Download Molecular events associated with acquisition of heat

FEMS Microbiology Reviews 11 (1993) 339-356
© 1993 Federation of European Microbiological Societies 0168-6445/93/$15.00
Published by Elsevier
339
FEMSRE 00319
Molecular events associated with acquisition of heat
tolerance by the yeast Saccharomyces cerevisiae
P e t e r W. P i p e r
(Received 11 March 1993; accepted 11 May 1993)
Abstract: The heat shock response is an inducible protective system of all living cells. It simultaneously induces both heat shock
proteins and an increased capacity for the cell to withstand potentially lethal temperatures (an increased thermotolerance). This
has lead to the suspicion that these two phenomena must be inexorably linked. However, analysis of heat shock protein function in
Saccharomyces cerevisiae by molecular genetic techniques has revealed only a minority of the heat shock proteins of this organism
having appreciable influences on thermotolerance. Instead, physiological perturbations and the accumulation of trehalose with heat
stress may be more important in the development of thermotolerance during a preconditioning heat shock. Vegetative S. cerevisiae
also acquires thermotolerance through osmotic dehydration, through treatment with certain chemical agents and when, due to
nutrient limitation, it arrests growth in the G1 phase of the cell cycle. There is evidence for the activities of the cAMP-dependent
protein kinase and plasma membrane ATPase being very important in thermotolerance determination. Also, intracellular water
activity and trehalose probably exert a strong influence over thermotolerance through their effects on stabilisation of membranes
and intracellular assemblies. Future investigations should address the unresolved issue of whether the different routes to
thermotolerance induction cause a common change to the physical state of the intracellular environment, a change that may result
in an increased stabilisation of cellular structures through more stable hydrogen bonding and hydrophobic interactions.
Key words: Saccharomyces cerevisiae; Heat stress; cAMP-dependent protein kinase; Trehalose; Heat shock protein; Membrane
protection; Intracellular water structurisation
Introduction
Cells given a mild, sublethal heat stress induce
the highly conserved heat shock response. One
important aspect of this response is the development of induced or 'acquired' thermotolerance,
manifested as increased capacity for survival dur-
Correspondence to: P.W. Piper, Department of Biochemistry
and Molecular Biology, University College London, London
WC1E 6BT, UK.
ing subsequent exposure to higher, potentially
lethal temperatures. However, a mild heat shock
is only one of a number of preconditioning treatments known to induce thermotolerance. Certain
chemical agents, osmotic dehydration and nutritional status have effects on yeast thermotolerance which are just as dramatic, as outlined in
this review. Indeed, so large is the number of
factors that appear to influence thermotolerance,
that even for the most genetically tractable microorganisms such as Escherichia coli and yeast
the events determining this property are not
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Department of Biochemistry and Molecular Biology, University College London, London, UK
340
Heat shock and growth arrest due to nutrient
limitation: two situations when thermotolerance
is strongly induced in yeast
The effects of heat stress on yeast
The heat shock response causes marked
changes in gene expression. Heat shock proteins
(HSPs) are strongly induced and most proteins
made prior to the heat stress are synthesised at a
greatly reduced level. The regulation of this response and the functions of the induced HSPs are
already reviewed extensively elsewhere [6-10].
This account seeks to emphasise the many other
physiological and molecular changes caused by
heat stress. Not only are these not as widely
publicised as the gene expression changes but, as
discussed later, there is increasing evidence that
these other changes must be considered together
with the altered gene expression pattern to
achieve a full picture of the events altering thermotolerance. Targeted HSP gene disruption has
shown only a minority of the HSPs of S. cerer,isiae
exerting appreciable effects on thermotolerance,
even though rather more of these proteins are
essential for proliferation at the highest temperatures of growth. Also, heat shock can induce
substantial increases in thermotolerance even in
the absence of protein synthesis, doubtless as a
result of physiological changes that occur independently of de novo HSP synthesis. Furthermore, while both thermotolerance acquisition and
the heat shock response can be triggered by a
number of chemical agents [6-9], not all chemical
inducers of thermotolerance are strong inducers
of HSPs.
Figure 1 summarises some of the events that
occur in vegetative yeast as the result of a sublethal heat stress. In addition to displaying induction of HSPs, the cells accumulate a large cytoplasmic pool of trehalose (a-D-glueopyranosyl aD-glucopyranoside). This trehalose is thought to
act primarily as a stress protectant rather than a
storage carbohydrate, its levels generally showing
a good correlative relationship with thermotolerance [14,15]. The trehalose accumulated very
rapidly in heat shocked yeast is mobilised equally
rapidly during a subsequent temperature shiftdown. This mobilisation resembles HSP synthesis
in being regulated by the levels of certain HSPs
(notably HSP70 [12]) (Fig. 1). Heat shock also
causes an increase in intracellular glucose, together with a partial inhibition of glycolysis [11].
Conversion of this glucose to trehalose may be
one way of preventing potential toxity problems
that may result when glucose is no longer totally
phosphorylated to glucose-6-phosphate immediately upon entry to the yeast cell. Heat also
causes perturbations to intracellular ion levels
and pH, these being attributable in part to an
enhanced permeability of membranes. Two important enzyme activities affected by these ion
and pH changes (Fig. 1) are the plasma membrane ATPase and cyclic AMP-dependent pro-
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clearly understood. This article seeks to bring
together the evidence from recent molecular genetic and biophysical experimentation concerning
causes of thermotolerance acquisition in the yeast
Saccharomyces cerevisiae, much of this evidence
being scattered among microbiology, molecular
biology, cell biology and biophysical journals. It is
also a summary of current knowledge of the physiological and molecular changes that occur within
heat stressed yeast.
