Molecular interaction of neutral trehalase with other
enzymes of trehalose metabolism in the fission yeast
Schizosaccharomyces pombe
Teresa Soto, Alejandro Franco, S. Padmanabhan, Jero Vicente-Soler, Jose Cansado and Mariano Gacto
Department of Genetics and Microbiology, Facultad de Biologı
´
a, University of Murcia, Spain
Trehalose metabolism is an essential component of the stress
response in yeast cells. In this work we show that the prod-
ucts of the principal genes involved in trehalose metabolism
in Schizosaccharomyces pombe, tps1
+
(coding for trehalose-
6-P synthase, Tps1p), ntp1
+
(encoding neutral trehalase,
Ntp1p) and tpp1
+
(that codes for trehalose-6-P phospha-
tase, Tpp1p), interact in vitro with each other and with
themselves to form protein complexes. Disruption of the
gene tps1
+
blocks the activation of the neutral trehalase
induced by heat shock but not by osmotic stress. We propose
that this association may reflect the Tps1p-dependent
requirement for thermal activation of trehalase. Data
reported here indicate that following a heat shock the
enzyme activity of trehalase is associated with Ntp1p dimers
or trimers but not with either Ntp1p monomers or with
complexes involving Tps1p. These results raise the possibility

by trehalose-6-P synthase (TPS1) from UDP-glucose and
glucose 6-P as substrates, and dephosphorylation of treha-
lose-6-P to trehalose by trehalose-6-P phosphatase (TPS2).
Studies based on two-hybrid analyses and on Western blot
analyses of complexes obtained by gel filtration fractiona-
tion concluded that TPS1 and TPS2 together with proteins
TSL1andTPS3(whichactasregulatorsofbothsynthase
and phosphatase activities) form part of a multimeric
protein complex of approximate molecular mass 800 kDa
called the trehalose synthase complex [5–7]. In this complex,
TPS1, TPS2, and TPS3 subunits interact with each other
and among themselves (as dimers or higher order oligo-
mers), whereas TSL1 interacts only with TPS1 and TPS2 [6].
Another component of trehalose metabolism is the hydro-
lysis of trehalose to glucose. This is catalyzed by the enzyme
neutral trehalase (NTH1), whose activity is regulated
by phosphorylation of the enzyme protein at serine residues
[8,9].
Comparatively much less is known about the regulation
of the trehalose synthesis in the evolutionarily distant yeast
Schizosaccharomyces pombe. In this fission yeast, the tps1
+
gene codes for trehalose-6-P synthase (Tps1p), that synthe-
sizes trehalose-6-P as occurs in S. cerevisiae [10]. However,
in contrast to the behaviour observed in S. cerevisiae, Dtps1
strains of S. pombe are able to grow on glucose or other
readily fermentable carbon sources, although disruption of
this gene does prevent spore germination [10]. Recently, we
characterized a second gene of the trehalose biosynthetic
pathway in S. pombe,namedtpp1

+
is accompanied by a
rise in enzyme activity, which is also regulated by the
Pka1p/Sck1p pathway during osmotic and oxidative
stress [16,17]. In a previous work, we showed that mutants
of S. pombe disrupted in trehalose-6-P synthase function
were unable to increase neutral trehalase activity under heat
shock conditions or after the addition of glucose or
nitrogen source. However, these Dtps1 strains still respond
to osmotic stress by increasing trehalase levels [18]. Thus, in
S. pombe, trehalose-6-P synthase appears to be involved in
the regulation of the thermal- and nutrient-induced activa-
tion of neutral trehalase but not during activation on
osmotic stress. These observations raised the question of
whether trehalose-6-P synthase and neutral trehalase would
interact in vivo. In this paper we demonstrate that both
Tps1p and Ntp1p interact in vivo and are part of distinct
trehalose-6-P synthase/trehalase complexes. Moreover,
trehalose-6-P phosphatase (Tpp1p) is likely a member of
these complexes suggesting that regulation of trehalose
synthesis and breakdown may be integrated mechanisms.
These findings indicate a divergence in the molecular designs
controlling trehalose metabolism in S. pombe from those in
S. cerevisiae.
MATERIALS AND METHODS
Strains and culture media
The S. pombe strains employed in this study are listed in
Table 1. They were routinely grown with shaking at 28 °C
in YES [19] or EMM2 with or without thiamin (5 mgÆL
)1

