes were exposed to autoradiographic film for periods varying between a few seconds to 5 rain.
RESULTS Thermotolerance
Strains VFP1-8C and Klg 102 initially exhibited similar levels of intrinsic thennotolerance to a 50~ heat challenge (Fig. 1). However, subsequent to 5 min heat stress exposure, resistance declined substantially in Klg 102, resulting in at least a 100-fold difference between the two strains for the remainder of the time course. Likewise, in the case of induced thermotolerance, for which an approximate 10-fold increase was observed in both strains, Klg 102 exhibited a more dramatic decline as compared with VFP1-8C. Maintenance of acquired thermotolerance
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10 2
101 tOo 9!
lOq 10-2 10-3
10-4 lO-S 10
20
30
40
50
60
Time (min) Fig. 1. Intrinsic (open symbols) and induced (closed symbols) thermotolerance in mid-log phase cultures of S. cerevisiae strains VFP1-8C (),#) and Klg 102 (, ,~). Intrinsic tolerance was assessed following incubation at 50~ for the times indicated. Induced tolerance was determined at 50~ following a 30 min heat shock at 37~ Induced tolerance was also monitored in cells incubated with cycloheximide (20 lag m1-1) fbr 20 rain prior to and during heat shock ("VFP18C, -~Klg 102).
was therefore substantially greater in VFPI-SC (Fig. t). The absence of protein synthesis during heat shock was found to inhibit development of thermotolerance in both strains. In addition, cell viability declined even more rapidly, throughout the duration of the 60 min time course, as compared with viability monitored for intrinsic tolerance. Trehalose content
Klg 102 did not exhibit substantial trehalose accumulation above the intrinsic value (0.75%) following either a 37~
or a 40~ heat shock (Fig. 2). However, the control strain
exhibited 2.5- and 3.5-fold increases in trehalose content subsequent to 37~ shock, respectively.
and 40~
heat
Incubation of cells prior to and during heat shock with 20 lag ml "1
cycloheximide was observed to affect a small reduction in the extent of heat shock induced trehalose accumulation in VFP1-SC and essentially no difference in Ktg 102 (Fig. 2).
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6.5 6.0 5.5 "~
5.0
4.0 3.5 ~
3.o
~
~
2.0 1.0 0.0
If
i .
.
.
.
VFP1-8C
Klg-102
Fig. 2. Trehalose levels in control (EZI) and heat shocked cultures ofS. cerevisiae strains VFP18C and Klg 102. Cells were grown to mid-log phase in glucose medium (25~ and heat shocked at 37~ (Ira) or 40~ ( t ~ ) for 30 min. Heat shock was also carried out in the presence of 20 ~tg m1-1 cycloheximide (~1). Error bars represent the standard deviation of measurements from three experiments.
Protein synthesis Fig. 3 shows autoradiograms of [35S]-methionine labelled protein extracts from VFP1-8C and Klg 102. Increased synthesis of typical heat shock inducible proteins at approximately 100, 90 and 70 kDa was noted in both strains, with particularly marked induction in Klg 102 (Fig. 3 indicated by arrows). Patterns o f de novo hsp synthesis induced during either 37~ or 40~ heat shock (results not shown) were consistent with immuno-complexes generated under the same conditions for hsps 104, 90, 70 and 60 (Fig. 4). Notably, levels of induction were similar, if not more intense, in Klg 102 following a 40~ heat shock as compared with heat shock at 37~
By
contrast, a 40~ heat shock was observed to elicit less intense induction in the control strain VFP1-8C, especially in the case of hsp 104. Overall, VFP1-8C exhibited greater constitutive expression of hsps 104, 70 and, to a lesser extent the inducible form (top band) of hsp 90 (Fig. 3). On the other hand, Klg 102 exhibited pronounced heat shock induced hsp synthesis, at both heat shock temperatures. Both strains also exhibited the presence of heat shock inducible proteins of approximately 48 and 35 kDa (Fig. 3), corresponding to enolase (21) and hsp 35 (22) respectively. 667
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DISCUSSION In the present studies, Klg 102 exhibited a significant level of heat shock induced thermotolerance, albeit lower than that acqtdred by the control strain VFPI-8C.
Previous
investigations conducted on these strains indicated a lack of tolerance acquisition in Klg 102 (17). However, the latter authors assessed both intrinsic and induced tolerance at only a single time point, 8 min, following exposure to 50.5~
Considering acquired tolerance declined
rapidly in Klg 102, these workers may have sampled the culture too late to observe its VFP1-8C kDa
1
2
/
3
97--
Kig 102 4
5
6
4
66~
45--
31~
Fig.3. SDS-polyacrylamide gel autoradiogram of [35S]-methionine labelled protein extracts from heat shocked (37~ / 30 min) cells ofS. cerevisiae strains VFPI-8C and Klg 102 in the presence or absence of protein synthesis. Lanes 1 and 2: control (~5~ and heat shocked samples of VFP1-8C. Lanes 4 and 5: control (25~ and heat shocked samples of Klgl02. Lanes 3 and 6: heat shock in the presence of 20 ~tg ml -l cycloheximide. Arrows indicate new or increased protein bands in heat shocked samples compared with controls. Molecular mass standards (kDa) are as indicated.