With the demonstration that the archaebacterium Sulfolobus acquires an increased thermotolerance with preconditioning heat stress [1], we
now know that heat shock induces thermotolerance in all the major microbial kingdoms. Several
of the mechanisms of this tolerance acquisition
are probably highly conserved in nature. Their
elucidation in genetically tractable organisms such
as E. coli and yeast promises therefore to lead to
real practical benefits for the use and control of
quite distantly related microbes. For example, it
may enable the manipulation of strains for altered robustness in industrial bioprocesses or after release into the environment. It will also
contribute to the design of food processing procedures for more effective inactivation of food-contaminating microbes. As a phenomenon wide
spread in industrially important, food-contaminating and pathogenic microorganisms (see,
for examples [2-5]), thermotolerance acquisition
has major implications for microbiological safety.
341
m
Sensing of
protein damage t
+
~
Induction of
heat shock proteins
I
I
I
I
I
cAMP-PK - i n d e p e n d e n t ~ ~
trehalose induction
HEAT
STRESS...
\
~" ~k pHi decline, partly as j
the result ot increased
membrane permeability.
~t
4" ,,=Stimulation of
" plasma membrane ATPase
~Stimulation
of RASadenylate cyclase pathway
/
m?
Fig. 1. Regulatory circuits known, or suspected of operating,
in the yeast response to heat stress. Events that also occur
during nutrient limitation-induced arrest in the G1 phase of
the cell cycle are underlined. The main sensor of protein
damage is probably the lack of free HSP70, as all the available
HSP70 binds to the accumulations of damaged protein in
heat-stressed cells [9]. The immediate trehalose increase with
heat shock may reflect changes to the kinetic properties of
enzymes of trehalose synthesis, although these enzymes may
be further induced by heat shock. Trehalose is also under
negative regulation by HSP70 level. The p H i decline with
heat stress stimulates both plasma membrane ATPase and the
RAS-adenylate cyclase pathway. The ATPase activation causes
a greater catatysed proton extrusion from the cell that helps
counteract the effects of stress-induced p H i decline.
tein kinase A (cAMP-PK). Finally, in addition to
the events shown in Fig. 1, heat shock causes a
transient arrest in the G1 phase of the cell cycle.
Also, biophysical studies show that it leads to
alterations to the physical states of both m e m branes and intracellular water.
The influence of cAMP-PK activity on both basal
and heat shock-acquired thermotolerance
Even in the absence of an inducing stress there
are large variations in the thermotolerance of S.
cerevisiae. This 'basal', uninduced thermotolerance is remarkably sensitive to physiological state
and especially to protein kinase A (cAMP-PK)
activity. In general, rapidly proliferating yeast cultures have high c A M P - P K activity levels and low
thermotolerance, while stationary cultures have
The acquisition of thermotolerance through arrest
in the G1 phase of the cell cycle
Starvation of yeast for specific nutrients, such
as carbon, nitrogen, phosphate or sulphate, causes
growth arrest at a specific point in the G1 phase
of the cell cycle called START. This G1 arrest is
associated with marked increases in thermotolerance [24]. The S T A R T point has two sites, the
nutrient-starvation site and a subsequent site for
pheromone-induced arrest (reviewed in [25-29]).
The activity of c A M P - P K is required for progression over the nutrient starvation site of START,
while activity of the p34 cDcz8 protein kinase (encoded by the CDC28 gene) is needed for progression over the p h e r o m o n e arrest site. Mutants
temperature-sensitive for c A M P synthesis arrest
at the nutrient-starvation site when shifted to
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,nhibi.ooo,
qlycolysis
low c A M P - P K activity and high thermotolerance.
Studies using S. cerevisiae mutants of the RASadenylate cyclase system and mutants of the catalytic (TPK) and regulatory (BCY) subunits of
c A M P - P K have clearly shown that a low cAMPPK activity correlates with a high basal thermotolerance, while high c A M P - P K activity results in
low basal thermotolerance [16-19]. In addition, a
cyrl mutant is considerably thermotolerant at a
semipermissive t e m p e r a t u r e (32°C) [20].
c A M P - P K activity also strongly influences the
extent of the increase in the thermotolerance of
vegetative yeast due to heat shock. C o m p a r e d to
wild-type cells, ras2 mutants (possessing a low
c A M P - P K activity) show a greatly enhanced induction of thermotolerance by mild heat shock,
while the bey1 mutation (causing constitutively
high c A M P - i n d e p e n d e n t activity of this kinase)
confers a much reduced thermotolerance acquisition with heat shock [21]. Claims that bey1 strains
totally lack thermotolerance acquisition with heat
shock [16], also that exogenous c A M P blocks heat
elevation of thermotolerance in wild-type strains
[16], have not been substantiated in later studies
(Cheng, L. and Piper, P.W., unpublished results).
Also, it has been reported that strains without
two of the three TPK genes (which encode
c A M P - P K catalytic subunits [26-29]) have a much
reduced thermotolerance acquisition with heat
shock as c o m p a r e d to wild-type cells [23].
342
Activation of systems for
catabolite derepression N ~
GLUCOSE
STARVATION
~
Induction of
many, but not all
he~t shock proteins
Inhibition of cAMP
synthesis
NITROGEN
Jq
/
STARVATION
"~ Loss of free, active
catalytic subunits of
~
Induction of trehalose
and glycogen
cAMP-PK
Inhibition of ,qlycolysis
Fig. 2. Important events in the yeast response to carbon and
nitrogen limitation. Changes also induced in heat-shocked
cells are underlined. In the presence of glucose there is a
basal level of cAMP synthesis, although the RAS-adenylate
cyclase pathway is subject to catabolite repression and therefore only transiently activated by glucose addition to derepressed cells [29]. Nitrogen source activation of cAMP-PK
requires glucose, occurs without any increase in cAMP, and is
therefore thought to act on free, active catalytic subunits of
cAMP-PK [26,29]. Loss of cAMP-PK activity, with upon starvation-induced GI arrest, leads to an inhibition of glycolysis
[37], an induction of storage carbohydrate synthesis [30-31],
and the induction of certain heat shock genes [26,32-34].
events are a consequence of the loss of cAMP-PK
activity. The trehalose accumulation with glucose
limitation is due to an inactivation of neutral
trehalase and a reversal of the catabolite inactivation of trehalose-6-phosphate synthase-phosphatase that both reflect lowered cAMP-PK activity [29,31,36]. Loss of cAMP-PK action also
leads to an inactivation, by dephosphorylation, of
phosphofructokinase 2. This downregulates glycolytic flux by reducing the formation of fructose
(2,6)-bisphosphate, the stimulator of phosphofructokinase 1 [37]. Loss of cAMP-PK action also
activates the genes for the trehalose-6-phosphate
synthase-phosphatase complex [36], as well as
genes for several HSPs (for literature see [26]). It
is not yet clear whether all of these heat shock
gene activations are directly under cAMP-PK
control, or whether some are responding to
catabolite derepression (Fig. 2), since low cAMPPK activity levels are essential for the operation
of catabolite repression in S. cerevisiae [35].