hybridizes at the 3¢ end of ntp1
+
ORF and incorporates a
NotI site placed immediately upstream of the TAA stop
codon). The restriction sites in both oligonucleotides are
underlined. PCR amplification employing the Expand high-
fidelity system (Roche Molecular Biochemicals) generated a
1.6 kb fragment that was cleaved with XhoIandNotIand
cloned into plasmids pIH-ura and pIH-LEU. These are
integrative plasmids derived from plasmid pDS472a [20],
without nmt promoter and ars1 sequences, and with ura4
+
or LEU2 as selectable markers, which allow the construc-
tion of vectors with Ha6H tag fusions at the C-terminus.
The resulting plasmids were digested at the unique SfiIsite
within the ntp1
+
coding region (at position 1665) and the
linear fragments transformed into haploid strains MM1 and
MM2, to target integration at the ntp1
+
locus. Uracil or
leucine prototrophs were selected for strains MM1 and
MM2, respectively. The identification of strains C3 and
C69, with one copy of Ntp1p–Ha6H expressed from the
genomic ntp1
+
promoter, was verified by Southern blot
analysis and immunoblot of whole-cell extracts with anti-
Ha Ig (see below).

Strain Genotype Source/Reference
MM1 h
+
ade6-M216 leu 1-32 ura4-D18 M. Yamamoto
MM2 h
–
ade6-M210 leu 1-32 ura4-D18 A. Dura
´
n
C3 h
+
ade6-M216 leu 1-32 ura4-D18 ntp1
+
:Ha6H (ura4
+
) This study
C69 h
–
ade6-M210 leu 1-32 ura4-D18 ntp1
+
:Ha6H (LEU2) This study
C4 h
)
ade6-M216 leu 1-32 ura4-D18 tps1
+
:GST (ura4
+
) This study
C5 h
)

ade6-M210 leu 1-32 ura4-D18 tpp1
+
:Ha6H (ura4
+
) ntp1
+
:Ha6H (ura4
+
) tps1
+
:Ha6H (ura4
+
) This study
3848 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002
expressed from the genomic tps1
+
promoter, was verified
by Southern blot analysis and immunoblot of whole-cell
extracts with anti-GST or anti-Ha Ig, respectively.
The double-tagged strain C694 (Ntp1p–Ha6H, Tps1p–
GST) was constructed by mating strains C69 and C4, and
selecting diploids in EMM2 medium without supplements.
Sporulation was performed in MEL medium and the spores
purified by glusulase treatment [21] were allowed to
germinate in minimal medium plus adenine. Strains with
the double-tagged genotype were identified by Southern and
immunoblot analysis with anti-HA and anti-GST Ig.
Double-tagged strain C33 (Ntp1p–Ha6H, Tpp1p–Ha6H)
was constructed by mating strains C3 and MMPI-3b, and
selecting diploids in EMM2 medium with leucine. After

by PCR with oligonucleotides NTP-5 (CCG
CTCGAGG
CTATCATTCGTGAATAG, whichhybridizesatsequences
upstream of the ATG start codon in the ntp1
+
ORF and
shows an internal XhoI site), and TAG-3. The 1.5 kb product
was cloned as above into pDS472M to create plasmid
pNGST, containing an in-frame fusion wherethe 3¢ endofthe
ntp1
+
ORF is followed by the GST epitope, and whose
expression is under the control of the medium strength nmt1
thiamin-repressible promoter. Both pNGST and control
plasmid pDS472M were transformed into strains C3, C5 and
MMPI-3, and leucine prototrophs were selected.
Purification of Ha6H- and GST-tagged proteins
by affinity chromatography
Total cell homogenates were prepared under native condi-
tions employing chilled acid-washed glass beads and lysis
buffer (10% glycerol, 50 m
M
Tris/HCl pH 7.5, 150 m
M
NaCl, 0.1% Nonidet NP-40, plus an specific protease
inhibitor cocktail for fungal and yeast extracts obtained
from Sigma Chemical Co.). The lysate was removed and
cleared by centrifugation at 10 000 g for 30 min. Ha6H-
tagged proteins were purified by using Ni
2+

