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J Biol Chem, Vol. 273, Issue 50, 33311-33319, December 11, 1998
From the In the yeast Saccharomyces
cerevisiae, trehalose-6-phosphate synthase (TPS) and
trehalose-6-phosphate phosphatase (TPP), which convert glucose
6-phosphate plus UDP-glucose to trehalose, are part of the trehalose
synthase complex. In addition to the TPS1 (previously also
called GGS1, CIF1, BYP1,
FDP1, GLC6, and TSS1) and
TPS2 (also described as HOG2 and
PFK3) gene products, this complex also contains a
regulatory subunit encoded by TSL1. We have constructed a
set of isogenic strains carrying all possible combinations of deletions
of these three genes and of TPS3, a homologue of
TSL1 identified by systematic sequencing. Deletion of
TPS1 totally abolished TPS activity and measurable
trehalose, whereas deletion of any of the other genes in most cases
reduced both. Similarly, deletion of TPS2 completely
abolished TPP activity, and deletion of any of the other genes resulted
in a reduction of this activity. Therefore, it appears that all
subunits are required for optimal enzymatic activity. Since we observed
measurable trehalose in strains lacking all but the TPS1
gene, some phosphatase activity in addition to Tps2 can hydrolyze
trehalose 6-phosphate. Deletion of TPS3, in particular in a
tsl1 In the yeast Saccharomyces cerevisiae, trehalose is
synthesized by a large enzyme complex comprising the two catalytic
activities of trehalose biosynthesis: trehalose-6-phosphate synthase
(TPS)1 and
trehalose-6-phosphate phosphatase (TPP) (1-3). Several genes encoding
subunits of the trehalose synthase complex have been cloned. The
TPS1/TSS1 gene has been cloned as encoding the smallest (56 kDa) subunit responsible for TPS activity (4, 5), the TPS2
gene as encoding the 100-kDa subunit responsible for TPP activity (6),
and the TSL1 gene as encoding the large (123 kDa) regulatory
subunit (5). The TPS3 gene is a homologue of TSL1, identified by systematic sequencing
(GenBankTM/EMBL Data Bank accession number M88172).
Two-hybrid analysis has indicated that it might also encode a
functional component of the trehalose synthase complex (7), and its
expression is coregulated with that of TPS1/TSS1 and
TPS2 (8). The TPS1/TSS1 gene has also been cloned
independently by complementation of the cif1 mutant (9) and
the fdp1 and byp1 mutants (10, 11). In the latter
case, we called it GGS1 for "component of a
general glucose sensing system"
since these mutants were unable to grow on glucose and were
deficient in a wide range of glucose-induced regulatory phenomena (10,
12-14).
The cloning of the TPS1 gene has revealed a novel connection
between trehalose metabolism and the control of glycolysis (for review,
see Ref. 15). Three hypotheses have been proposed to explain this
connection. 1) The Tps1 protein has, in addition to its function in
trehalose 6-phosphate (Tre-6-P) synthesis, a separate regulatory
function that is responsible for the restriction of glucose influx by
interaction with glucose transport and sugar kinase activity (14). 2)
Trehalose metabolism prevents overflow of glycolysis during the
initiation of glucose fermentation by deviation of sugar phosphates
into trehalose synthesis with concomitant recovery of free
Pi required by the glyceraldehyde-3-phosphate dehydrogenase
reaction (16). 3) Tre-6-P, the intermediate of trehalose biosynthesis,
restricts sugar influx into glycolysis by inhibition of hexokinase
activity (17).
All genes encoding subunits of the trehalose synthase complex display
significant sequence homology to the TPS1 gene (5, 6). In
this study, we show that despite this homology, none of the other
subunits of the trehalose synthase complex, singly or in combination,
can take over the function of Tps1 in synthesizing Tre-6-P or in
controlling glucose influx into glycolysis. Using a set of strains with
all possible combinations of deletions in the four genes encoding the
trehalose synthase complex, we report on the contribution of the
different gene products to the two catalytic activities of the
trehalose synthase complex in vitro, to regulatory
properties of TPS activity in vitro, and to trehalose accumulation in vivo. In addition, we show that part of the
Tps1 protein is apparently present as free protein, that this monomeric Tps1 protein has catalytic activity, and that it displays different regulatory properties compared with the Tps1 protein in the trehalose synthase complex.