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BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL VFP1-SC C'
37
40
Klg 102 C'
37
A
....
~
B
~ ~ ~ : ~ i l l l ~ ' ~
40
hsp 104 hsp 90 hsp 70
D
hsp 60
Fig. 4. Western immunoblot analysis of protein extracts from 25~ (C) and heat shocked cells ofS. cerevisiae strains VFP1-8C and Klg 102. Heat shock was carried out at 37~ for 30 min. Immune complexes were generated for anti-hsp 104, 90, 70 and 60 antibodies.
acquisition, which, in addition, remained significantly higher than intrinsic values for the entire duration of the time course (Fig. 1). It should also be noted that previous studies employed a 40~
heat shock for 60 min (17). Consequently, further experiments were conducted where
cultures of both strains were divided and exposed to 37~ and 40~ pre-treatments in parallel. The difference in heat shock temperature was found to have negligible affect on the extent or kinetics of induced tolerance to a subsequent 50~ challenge (results not shown). A notable difference in the kinetics of tolerance between the two strains however, was that of prolonged durability in VFP1-SC (Fig. 1). A tenuous correlation may thus be inferred between trehalose and the maintenance of thermotolerance, as distinct from conferral, throughout the time course. However, in the case of Klg 102 the pronounced decline in acquired tolerance and loss of viability at 50~ may be attributed not only to a trehatose deficiency but also, low constitutive expression ofhsp 104 (Figs. 3, 4). Pre-existing hsp 104 in VFP1-SC may be sufficient to confer the extra margin of intrinsic tolerance upon this strain, although not as effective as its possible activation and increased synthesis at 37~ (13). It was not surprising therefore, that an absence of protein synthesis during the 37~
pre-treatment was found to inhibit development of
thermotolerance in both strains (Fig. 1). However, heat shock protein profiles and the effect of cycloheximide on thermotolerance acquisition have not been previously described in these strains and consequently, this was a key result. Trehalose content of cells under control and heat shock conditions (Fig. 2) was found to be in general agreement with those reported previously (17). However, significant accumulation
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was still observed in heat shocked cells of VFP1-8C in the absence of protein synthesis, despite the obvious compromise in tolerance acquisition (Fig. 1).
It has been suggested that a
'threshold' level of trehalose is required to induce significant levels of stress tolerance (23). This concept suggests that the protective effect of trehalose is influenced by its intracellular concentration, but that above a critical value, in the order of 4 to 5 % w/w in S. cerevisiae (24), further accumulation will not further increase stress tolerance. This may explain why induction of tolerance in VFP1-SC in the absence of protein synthesis is abolished while the concentration of trehalose is only slightly reduced, to approximately 3 % w/w, presumably below the critical value. In support of the 'threshold' concept, trehalose accumulation following a 40~ heat shock was observed to be significantly greater than the level induced at 37~
(Fig. 2) but was not
accompanied by an increase in the degree of induced tolerance (results not shown).
Heat
shocked induced trehalose accumulation in excess of the threshold value may therefore be a consequence of the kinetic affect of temperature (25), which would otherwise appear to comprise a futile investment in energy expenditure for the cell. The salient feature of the present results however, was the acquisition of thermotolerance by trehalose deficient Klg 102 and its total inhibition in the absence of protein synthesis (Fig. 1). Clearly, de novo protein synthesis, and by inference induction of hsps, is a crucial component of the tolerance mechanism in this strain. Moreover, threshold theory aside, VFP1-8C was also compromised in acquisition of tolerance, even in the presence of significant trehalose accumulation. It seems unlikely that the small difference in trehalose content in cells heat shocked in the absence or presence of protein synthesis would be solely responsible for conferral of thermotolerance. Thus, hsps appear to be of greater significance. However, the concept that the combined action of hsps and trehalose confer thermotolerance remains valid in the case of VFP1-SC, which exhibited a greater extent of intrinsic and heat shock induced thermotolerance as compared with Klg 102. A lack of correlation between trehalose and acquired thermotolerance extends to other stresses as well. For example, exposure of yeast to some toxic chemicals has been observed to induce hsp synthesis but not trehalose accumulation (26).
Furthermore, hsp induction, in
contrast to trehalose accumulation, has been reported in yeast cells exposed to the potential mutagen tetrachloroisophthalonitrile (TPN) (27).
It was demonstrated that the minimal
concentration of TPN required for induction of hsp synthesis leads to acquisition of tolerance to a 51 ~ challenge without an increase in trehalose. Taken together, the present results and published accounts indicate that while relative contributions of the two factors are difficult to separate, trehalose may only be required for 670
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prolonged stress protection while hsps clearly play a predominant role in conveying thermotolerance. ACKNOWLEDGMENTS
This work was supported by an Australian Postgraduate Scholarship (C.G.) and internal research grants from the University of New England. REFERENCES
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