At the same time that S. cerevisiae cells are
inducing trehalose, many HSPs and thermotolerance during nutrient limitation-induced G1 arrest
they are losing their capacity for further induction of HSPs, trehalose and thermotolerance in
response to a sublethal heat shock [38,39]. A heat
stress-inducible heat shock response is therefore
a manifestation of vegetative yeast, the reason
that cultures in exponential growth are the starting point for most heat shock studies.
It is noteworthy that E. coli, like yeast, also
accumulates trehalose at stationary phase. This
trehalose is important for the thermotolerance of
stationary phase E. coil cells [40]. Trehalose is
also accumulated by E. coil subjected to an osmotic stress but, unlike in yeast, it is not strongly
induced in E. coli by heat and the enzymes of
trehalose synthesis do not contribute to heat
shock-acquired thermotolerance in E. coil [40].
Cell cycle arrest due to heat shock
The transition over the pheromone-sensitive,
CDC28-dependent step of S T A R T requires activation of the p34 c°cz8 protein kinase. This activation occurs when p34 cdc28 complexes with other
unstable proteins termed the G1 cyclins. These
cyclins are the products of a family of functionally
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their restrictive temperatures, while mutants with
constitutively high cAMP-PK activity fail to arrest
at this point even under starvation conditions.
Progression over this nutrient starvation site requires a basal level of cAMP, but does not appear
to be stimulated by further activation of the
RAS-adenylate cyclase pathway, as occurs with
glucose addition to derepressed cells. Furthermore, nitrogen source-induced progression over
the nutrient starvation site of S T A R T can occur
with no increase in cAMP by a nitrogen sourceinduced signalling pathway, a system which is
dependent on a relatively high concentration of
glucose and which therefore appears to operate
via the free catalytic subunits of cAMP-PK [26,29].
Yeast cells entering G1 arrest through nutrient
limitation share not just their acquisition of high
thermotolerance with vegetative cells given a preconditioning heat shock, but also certain of the
same molecular and metabolic changes. A high
trehalose pool is accumulated [30,31], most HSPs
are synthesised [31-33] and glycolytic flux is reduced (Fig. 2). In nutrient limitation-induced
growth arrest, but not heat shock, these three
343
Thermotolerance and the general control system of
yeast
There is some evidence for a correlation between basal thermotolerance levels and the activity of the S. cerevisiae general control (GC) system for amino acid biosynthesis. In the G C system the undercharging of one transfer R N A
causes a translational induction of GCN4 protein,
the transcriptional transactivator of aminoacid
biosynthetic pathway genes. GCN4 protein itself
is not induced by heat shock [20]. It is, however,
present at much higher levels, with commensurate activation of the G C system, during extended
growth of wild-type cells at 37°C as compared to
29°C [20]. The starvation of leaky amino acid
auxotrophs for amino acids, the growth of cdc
mutants at temperatures semipermissive for
growth, or the growth of wild-type ceils at 37°C
instead of 29°C, are all conditions that cause an
intrinsically high thermotolerance together with
an induction of the GC system [20]. Also, the
gcnl, gcn3 and gcn4 mutations of the GC system
cause a small reduction in resistance to 48°C
exposure after a heat adaptation at 37°C [20].
Heat shock gene expression and thermotolerance
Evidence for a limited role of protein synthesis and
of a minority of HSPs in the thermotolerance induction with heat shock
It has often been assumed that HSP levels
must be a major determinant of acquired thermotolerance, since induction of HSPs generally occurs simultaneously with the induction of a greatly
increased thermotolerance during the heat shock
response. It is noteworthy, therefore, that induction of the full spectrum of HSPs in E. coli in the
absence of heat shock, using a plasmid with the
rpoH gene under the control of the IPTG-inducible Ptac promoter, is not sufficient for E. coli cells
to develop thermotolerance [48].
It is well-documented that HSP levels in yeast
cells do not always correlate well with levels of
thermotolerance; also that numerous studies employing the protein synthesis inhibitor cycloheximide have given a confusing picture of whether
protein synthesis is actually needed for the thermotolerance increase with heat shock (reviewed
in [6-8,49-52]). There is a major problem when
using inhibitors of protein synthesis in unravelling
the role of de novo protein synthesis in the heat
shock response. If, as is widely assumed, the
response is triggered by the accumulation of abnormal proteins [9], then the presence of cycloheximide may partly abolish this signal. Newly or
partially synthesised proteins are expected to be
particularly prone to thermal denaturation. By
preventing their synthesis, cells possibly do not
'feel' as stressed as they would otherwise. Therefore, even if a decrease in thermotolerance is
found when protein synthesis is prevented, this
might reflect attenuation of the stress signal
rather than any necessity for protein synthesis in
increased stress protection.
Leaving cycloheximide experiments aside, it is
noteworthy that thermotolerance is induced most
quickly in vegetative 25°C S. cerevisiae cultures
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redundant genes (CLN1, CLN2 and CLN3(WHI1
/DAF1) [41-44]. The failure of this p34 cDc28
protein kinase activation causes yeast cells to
arrest at START.