Yeast Strains and Growth Conditions--
The S. cerevisiae strains used in this study are shown in Table I.
Overexpression of TPS1, TPS2, TPS3,
and TSL1 was carried out with a YEplac195
(URA3) plasmid containing the respective genes behind their
own promoter. The cells were grown at 30 °C, as indicated, either to
exponential phase or to stationary phase and on rich or synthetic
medium with glucose (2%, w/v), galactose (2%, w/v) or glycerol (3%,
v/v) as carbon source. Rich medium contained 1% (w/v) yeast extract
and 2% (w/v) Bacto-peptone. Synthetic medium was prepared according to
Sherman et al. (18). The growth measurements on solid agar
medium (see Fig. 7) were performed on rich medium with 1% (w/v) yeast
extract, 2% (w/v) Bacto-peptone, and the respective sugar as indicated.
Construction of Deletion Strains--
The deletion approach for
TPS1 and TPS2 has been described previously (16,
19). The coding region of the TSL1 and TPS3 genes
was deleted completely using the same polymerase chain reaction approach (20) and replaced by the marker genes as indicated in Table
I.
Determination of Trehalose and Tre-6-P Content and TPS and TPP
Activities--
Trehalose was determined as described by Neves
et al. (21). Tre-6-P was quantitated by high pressure liquid
chromatography as described previously (22). For the expression of
trehalose and Tre-6-P content as estimated intracellular concentration, the following conversion factor was used: 7 mg of protein equals 100 µl of intracellular volume. TPS activity in crude cell extracts or
after purification by Superose 6 FPLC (see below) was determined according to Hottiger et al. (23), and TPP activity
according to De Virgilio et al. (6). Activity is expressed
as microkatals/g of protein (1 microkatal/g of protein = 1 µmol/s/g of protein). Protein was determined by the biuret method
(24).
Superose 6 FPLC of Cell Extracts and Western Blot
Analysis--
Extracts of stationary phase cells were loaded on a
Superose 6 HR 16/50 FPLC column (Amersham Pharmacia Biotech) and eluted with buffer (pH 8.0) containing 150 mM
NH4HCO3, 0.5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/ml pepstatin
A. The flow rate used was 12 ml/h, and the volume of the fractions
collected was 0.8 ml. The proteins in the samples from the gel
filtration column were precipitated with trichloroacetic acid and
washed once with cold acetone. The precipitate was dissolved in 0.1 M NaOH before addition of SDS-polyacrylamide gel
electrophoresis loading buffer. Conditions for SDS-polyacrylamide gel
electrophoresis were as described by Laemmli (25). After
electrophoresis, the proteins were blotted by electroporation from the
SDS gel onto nitrocellulose membranes. After transfer, the membranes
were treated with blocking solution and incubated with the primary
antibody (polyclonal rabbit anti-trehalose synthase complex antibody,
kindly provided by Dr. J. Londesborough, VTT, Espoo, Finland) overnight at room temperature in a 1:2000 dilution. After washing, the bound primary antibodies were detected using monoclonal anti-rabbit alkaline
phosphatase-conjugated immunoglobulin.
TPS Activity in Multiple Deletion Strains--
We have constructed
a set of 16 isogenic strains in the W303-1A background displaying all
possible combinations of deletions in the four known genes encoding the
trehalose synthase complex (Table I). For
all genes, the entire open reading frame was deleted. The
TPS1 gene was essential to detect significant TPS activity whatever the combination of other genes present (Fig.
1). This was true in exponential as well
as in stationary phase. However, in the presence of a functional
TPS1 gene, deletion of the other genes also affected TPS
activity, both in exponential and in stationary phase. The single
deletion of TPS3 or TSL1 had the least effect, but the double deletion caused a strong decrease in TPS activity. This
is the first evidence that TPS3 encodes an active functional component of the trehalose synthase complex. In the triple deletion strain tps2 TPP Activity in Multiple Deletion Strains--
TPP activity was
entirely absent in all strains lacking TPS2, both in
exponential and in stationary phase (Fig.
2). Deletion of TSL1 and/or
TPS3 had only a limited effect on TPP activity, especially
in exponential phase, but deletion of both genes reduced TPP activity
under either growth condition. Deletion of TPS1 severely reduced TPP activity, whatever other gene combination present.
Trehalose and Tre-6-P Content in Multiple Deletion
Strains--
Trehalose and Tre-6-P levels were determined only in
stationary phase cells because, in wild-type strains, trehalose content is very low in exponential phase. In strains lacking TPS1,
no trehalose or Tre-6-P was detected (Fig.
3). Deletion of TSL1 or TPS3 slightly reduced trehalose content, whereas the double
deletion strain had <50% of the trehalose level in the wild-type
strain. In addition, the tsl1 Superose 6 FPLC of Cell Extracts--
We fractionated yeast cell
extracts according to molecular mass of the proteins using Superose 6 FPLC, and we used Western blot analysis to detect the presence of the
protein components of the trehalose synthase complex after separation
of all proteins in each fraction by SDS gel electrophoresis (Figs.
4-6). Antibodies raised against the
whole trehalose synthase complex (kindly provided by Dr. J. Londesborough) were used. We consistently detected the smallest
component (Tps1) both as part of the trehalose synthase complex and as
apparently free monomeric protein, at least not bound to the complex.
The other subunits were mainly or only detected in the trehalose
synthase complex. They seem to be present at lower levels than Tps1,
although it cannot be excluded that the reactivity of the antibodies
for the other components of the trehalose synthase complex is
lower.
To check whether the presence of the free Tps1 protein might be the
result of unspecific breakdown of the trehalose synthase complex, we
incubated cell extracts for an extended period of time (30 min) at
30 °C. However, this treatment did not cause any change in the
protein pattern of the FPLC-fractionated cell extracts (Fig. 4),
indicating that the trehalose synthase complex is quite stable and that
free Tps1 protein is probably also present in vivo.
Deletion of either TSL1 or TPS3 appeared to
destabilize the trehalose synthase complex to some extent since more
free Tps1 protein and more breakdown products were detected (Fig.
5, A-C). However, in both
cases, Tps2 and a strong band of Tps1 could still be detected at the
normal elution position of the trehalose synthase complex. Deletion of
TSL1 produced a more dramatic effect than deletion of
TPS3, with a higher proportion of Tps1 and also Tps2 being
detected free from the complex. In cell extracts of a
tsl1
We also investigated whether overexpression of TPS2, TSL1,
and TPS3 separately or TPS2 and TSL1
simultaneously could reduce the level of free Tps1 protein. In none of
these cases, however, was a reduction observed (data not shown). This
also indicates that the expression of neither of these subunits is
limiting for the formation of trehalose synthase complexes.
Overexpression of TPS2 and TSL1, both separately
and combined, was clearly observed on the Western blots (data not
shown). Overexpression of TPS3, on the other hand, did not
result in the appearance of an additional band, probably because of the
failure of the antibodies to react with the Tps3 protein. In a control
experiment, the TPS3-containing plasmid was introduced into
a tsl1
In FPLC-fractionated extracts of wild-type cells, TPS activity could be
measured only at the position of the trehalose synthase complex and not
at the position of the free Tps1 protein (data not shown). However, in
extracts from a tps2 Growth of Multiple Deletion Strains--
The growth properties of
the 16 different deletion strains on galactose and fructose are shown
in Fig. 6A. All strains
lacking the TPS1 gene were unable to grow on fructose
whatever other combination of genes present. In addition, the presence
of the TPS1 gene was sufficient for growth on fructose
whatever other combination of genes present (Fig. 6A).
Similar results were obtained for growth on glucose, except that the
growth defect was somewhat less stringent than for growth on fructose
(data not shown). Overexpression of any one of the four genes in a
wild-type strain did not cause a noticeable growth defect on glucose or
fructose (data not shown). In addition, overexpression of
TPS2, TPS3, or TSL1 in a
tps1 Regulation of TPS Activity in Multiple Deletion Strains--
We
also investigated the effect of deletion of the subunits encoded by
TPS2, TSL1, and TPS3 on the regulation
of TPS1-encoded TPS activity. The enzyme from wild-type
cells is inhibited by inorganic Pi with a
Ki of 1-2 mM (Fig.