Proliferating wild-type S. cerevisiae arrest at
S T A R T when heat-shocked, transiently accumulating as unbudded ceils [45]. A slow resumption
of growth then occurs even under heat shock
conditions, provided that the temperature is not
more than about 38-39°C. Both pheromone signalling and heat shock decrease the abundance of
the CLN1 and CLN2 (but not CLN3) transcripts
[46,47]. The continuous expression of cyclin from
a heterologous promoter, or the synthesis of more
stable cyclins during a heat shock, are both sufficient to eliminate heat-induced S T A R T inhibition without affecting other aspects of the heat
shock response [47]. A temporary lack of cyclin
appears therefore to be the reason for the transient arrest of yeast growth with heat shock [47].
However, this may not be the complete explanation as, for reasons that are not yet clear, this
heat shock-induced arrest at S T A R T is prevented
by the constitutive high cAMP-independent protein kinase A activity of bcyl point mutants [16].
Also, levels of the CLN1 and CLN2 transcripts
still decrease with heat shock of bcyl strains [47].
344
HSPI04 in therrnotolerance
A S. cereuisiae strain lacking HSP104 grows at
the same rate as wild-type cells on glucose at
25°C or 37°C and still acquires thermotolerance
with heat shock, yet not to the same extent as an
isogenic wild-type [62-64]. Its lowered capacity
for thermotolerance acquisition is especially
marked if cells are left at 50°C for more than 8
min, but much less apparent during a shorter
50°C exposure [62]. Increasing the preconditioning mild heat treatment from 30 to 60 min diminishes the importance of HSP104 for acquired
thermotolerance [52]. While HSP104 does not
influence the basal thermotolerance of fermentative yeast it does have an effect on the basal
thermotolerance of vegetative respiratory cells,
stationary cultures and spores [64]. Also, in strains
lacking HSP104 the levels of another major HSP
(HSP70) can be shown to affect thermotolerance
[64], whereas HSP70 levels cannot be demonstrated to influence thermotolerance in strains
possessing HSP104 [58].
S. cereL'isiae HSP104 is an ATPase homologous
to the E. coli heat-inducible ClpA protease
[63,64]. It also has two nucleotide binding sites
essential for its contribution to acquired thermotolerance [63]. HSP104 does not influence the
trehalose accumulated during a heat shock [52],
although it does have a small effect on mobilisation of this trehalose during a subsequent temperature shift-down [12].
Heat shock stimulates the enzymatic anti-oxidant
defences of yeast
The anti-oxidant defences of yeast are both
non-enzymatic (as the glutathione pool) and enzymatic (as the catalase and superoxide dismutase enzymes that catalyse degradation of hydrogen peroxide ( H 2 0 2) and superoxide radicals,
respectively). Heat shock does not alter glutathione levels [68], yet it increases the activities
of at least two enzymes important for protection
against oxidative damage. These are cytoplasmic
catalase T, the product of the CTT1 gene [65-67],
and the mitochondrial manganese form of superoxide dismutase (MnSOD) [68]. MnSOD is a nuclear-encoded enzyme that is translocated to the
mitochondrial matrix during maturation. Its elevation by heat and ethanol may allow more efficient trapping of superoxide radicals within mitochondria, limiting their escape to the cytosol. In
contrast to MnSOD, the cytoplasmic CuZn form
of superoxide dismutase in yeast is not elevated
by heat shock [68].
CTT1, unlike most other HSP genes of yeast, is
under a negative regulation by cAMP-PK, with
the result that catalase T is not induced by heat
shock in cells with high cAMP-PK activity [67].
Lack of catalase T causes a small thermotolerance reduction in both proliferating and stationary cells, except when cAMP-PK activity is high
[67]. It is almost certain that the primary role of
this enzyme is to protect against the damage to
cellular proteins, nucleic acids and lipids caused
by H 2 0 2 and that its influence on thermotoler-
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rapidly heat-shocked to 45°C, a temperature too
high for significant protein synthesis in this organism [13]. Furthermore, osmotic dehydration
causes a very rapid induction of thermotolerance
under conditions where most HSPs are not
strongly induced. Also, certain chemical inducers
of thermotolerance in yeast, such as weak organic
acid preservatives (discussed further below) and
N6-(a2-isopentenyl)adenine [13] are not inducers
of HSPs (Cheng, L. and Piper, P.W., unpublished
results). In balance, therefore, it would seem that
thermotolerance induction in yeast can often occur largely independently of de novo protein synthesis. Consistent with this, targeted disruption of
a number of the HSP genes of S. cereuisiae has
been found not to alter basal or heat-acquired
thermotolerance levels [53-59]. Even when, as
with disruptions of UBI4 [57], SSA1 plus SSA2
[58], or HSP82 [59], the growth or viability of
yeast at 37-38.5°C was reduced, the extreme temperature (50-52°C) thermotolerance was not similarly affected and was even, in certain instances,
increased. For HSP60 and KAR2 disruptions,
inactivation produced a lethal phenotype [60,61],
preventing determination of whether the encoded
protein contributes to thermotolerance from a
gene disruptant. Nevertheless, inactivations of two
heat shock genes, HSPI04 and CTT1, have been
shown to reduce the thermotolerance acquired
with heat shock [62-67].
345
The activation elements for yeast heat shock genes,
their differing controls and dependency on cAMPPK activity
Two separate gene promoter elements have
been identified causing gene activation by heat
shock in S. cerevisiae. Heat induction of most S.
cerevisiae HSP genes is due to the same heat
shock element sequence (HSE) as is used in
higher eukaryotes, a H S E consisting of contiguous arrays of the five base-pair sequence n G A A n
arranged in alternating orientations at each halfturn of the D N A helix [71]. This HSE is the
binding site for the well-characterised yeast heat
shock factor, a trimeric trans-acting transcriptional activator whose activity is needed at all
growth temperatures. This factor has two physically separable transcriptional activation domains; a N-terminal activator domain mediating
the transient heat shock response and a C-terminal activator domain that mediates a sustained
HSP expression at elevated temperatures [71]. A
subset of the heat-inducible sequences of S. ceret,isiae appear not to be activated by this factor
and HSE sequences, but to use an alternative
stress-control element. Characterised best as the
UAS.360 element of CTT1, this alternative element directs the induction of catalase T by both
nitrogen starvation and heat shock in a heat
shock factor and HSE-independent mechanism
[21,65-67]. A sequence similar to the UAS360
element of CTT1 is to be found in the promoters
of a number of S. cerevisiae HSP genes which
also have the HSE sequence [21], raising the
possibility that the heat activation of some of
these may be under a dual regulation by both this
alternative element and heat shock factor bound
to the HSE.