7) (2, 26). Deletion of TSL1,
but not TPS3, slightly reduced the inhibition by
Pi (Fig. 7). This was observed both with cells grown on
glucose until stationary phase and with exponential phase cells grown on glycerol (Fig. 7). Deletion of both TSL1 and
TPS3, deletion of TPS2, or deletion of all three
genes eliminated Pi inhibition and switched it into
Pi stimulation (Fig. 8). This
was observed both for cells grown on glucose until stationary phase
(Fig. 8) and with exponential phase cells grown on glycerol (data not
shown). In the latter case, the activities in the strains
tsl1
TPS activity is strongly stimulated by fructose 6-phosphate (26). This
effect can be observed clearly only in extracts from cells of a
phosphoglucoisomerase mutant. In extracts of wild-type strains,
phosphoglucoisomerase activity converts part of the TPS substrate
glucose 6-phosphate into fructose 6-phosphate, which then acts as a
stimulator of TPS activity. Our measurements of inhibition by
Pi were carried out in cell extracts of the wild-type strain without added fructose. However, because of the presence of
phosphoglucoisomerase, an equilibrium mixture of glucose 6-phosphate and fructose 6-phosphate was rapidly formed in the test medium. Probably because of this reason, further addition of fructose 6-phosphate only slightly stimulated TPS activity (±20%). This additional activation did not appear to be affected by deletion of the
other subunits of the trehalose synthase complex (data not shown).
Subunits of the Trehalose Synthase Complex--
In the yeast
S. cerevisiae, trehalose is synthesized by a large
multisubunit complex comprising the two catalytic activities TPS and
TPP (1-3). This is different from Escherichia coli, where the two enzymatic activities reside in separate enzymes encoded by the
genes otsA (responsible for TPS activity) and
otsB (responsible for TPP activity) (27-29). Up to now,
four genes have been identified that appear to encode components of the
trehalose synthase complex in S. cerevisiae:
TPS1, TPS2, TPS3, and TSL1.
TPS1 is homologous to otsA from E. coli and
remarkably also over its entire sequence to each of the other yeast
subunits (~35% identity in each case). Interestingly,
otsB shows homology only to the C-terminal part of the
TPS2 gene product, which is therefore probably responsible for the TPP activity of Tps2 (29). The comparison between the yeast and
E. coli enzymes shows that the homology among the four genes
in yeast might play a role in the formation of the trehalose synthase
complex. Why yeast employs a complex to synthesize trehalose is not
clear, but may have to do with improved efficiency and/or control of
trehalose synthesis by a complex of the two enzymatic activities. In
addition, it might also have to do with the role played by the
TPS1 gene product and/or the intermediate Tre-6-P in the
control of glucose influx into yeast glycolysis (15). Possibly,
trehalose synthesis by the complex allows a tighter control of the
cellular Tre-6-P level, which otherwise might cause too much inhibition
of hexokinase activity. Normally, one would expect most of the Tre-6-P
to be channeled inside the complex and only small amounts to leak out
into the cytosol. In this respect, it is remarkable that after addition
of glucose to yeast cells, there is a large transient overshoot of the
cellular Tre-6-P level up to 1-2 mM (22), which is
severalfold higher than the steady-state level of 100-200
µM (17, 22). If trehalose synthesis were carried out only
by the complex, it would indicate uncoupling of TPS and TPP activities,
resulting in strong deviation of Tre-6-P into the cytosol rather than
further hydrolysis to trehalose. However, our discovery of free
monomeric Tps1 protein offers a new explanation for the rapid increase
in Tre-6-P after glucose addition (see below).
It has been shown that the products of three of the four genes,
TPS1, TPS2, and TSL1, are subunits of
the trehalose synthase complex (4-6). For the TPS3 gene,
which has been identified by systematic sequencing, we have provided
evidence using the yeast two-hybrid system that it encodes a structural
subunit of the trehalose synthase complex. We showed that both Tps3 and
Tsl1 interact in vivo with Tps1 and Tps2, whereas the latter
two proteins also interact with each other (7). Now we provide evidence that TPS3 encodes an active functional subunit of the
trehalose synthase complex. The double deletion of TSL1 and
TPS3 caused a stronger reduction in TPS activity in
vitro and a stronger reduction in trehalose accumulation in
vivo compared with the single deletion mutants (Figs. 1 and 3).
The single deletion of TSL1 reduced Pi inhibition of TPS activity, whereas the single deletion of
TPS3 had no effect in this respect (Fig. 7). On the other
hand, the double deletion of TSL1 and TPS3
switched Pi inhibition into Pi stimulation
(Fig. 8). The double deletion mutant displayed a strong destabilization
of the trehalose synthase complex as opposed to the single deletion
mutants, providing further evidence for a role of Tps3 as structural
subunit. These results support the conclusion that TSL1 and
TPS3 encode redundant genes, with TSL1 playing a
somewhat more important function, at least for the parameters that we
have measured. Previously, Ferreira et al. (30) were unable
to detect a significant effect of TPS3 deletion, as opposed to TSL1 deletion, on the increase in TPS activity that
occurs after glucose exhaustion. This fits with our observation that in
stationary phase cells, the effect of TPS3 deletion is
minimal (Fig. 1). Since the expression of TSL1 differs from
that of the other three genes during growth on glucose, the function of
Tsl1 and Tps3 might be different under these conditions (8). The relative differences in trehalose and Tre-6-P content and the activities of TPS and phosphatase in the same 16 deletion strains were
also observed after heat shock, supporting our conclusions and
indicating that if Tsl1 has a specific role, it is not associated with
the response to heat shock (7).
Although all genes of the trehalose synthase complex display striking
homology, the other gene products are not capable of taking over the
function of Tps1 in Tre-6-P synthesis and in conferring ability to grow
on glucose or fructose. This is true for whatever combination of genes
present. In addition, overexpression of the other genes is unable to
restore the growth on glucose or fructose of a strain lacking
TPS1. The inability of the other subunits to take over the
function of Tps1 suggests that the only function of the parts with
homology to Tps1 in the other subunits might be the formation of the
trehalose synthase complex. We did not investigate whether the parts of
the other genes homologous to Tps1 could take over the function of Tps1
when separated from the non-homologous part. If they were able to do
so, however, the physiological meaning would be rather unclear.
The actual subunit composition of the trehalose synthase complex is not
clear. Since its total estimated molecular mass (600-800 kDa) exceeds
the sum of the molecular mass of the known gene products (3), at least
one of the genes is expected to encode more than one subunit. The
antibodies used for the Western blot analysis of the FPLC-purified cell
extracts were raised against the whole trehalose synthase complex.
Apparently, these antibodies are capable of reacting with all
components, except for the Tps3 subunit. This is indicated by the
failure to detect Tps3 in a tsl1 Presence of Tps1 Not Bound to the Trehalose Synthase
Complex--
The Superose gel fractionation experiments indicated that
a significant part of the Tps1 protein might be present in the cells as
free monomeric protein or at least not bound to the trehalose synthase
complex. This was an unexpected finding. The appearance of the two
pools was not abolished by single overexpression of the other subunits,
Tps2, Tsl1, or Tps3, or by combined overexpression of Tps2 and Tsl1.
This indicates that the amount of one of the other subunits is not
simply limiting for complex formation and that the separation of Tps1
into two pools might be controlled in a more precise way. The dual
location of Tps1 might reflect a dual function. Possibly, the Tps1
protein in the trehalose synthase complex is involved only in trehalose
synthesis, whereas the free Tps1 protein might have a function in
controlling the influx of glucose into glycolysis, e.g. by
interaction with hexokinase and/or sugar carriers (14, 15). The
presence of the free Tps1 protein provides an alternative explanation
to uncoupling of TPS and TPP activities in the trehalose synthase
complex for the large increase in cellular Tre-6-P content after
addition of glucose. The free Tps1 protein might be responsible for
this increase.
The presence of free Tps1 protein does not appear to be an artifact of
the extraction procedure since incubation of the extract for an
extended period of time at 30 °C did not change the proportion of
free and complex-associated Tps1 protein. Hence, the trehalose synthase
complex appears to be quite stable. This agrees with previous reports
on the purification of the complex. Vandercammen et al. (2)
were unable to separate TPS and TPP activities using several
purification procedures. In addition, Londesborough and Vuorio (3) were
able to purify a complex in which the TPP subunit and the regulatory
subunit were proteolytically reduced in size, although the complex
still retained all subunits and both enzymatic activities. (Simple
addition of a protease inhibitor, as was also done in our experiments,
prevented the limited reduction in size.) This shows that the subunits
remain together even after limited proteolysis. This stability of the
trehalose synthase complex supports the conclusion that the presence of
free Tps1 protein in the cell extracts is not due to the extraction
procedure and that therefore free Tps1 protein is also present in
vivo.