It is important to note that the CTT1 UAS.360
element and the HSE differ in the extents to
which they are influenced by cAMP-PK activity.
The activity of the CTT1 UAS.36o element is
strongly influenced by cAMP-PK, whereas HSE
activity appears to be largely independent of
cAMP-PK. In vegetative cells wide variations in
cAMP-PK activity do not greatly alter the heat
inducibility of HSE sequences in reporter gene
constructs ([21]; Cheng, L. and Piper, P.W., unpublished results). These variations also do not
change the heat-inducibility of the major S. cerevisiae HSP genes, the extremely low cAMP-PK
activities of the cyrT1 [18] and tpk2 wl [35] mutants causing only a slight reduction in sustained
expression of these genes following a 25°C-39°C
heat shock (Cheng, L. and Piper, P.W., unpublished results). In view of the dramatic influences
of cAMP-PK activity on thermotolerance, this is
an indication that gene expression from HSE
sequences is not tightly linked to heat-induced
increases in thermotolerance. In addition, a mutant (hsrl-m3) which cannot activate HSE sequences with heat shock through its possession of
an altered heat shock factor still displays normal
thermotolerance induction with heat [50]. This
was originally reported as evidence that HSPs are
dispensible for thermotolerance induction, most
major heat shock genes not being heat-inducible
in this hsrl-m3 mutant. However, the recent discovery of a heat shock factor-independent heat
activation sequence (the UAS36 o element of
CTT1) in certain S. cerevisiae genes raises the
possibility that a subset of the heat shock genes
of yeast remain heat inducible in hsrl-m3 cells.
As mentioned earlier, at least one member of this
subset (CTT1) is now known to contribute to
thermotolerance acquisition in cells of low
cAMP-PK activity.
Besides acquiring high thermotolerance, cells
that are entering G1 arrest through nutrient limitation also activate most, but not quite all, of
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ance reflects a tendency for more severe oxidative
damage at higher temperatures, especially in respiratory cultures. The heat induction of the mitochondrial MnSOD probably provides further protection against such damage.
H 2 0 2 and mild heat shock independently induce resistances to both heat and the lethal effects of H20 2 [7,69], except in a strain lacking
catalase T [67]. Certain HSPs of S. cerevisiae
appear to be induced by H 2 0 z treatment [69].
However, H2Oz-inducibility is not a property of
all yeast HSP genes since the UBI4 polyubiquitin
gene is not induced by H 2 0 2 treatment even
though its disruption in yeast cells leads to increased H 2 0 2 sensitivity (Kirk, N., Watt, R. and
Piper, P.W., unpublished results).
346
their HSP genes and accumulate high levels of
trehalose (see above and Fig. 2). A few heat
shock genes (e.g. SSA4 [72]) fail to be activated at
G1 arrest. Starvation-induced activation of the
remainder is sometimes a result of low c A M P - P K
activity, many HSPs being expressed constitutively in the cyrl.2 mutant [16]. For certain heat
shock genes this stationary phase activation has
been shown to use p r o m o t e r elements distinct
from the H S E [73]. The UAS_36o element of CTT1
is one element causing gene activation in response to nitrogen starvation [21,65-67], while
the H S E itself loses its heat inducibility at the
approach to stationary phase [39].
and thermotolerance
Trehalose content of yeast and resistance to temperature extremes, freeze-thaw and dehydration
A large pool of trehalose accumulates in the
cytosol of yeast during both heat shock (see above
and Fig. 1) and starvation-induced growth arrest
in G1 (see above and Fig. 2). Trehalose is synthesised from UDP-glucose and glucose-6-phosphate
by a two-step reaction catalysed by the trehalose6-phosphate synthase-trehalose-6-phosphate
phosphatase complex [11,31,36,85-87]. It is broken down to glucose by two distinct trehalase
enzymes; an acid trehalase confined to the vacuole and a cytoplasmic neutral trehalase which is
activated by c A M P - P K - d e p e n d e n t phosphorylation [11,31,36,85-87]. Mobilisation of trehalose is
generally due to an activation of the neutral
trehalase [31,85-87].
There exists a strong correlation between the
trehalose content of yeast cells and their tolerance of t e m p e r a t u r e extremes, freezing-thawing
cycles and dehydration (reviewed in [14,15,7476]). It has therefore been proposed that the
primary function of trehaiose in yeast (also other
fungi, nematodes and insects) is not as an energy
source, but as a protectant of the structure of cell
m e m b r a n e s and proteins under conditions that
deplete intracellular water activity [14,15,74-76].
Trehalose is also thought to influence m e m b r a n e
structure (Table 1) and the strength of hydrogen
bonding (H-bonding) interactions.
Alterations to yeast membranes resulting from a preconditioning heat stress
(a) Changes to lipid composition. The degree of saturation of
membrane lipids increases, as part of a long-term acclimation
to growth at higher temperatures seen in many organisms
[49,78]. In vivo 3H and 13C nuclear magnetic resonance (NMR)
reveals that mild heat shock treatment substantially reduces
the disordering of yeast lipid bilayers during subsequent freezing and thawing [79,80].
(b) Changes to membrane proteins. Plasma membrane ATPase levels are reduced [81]. The plasma membrane also
acquires an integral membrane HSP (HSP30) [81,82].