Regulation of Tps1 Bound to and Free from the Trehalose Synthase
Complex--
The finding that deletion of TPS2, of both
TSL1 and TPS3, or of all three genes switches
Pi inhibition of TPS activity into Pi
stimulation without compromising the capacity to grow on glucose and
fructose indicates that Pi inhibition of TPS is not
essential for growth on rapidly fermented sugars. It also indicates
that any free Tps1 protein (synthesizing Tre-6-P) would be stimulated by Pi, whereas the Tps1 protein present in the trehalose
synthase complex (synthesizing trehalose in combination with Tps2)
would be inhibited by Pi. Free Pi could play a
role in the control of glucose influx into glycolysis (15).
Pi is a substrate for glyceraldehyde-3-phosphate dehydrogenase in glycolysis, and it is precisely at this point that
glycolysis becomes blocked in the tps1 mutants after
addition of glucose (11, 31). A high free Pi level is
indicative of slow sugar influx into glycolysis (slow sequestration of
Pi into sugar phosphates), whereas a low Pi
level indicates rapid sugar influx (rapid sequestration of
Pi into sugar phosphates). The highest free Pi
level is observed before the initiation of sugar fermentation. Addition
of fermentable sugar to wild-type cells is always followed by a rapid
decline in the free Pi level to ~40% of the initial
concentration (11). A rapid increase in the Tre-6-P level has also been
observed during the initiation of fermentation (22), whereas at the
same time, the trehalose level rapidly decreases (32). Hence, under
these conditions, the synthesis of trehalose and Tre-6-P is clearly
regulated in an opposite way in vivo. The opposite control
by Pi of Tps1 in the trehalose synthase complex and of free
Tps1 might offer an explanation for this opposite behavior of Tre-6-P
and trehalose in vivo after addition of glucose. Activation
of the free Tps1 protein by Pi would result in accumulation
of Tre-6-P, whereas at the same time, the inhibition of Tps1 in the
complex by Pi would result in the down-regulation of
trehalose synthesis.
Phosphatase Activity Not Encoded by Tps2--
We have observed
that all tps2
In conclusion, our results have more precisely established the
structural and functional importance of the four subunits of the
trehalose synthase complex, Tps1, Tps2, Tsl1, and Tps3, for trehalose
synthesis. In addition, they have revealed that the enzymatic machinery
of trehalose metabolism contains additional components and regulatory
controls than previously known. These novel controls might at least in
part be responsible for some of the previous unexpected observations
concerning trehalose metabolism, such as the opposite behavior of the
trehalose and Tre-6-P levels after addition of glucose and the
uncoupling of glucose influx control and the capacity to grow and
ferment on glucose.
We thank J. Rosseels and W. Verheyden for
excellent technical assistance.
*
This work was supported by fellowships from the Research
Fund of the Katholieke Universiteit Leuven (to W. B.) and from the Fund for Scientific Research, Flanders (to S. W.), by European Commission Human Capital and Mobility Grant CHRX-CT93-0265, by grants
from the Belgian National Fund for Scientific Research and the Research
Fund of the Katholieke Universiteit Leuven (to J. M. T.), and by
Swiss National Science Foundation Grant 42535.94 (to A. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Cell and Molecular Biology/Microbiology,
Lundberg Laboratory, Göteborg University, P. O. Box 462, S-40530
Göteborg, Sweden.
The abbreviations used are:
TPS, trehalose-6-phosphate synthase; TPP, trehalose-6-phosphate phosphatase; Tre-6-P, trehalose 6-phosphate; FPLC, fast protein liquid chromatography.