(c) Protective effects of the induced trehalose on the lipids and
protein structure of membranes. Trehalose binds to the polar
head groups of lipids, where it replaces water, and acts to
preserve the properties of a hydrated membrane during dessication [74,83]. In vitro experiments show trehalose can inhibit
membrane fusion [84]. Trehalose and polyols also increase the
thermal stability of proteins [74-77], and probably exert a
similar action also on membrane proteins.
The thermotolerance inductions in S. cerevisiae with declining c A M P - P K activity (as in G1
arrest) or with heat shock both occur in parallel
with the accumulation of trehalose. However,
certain chemical treatments are known to induce
thermotolerance in yeast and yet to cause either
no induction, or only slight induction, of trehalose. Such treatments include exposure to weak
organic acid preservatives (discussed further below), exposure to N6-(AZ-isopentenyl)adenine [23]
and salt challenge. It appears therefore that while
trehalose induction may be a major way for the
yeast cell to acquire increased thermotolerance, it
may not be the only means of achieving this
objective.
The mechanisms of trehalose induction with heat
shock and trehalose mobilisation following temperature shift-down
Trehaiose does not accumulate in unstressed
vegetative yeast because neutral trehalase is predominantly in its activated (phosphorylated) form
and the activity of the trehalose-6-phosphate syn-
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Trehalose
Table 1
347
[12,38,93,94]. At least in vegetative cells, this trehalose mobilisation is controlled by the levels of
certain HSPs synthesised during the period of
heat shock. HSP70 levels are the major influence,
the levels of HSP90 and HSP104 exerting comparatively more minor effects ([12]; Cheng, L. and
Piper, P.W., unpublished results). Levels of 'free'
HSP70 may therefore be the major control modulating the heat induction of trehalose, in much
the same way as they act to downregulate the
synthesis of HSPs [9] (Fig. 1). This recent discovery of HSPs controlling trehalose levels raises the
possibility that these HSPs might, in certain circumstances, influence thermotolerance indirectly
through their effect on trehalose.
Membrane protection, plasma membrane ATPase
action and extracellular/intracellular pH in
thermotolerance
Stress-induced alterations to the structure and permeability of membranes
Heat stress is known to lead to changes to the
lipid and protein compositions of yeast membranes, while the induced trehalose is suspected
of exerting protective effects on membrane structure that may be even more important in stress
survival (see Table 1).
Heat, and certain other stress agents such as
ethanol, also act to increase membrane permeability. Any factor which can limit this membrane
permeabilisation should help the cell maintain
homeostasis under stress. One effect of increased
membrane permeabilisation in yeast at high temperature should be an increased sensitivity to
membrane-impermeant drugs. Another is an increased proton influx, a rapid decrease in intracellular pH (pHi) being an almost immediate
effect of mild heat, a number of chemical agents
and exposure to acid external pH [95-97].
Plasma membrane ATPase in thermotolerance and
the heat shock response
The decline in pH i with stress-increased proton influx to the cell causes a stimulation of
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thase-phosphatase complex is only 20% of that
found at stationary phase [36,88]. When such cells
are heat-shocked, trehalose immediately starts to
accumulate, even though there is a lag period of
about 10 min before any increase in the activities
of trehalase or trehalose-6-phosphate synthase/
phosphatase [11,38]. Induced synthesis of the enzymes of trehalose metabolism is not therefore
the cause of the immediate trehalose increase
with heat. Instead, not only does heat shock
cause a sudden rise in intracellular glucose, simultaneous with decreases in the concentration
of hexose phosphates and fructose 2,6-bisphosphate in vivo, but it also changes the in vitro
kinetic properties of partially purified enzymes of
trehalose metabolism [11]. In vitro, the affinity of
both the unactivated and cAMP-PK-activated
forms of trehalase for Ca 2+ ions was decreased
more than 20-fold at 40°C as compared to 30°C,
while the activities of the trehalose-6-phosphate
synthase-phosphatase complex were three times
more active at 40°C [11].
While these changes in the catalytic properties
of trehalose-6-phosphate synthase/phosphatase,
and possibly Ca 2+ level, may be responsible for
the immediate trehalose induction in heatshocked yeast, there is nevertheless evidence for
a slower induction of these enzymes of trehaiose
metabolism in the same cells. Using cycloheximide [11,23,89] or a temperature-sensitive mutation [52] to block protein synthesis during heat
shock, it has been reported that the trehalose
induction with heat stress of S. cerevisiae is partially inhibited. In contrast, the trehalose induction of Schizosaccharomyces pombe with heat
shock is unaffected by cycloheximide [90]. Heat
stress appears therefore to rapidly stimulate the
activity of pre-existing trehalose-6-phosphate synthetase/phosphatase and, in S. cerevisiae, to also
cause a slower increase in de novo synthesis of
this enzyme complex.
The trehalose accumulated in S. cerevisiae during heat shock is mobilised very rapidly during a
subsequent temperature shift-down. Cooling after
heat shock causes rapid reactivation of the neutral trehalase, both in yeast ascospores [91] and in
vegetative cells [92]. This trehalase reactivation is
c o m m e n s u r a t e with rapid t r e h a l o s e loss
348
an integral membrane protein that might conceivably regulate or stabilise plasma membrane components during stress.
Thermotolerance effects of external pH
External pH has been reported to affect thermotolerance [13]. This is perhaps not surprising
since it is known that even in unstressed yeast
cells pH i can be affected by external pH [100].
Results obtained in the author's laboratory indicate that basal thermotolerance is remarkably
sensitive to several manipulations of cells, varying
with the centrifugation steps of medium changes
and, as found earlier [13], with adaptation to
media of different pH. However, provided the
lethal heating is conducted under defined conditions of temperature and pH, heat shock-acquired
thermotolerance is not markedly influenced by
the pH of the medium during the preconditioning
heat stress (Cheng, L. and Piper, P.W., unpublished results).