Composition and Functional Analysis of the Saccharomyces
cerevisiae Trehalose Synthase Complex*
,,§,,
Laboratorium voor Moleculaire Celbiologie,
Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001
Leuven-Heverlee, Flanders, Belgium and the ¶ Botanisches Institut
der Universität, Hebelstrasse 1, CH-4056 Basel, Switzerland
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
background, reduced both TPS and TPP activities and
trehalose content. Deletion of TPS2, TSL1, or
TPS3 and, in particular, of TSL1 plus
TPS3 destabilized the trehalose synthase complex. We
conclude that Tps3 is a fourth subunit of the complex with functions
partially redundant to those of Tsl1. Among the four genes studied,
TPS1 is necessary and sufficient for growth on glucose and
fructose. Even when overproduced, none of the other subunits could take
over this function of Tps1 despite the homology shared by all four
proteins. A portion of Tps1 appears to occur in a form not bound by the
complex. Whereas TPS activity in the complex is inhibited by
Pi, Pi stimulates the monomeric form of Tps1.
We discuss the possible role of differentially regulated Tps1 in a
complex-bound or monomeric form in light of the requirement of Tps1 for
trehalose production and for growth on glucose and fructose.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
tsl1 tps3,
significant TPS activity was detected, indicating that the
TPS1 gene product displays TPS activity in the absence of
all other (known) subunits.
S. cerevisiae strains used in this study All strains are isogenic to W303-1A and carry the same markers.
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Fig. 1.
TPS activity in galactose-grown exponential
(A) and stationary phase (B) cells of yeast
strains containing all possible combinations of deletions in the four
genes (TPS1, TPS2, TPS3, and
TSL1) encoding the trehalose synthase complex.
µkat, microkatals.
View larger version (24K):
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Fig. 2.
TPP activity in galactose-grown exponential
(A) and stationary phase (B) cells of yeast
strains containing all possible combinations of deletions in the four
genes (TPS1, TPS2, TPS3, and
TSL1) encoding the trehalose synthase complex.
µkat, microkatals.
tps3 and
tps3 strains showed a somewhat elevated Tre-6-P level
compared with the wild-type strain. Very high increases in Tre-6-P were
observed in all strains containing tps2. Additional
deletion of TSL1 or TPS3 had only a little effect on this elevated Tre-6-P level. All tps2 strains,
however, still contained high levels of trehalose.
View larger version (23K):
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Fig. 3.
Trehalose (A) and Tre-6-P
(B) content in galactose-grown stationary phase cells of
yeast strains containing all possible combinations of deletions in the
four genes (TPS1, TPS2, TPS3, and
TSL1) encoding the trehalose synthase complex.
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Fig. 4.
Western blot analysis of subunits of the
trehalose synthase complex in cell fractions first separated by
Superose 6 FPLC and subsequently submitted to SDS-gel
electrophoresis. Antibodies raised against the purified trehalose
synthase complex were used. Tps1, Tsl1, and Tps2 were detected in
fractions 16 and 17 (±600 kDa). Dimers of Tps2 and/or Tps1 and Tps2
might be present in fractions 20 and 21 (±200 kDa). Free Tps1 was
present in fractions 23 and 24 (±50 kDa), with possible Tps1 dimers in
fraction 22. The positions of Tps1, Tps2, and Tsl1 are indicated. (Tps3
is most probably not detected by the antibodies (see Fig. 5 and
"Results").) A, extract of the wild-type strain;
B, part of the same extract incubated for 30 min at 30 °C
before separation by FPLC. The molecular mass standards used for
calibration are indicated on the right.
tps3 strain, on the other hand, no
evidence for the presence of the trehalose synthase complex was
observed (Fig. 5D). All Tps2 was detected free from the
complex. The clear difference in complex formation in the presence and
absence of Tps3 in the tsl1 strain confirms that it is
part of the trehalose synthase complex. In all samples, a slight
background of Tps1 was also detected, possibly caused by unspecific
retention by the Superose beads. Deletion of TPS2 caused the
most dramatic effect, with only Tps1 free from the complex being
present (Fig. 5E).
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Fig. 5.
Western blot analysis of subunits of the
trehalose synthase complex in cell fractions first separated by
Superose 6 FPLC and subsequently submitted to SDS-gel
electrophoresis. Antibodies raised against the purified trehalose
synthase complex were used. The trehalose synthase complex (±600 kDa)
was present in fractions 16 and 17, whereas the free Tps1 protein (±50
kDa) was detected mainly in fractions 23 and 24. A,
wild-type strain; B, tps3 strain;
C, tsl1 strain; D,
tsl1 tps3 strain; E,
tps2 strain. The molecular mass standards used for
calibration are indicated on the right.
tps3 strain. This restored the
presence of the trehalose synthase complex in the high molecular mass
fractions, confirming that TPS3 was truly expressed from this plasmid (data not shown).
tsl1
tps3, strain TPS activity was clearly detected (see
below), which fits with the presence of trehalose and Tre-6-P in
vivo in cells of such a strain.
strain was unable to restore or even improve growth
on glucose or fructose (Fig. 6B).
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Fig. 6.
A, growth of yeast strains containing
all possible combinations of deletions in the four genes
(TPS1, TPS2, TPS3, and
TSL1) encoding the trehalose synthase complex on medium
containing either galactose or fructose as carbon source; B,
growth of a tps1 strain (YSH6.127.-17C) either with the
YEplac195 plasmid without insert or with overexpression
using the same plasmid of TPS1, TPS2, TPS3, or
TSL1 on medium containing galactose, glucose, or fructose as
carbon source.
tps3, tps2, and
tsl1 tps3 tps2 in the absence
of Pi were 0.049, 0.130, and 0.024 microkatals/g,
respectively, and they increased to 344.7, 261.3, and 438.8% of these
values in the presence of 10 mM Pi. TPS
activity as a function of the Pi concentration in the assay
is shown in Figs. 7 and 8 as relative activity compared with the
activity in the absence of Pi, which was taken as 100%. The absolute values for the activity in the absence of Pi
are indicated in the legends of Figs. 7 and 8. We also measured
phosphate inhibition of trehalose-6-phosphate synthase in crude cell
extracts and after FPLC purification of the complex from cells grown on rich yeast extract/bacto peptone/glucose medium, and we found that, in
this case, the activity was still inhibited by phosphate, but much less
(maximum 50%) compared with enzyme from cells grown on synthetic
glucose medium (data not shown).
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Fig. 7.
Inhibition of TPS activity by
Pi. 
and 
, wild-type strain; 
and 
,
tsl1 strain; 
and 
, tps3 strain. ,
, and , cells grown on synthetic medium with glucose until
stationary phase; , , and , exponential phase cells grown on
synthetic medium with glycerol. The absolute values for the activity
(expressed as microkatals/g of protein) in the absence of
Pi for glucose-grown stationary phase cells were 0.518 (wild-type strain), 0.498 (tsl1), and 0.391 (tps3), and those for exponential phase cells grown on
glycerol were 0.713 (wild-type strain), 0.372 (tsl1), and
0.779 (tps3).
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Fig. 8.
Stimulation of TPS activity by Pi
in strains with a deletion of both TSL1 and TPS3
( ), a deletion of TPS2 (), or a deletion of
TPS2, TSL1, and TPS3 ().
The cells were grown on synthetic medium with glucose until stationary
phase. The absolute values for the activity (expressed as microkatals/g
of protein) in the absence of Pi were 0.036 (tsl1 tps3), 0.123 (tps2),
and 0.097 (tps2 tsl1
tps3).
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
strain and in a
TPS3 overexpression strain. Because the reactivity of the
antibodies with the different subunits is not known, it is not possible
to draw firm conclusions concerning the actual composition of the
trehalose synthase complex. However, since the Tps1 band was always
much stronger than the others, it appears to be the first candidate for
being present with more than one molecule in the trehalose synthase complex.
strains, including the tps2
tsl1 tps3 strain, still accumulate high
levels of trehalose. This indicates that other phosphatases apparently
independent of the trehalose synthase complex are able to hydrolyze
Tre-6-P with relatively high efficiency. The finding that a significant part of the TPS1 gene product appears to be present as free
protein gives this result more importance. Possibly,
TPS2-encoded TPP activity is responsible only for the
hydrolysis of the Tre-6-P produced "internally" in the trehalose
synthase complex. The free Tps1 protein displays less catalytic
activity than the Tps1 subunit in the complex, and the Tre-6-P that it
produces might be hydrolyzed in vivo by other phosphatases.
Strong stimulation of TPS activity by the other subunits in the
trehalose synthase complex is supported by the phenotype of the single
and multiple TPS2, TSL1, and TPS3 deletion strains. They always display less TPS activity in
vitro and accumulate less trehalose in vivo.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.:
32-16-321507/321500; Fax: 32-16-321979; E-mail:
johan.thevelein{at}bio.kuleuven.ac.be.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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