Thermotolerance effects of weak acids
It is possible to induce a rapid pH i decline in
cells in the absence of stress by the addition of
weak organic acids preservatives (e.g. benzoic acid
or sorbic acid, both widely used in food preservation). At low medium pH values, the undissociated forms of these acids readily cross the cell
membrane and reduce pH i by dissociating in the
higher pH environment of the cytosol [101]. In S.
cerevisiae such weak acids inhibit glycolysis,
through a reduction in fructose-2,6-bisphosphate
level, and cause transient increases in cAMP and
trehalase activity [102]. Sorbic acid (pK~ 4.75) has
been variously reported as either having no effect
on [103], or increasing [13], the thermotolerance
of S. cerevisiae. A more recent study has shown
that its effects are strongly dependent on medium
pH (Cheng, L. and Piper, P.W., unpublished results). When present during the lethal heat treatment it increases thermotolerance at pH values
above 6.5. However, at lower pH values it greatly
decreases thermotolerance, and especially heatinduced thermotolerance, causing a strong selection for cytoplasmic petites amongst the surviving
cells (Cheng, L. and Piper, P.W., unpublished
results).
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plasma membrane ATPase activity. This in turn
results in an increased catalysed proton extrusion
that acts to counteract the drop in pH~ (Fig. 1).
This increased proton extrusion by intact cells is
demonstrable as an increased acidification of the
medium in response to heat [13]. The necessity
for increased plasma membrane ATPase action,
and for a general restoration of homeostasis,
probably represent one of the major energy demands imposed by heat stress [13,81,98]. It is not
surprising, therefore, that plasma membrane ATPase action is an important influence over the
thermotolerance of S. cerevisiae and S. pombe, as
well as toleran6es to a number of other stresses
[98]. Also, inhibition of this ATPase using diethylstilbestrol hypersensitises yeast to thermal death
[13]. Plasma membrane ATPase action is also
vital for the ability of S. cerevisiae cells to synthesise HSPs in response to heat shock. A reduction
in this ATPase activity reduces HSP induction
with heat shock [98], while total inhibition of this
ATPase by diethylstilbestrol treatment has the
almost immediate effect of rendering cells incapable of any induction of HSPs by heat shock,
even though these cells still retain a capacity for
protein synthesis (Cheng, L. and Piper, P.W.,
unpublished results).
Plasma membrane ATPase activity is vital in
maintenance of homeostasis [99] and in the general restoration of homeostasis during recovery
from stress. It is surprising therefore that the
levels of this, the most abundant enzyme of the
plasma membrane, decline rapidly in the plasma
membrane of heat-shocked yeast [81]. However,
this ATPase alone is thought to consume 10-15%
of the ATP produced during normal yeast growth,
this increasing to 50% of ATP produced under
certain situations, as during glucose fermentation
in the absence of growth [99]. Stress survival may
therefore require a fine balance between the expenditure of ATP for maintenance of homeostasis and the ability of the stressed cell to maintain
ATP levels. The rapid loss of plasma membrane
ATPase in heat-shocked cells may be influenced
by proteins induced by the heat shock response.
In this regard it is interesting to note that S.
cerevisiae has recently been found to have a HSP
targeted to the plasma membrane (HSP30) [81,82],
349
The thermotolerance induced by sorbic acid
occurs with no accumulation of trehalose [23].
Remarkably, sorbic and benzoic acids are inducers of many HSP genes, yet they also render
cultures of low medium pH incapable of responding to a heat shock by the normal heat induction
of HSPs. This is despite the fact that the treated
cells are still competent of transcription and protein synthesis (Cheng, L. and Piper, P.W., unpublished results). Many chemical agents are inducers of HSPs [6-9], but weak acid preservatives
(and diethylstilbestrol) are amongst the few
chemical agents known to selectively prevent induction of these proteins by heat.
The thermotolerance induction with osmostress
Sudden exposure of ceils to high osmolarity
causes rapid water loss. Many microorganisms
counteract this reduced water activity and turgor
pressure by accumulating osmoprotective compounds. Fungi combat osmotic dehydration by
the production of polyol compounds, S. cerevisiae
synthesising glycerol as its major osmoregulator
[108,109]. The induction of trehalose by S. cerevisiae in response to osmostress is small [110], in
contrast to the substantial trehalose induction
with osmostress of E. coli [40]. Osmostress also
leads to a very rapid increases in thermotolerance, both in yeast [111] and in certain important
bacterial pathogens [112]. In S. ceretqsiae a 0.7 M
NaC1 challenge does not strongly induce the synthesis of most major HSPs, although it does lead
to rapid induction of HSP12 and HSP26 [113].
However, the loss of these two proteins appears
to have no effect on either osmotolerance [113] or
heat-induced thermotolerance [53-55].
The question arises of whether the rapid thermotolerance increase of osmostress is due to intracellular glycerol. Glycerol is a well-known protector of mammalian cells during hyperthermia
(for literature see [114]), causing an increased
interaction between solvent and solute macromolecules that acts to strengthen hydrophobic
bonding (discussed below). However, glycerol
production is inhibited during the initial period
of adaptation of S. cerevisiae to osmostress, since
it takes more than 6 h for intracellular glycerol to
increase after transfer to 1.4 M NaCI [110] and
60-80 h for a similar increase after transfer to 1.7
M NaC1 [115]. Giycerol-3-phosphate dehydrogenase activity does not increase until 45-60 h after
the latter salt transfer [115], although the mRNA
for this enzyme is induced very transiently 10-30
min after a 0.7 M NaCt challenge [113].
The slowness of intracellular glycerol accumulation by S. cerevisiae in response to salt challenge cootrasts strongly with the rapidity of the
thermotolerance increase [111]. This indicates osmotic dehydration as the cause of the thermotolerance rapidly acquired with osmostress, al-
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Intracellular acidification activates cAMP synthesis
A decrease in pH~ is one of the consequences
of heat shock [96]. Such intracellular acidification
stimulates the RAS-adenylate cyclase pathway
(Fig. 1) irrespective of the catabolite repression
status of the yeast [26], intracellular cAMP levels
increasing approximately two-fold with 24°C-36°C
[104,105] or 30°C-42°C [23] heat shocks. This
cAMP increase is possibly too small to be physiologically important. Even though it may belie a
rather greater stimulation to cAMP synthesis, as
much of the cAMP synthesised by yeast is lost to
the medium [106], there appears to be no transient peak of intracellular cAMP immediately
following heat shock. However, any increase in
cAMP-PK activity should help to counteract the
inhibition of glycolysis with heat stress (Fig. 1). In
unstressed yeast phosphofructokinase 2 is activated by cAMP-PK-catalysed phosphorylation,
the resultant increase in fructose (2,6)-bisphosphate levels acting to increase glycolytic flux
through its stimulation of phosphofructokinase 1
[37]. Reversal of this activation of phosphofructokinase 2 in response to low cAMP is thought to
be a major factor reducing glycolytic flux on starvation (Fig. 2). Another effect of an increased
cAMP-PK action with heat shock might be a
stimulation of the plasma membrane ATPase,
although the regulation of this enzyme by phosphorylation at its C-terminal regulatory domain is
now thought to involve the calmodulin-dependent
multiprotein kinase and not cAMP-PK [107].
Effects of osmotic dehydration, cryoprotectants
and the structurisation of intraceilular water
350
though glycerol might still contribute to the thermotolerance of S. cereL'isiae adapted to extended
growth in salt medium. Sublethal heat shock of
yeast does not provide significant protection
against osmostress [111,113], heat stress-acquired
thermotolerance or high trehalose levels not apparently protecting against the effects of osmotic
dehydration.
Influence of the physical state of intracellular water
on thermotolerance
Conclusion
Integration of biochemical, molecular genetic and
biophysical studies will be needed for a complete
understanding of thermotolerance
This account has discussed several factors
known, or suspected of influencing thermotolerance in yeast, but other important influences may
yet remain unidentified. For example, heat stress
of mammalian cells causes a rapid build-up of
intracellular inositol triphosphate and Ca 2÷ [119].
Similar effects on the phosphoinositide signalling
system probably occur in yeast and might be
important for the stress response and tolerance
determination. Also, in most of the cited yeast
studies there has been no systematic attempt to
distinguish respiratory from fermentative cells.
This is despite the fact that it has long been
known that growth of yeast at elevated temperatures provides strong selection for cytoplasmic
petites [120], reflecting the high sensitivity of mitochondria to heat damage [103].
It is interesting to compare studies on thermotolerance with studies on other kinds of stress
tolerance. As discussed earlier, there is a partial
overlap between the heat and oxidative damage
responses of yeast. The non-equivalence of the
osmotolerant and thermotolerant states has been
mentioned above. Counteracting osmotic dehydration requires a different conditioning (partly
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Biophysical investigations have revealed that a
preconditioning heat shock of yeast changes the
physical state o]~ intracellular water [116-118]. At
least three physical properties of intact ceils undergo change. A decrease in intracellular water
mobility, as measured by NMR, and an increased
cytoplasmic viscosity [118], are both indicative of
increased water structurisation and a reduction in
the 'free' freezable water of the cell. Finally an
approximate 7°C reduction in the freezing temperature of intracellular water, as measured by
differential scanning calorimetry (DSC), is an indication that 'free' water regions of the cytosol
have undergone substantial reduction in size. This
increased structurisation of intracellular water
with heat shock is possibly a reflection of more
stable H-bonding interactions between solvent
and solute [116]. It correlates well with increases
in thermotolerance, pressure tolerance (barotolerance) and freeze-thaw tolerance [117].
Cryoprotectants (e.g. deuterium oxide, dimethylsuphoxide and glycerol) act to increase water
structurisation in the absence of heat shock. In
N M R studies they can be seen to produce in vitro
changes similar to those seen in vivo with heat
shock of intact yeast cells. By strengthening hydrogen bonding and, indirectly, hydrophobic interactions, such cryoprotectant compounds help
to stabilise proteins and membranes from thermal denaturation in vitro. Probably they exert a
similar action in vivo, increasing the amount of
unfreezable (tightly bound) water [116]. Although
heat shock of yeast produces similar changes [116],
it is still not clear whether it is increased level of
HSPs or increased trehalose that confers these
properties by acting as an intracellular protector
against thermal denaturation in vivo. Simple in
vitro studies show that those sugars with the most
equatorial hydroxyl groups, notably trehaiose, are
most efficient at stabilising membranes and proteins [75-77]. HSPs may also help to maximise
such interactions, but even with a severe heat
shock of S. cerevisiae they might not accumulate
to sufficient level to account for the major changes
in the structural state of intracellular water with
heat shock observed in NMR studies, even though
it is reported that these changes are blocked by
cycloheximide [117]. The availability of yeast mutants that accumulate either trehalose or HSPs,
but not both, with heat shock should soon resolve
the issue of whether either trehalose or HSPs are
contributing to increased structurisation of intracellular water in vivo.
351
mutants defective in either the trehalose or the
HSP inductions of the heat shock response
promise to greatly clarify the relative contributions of HSPs and trehalose to the thermotolerance acquisition with heat shock. Other evidence
for the role of trehalose in stress tolerance will
come from mutants defective in the nutrient limitation-induced trehalose accumulation. Meanwhile, biophysical methods will be valuable in
addressing the unresolved issue of whether the
different routes to thermotolerance induction
cause a common alteration to the physical state
of the intracellular environment, possibly an increased structurisation of the intracellular water
through formation of more stable hydrogen bonding.
Acknowledgements
I am indebted to Lili Cheng, Peter Coote,
Claudio DeVirgilio, Jean Francois, Thomas Hottiger, Martin Jones, Pim Mager, Pedro MoradasFerreira, Sunil Kaul, Yasuhiko Komatsu, Johan
Thevelein and David Wilkie, all of whom have
made valuable contributions to this review. Projects in my laboratory have been supported by the
UK Science and Engineering Research Council,
the British Council and the Ciba Geigy Fellowship Trust.
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