Mes pH 6 plus 200 m
M
trehalose
(Sigma Chemical Co.) for 2 h at 30 °C. After washing with
distilled water, active neutral trehalase proteins were
detected in situ by incubating the gels with 0.1% TTC in
0.5
M
sodium hydroxide at 80 °C. Color development was
stopped with a 7.5% acetic acid solution.
Gel filtration
A Superdex-200 column (Amersham-Pharmacia) equili-
brated with buffer A (10 m
M
Mes, pH 6.0, 150 m
M
NaCl)
was used for size-exclusion analysis in an AKTA HPLC
system (Amersham-Pharmacia). Lower salt concentrations
were not used in order to minimize nonspecific electrostatic
interactions with the column matrix. The column was
calibrated using vitamin B
12
(1.3 kDa), cytochrome c
(12.4 kDa), carbonic anhydrase (29 kDa), ovalbumin
(43 kDa), BSA (66 kDa), yeast alcohol dehydrogenase
(150 kDa), b-amylase (200 kDa), apoferritin (430 kDa)
and thyroglobulin (670 kDa) (all from Sigma Chemical
Co.) at the concentrations recommended by the manufac-
turer. One-hundred microliters (1 mg total protein) of the

were detected by Western blot analysis with anti-Ha Ig (see
below).
SDS/PAGE and Western blotting
Proteins were resolved in 8 or 10% SDS/PAGE gels as
previously described [11], transferred to nitrocellulose filters
(Amersham-Pharmacia), and incubated with mouse anti-
Ha or sheep anti-GST Ig. The immunoreactive bands were
revealed with HRP-conjugated secondary Ig [anti-(mouse
IgG) Ig or anti-(sheep IgG) Ig; Sigma Chemical Co.] and the
ECL system (Amersham-Pharmacia).
Enzyme assays and trehalase activation
Trehalase activity was assayed after cell breakage as
described previously [23]. Activation of trehalase by heat
treatment or osmotic shock was carried out as indicated
earlier [17]. Enzyme activity in eluates was expressed as
nmol glucose produced per min. All trehalase determina-
tions were repeated at least three times with consistent
results. Representative results are shown. Specific activity of
trehalase in slab gels was expressed as enzyme units per mg
protein. Protein determination was performed by absor-
bance measurement at 280 and 205 nm according to the
method described in previously [24].
RESULTS
Neutral trehalase and trehalose-6-
P
synthase
association
in vitro
In order to analyse possible interactions between treha-
lose-6-P synthase and neutral trehalase interaction

domain fused to Tps1p.
Ntp1p–Tps1p association takes place during
normal yeast growth and thermal shock, but might not
occur when the cells are stressed by an osmotic upshift [18].
To test this possibility, we performed the same experiment
described above by subjecting strain C3 plus plasmid
pTGST either to osmotic stress or to a thermal one. As
shown in Fig. 1, Ntp1p–Tps1p association was observed
not only in control, exponentially growing cells but also for
both thermal as well as osmotic stresses (lanes 7 and 8).
Thus, in S. pombe, Ntp1p–Tps1p interaction does not
appear to be transient but is stable, and is maintained even
under conditions where the presence of Tps1p is not needed
for neutral trehalase activation (osmotic shock). These
results, however, do not exclude by themselves the possibi-
lity that some Ntp1p might be in a free, nonassociated state
(see below).
We employed a medium-strength thiamin-repressible
promoter for Tps1p–GST expression in Ntp1p–Ha6H cells
to achieve low levels of Tps1p–GST synthesis. Although
Ntp1p–Ha6H was not detectable in the absence of the
Tps1p–GST fusion, it was conceivable that the described
Tps1p–Ntp1p interaction could be due, in part, to the
presence of nonphysiological levels of Tps1p–GST. In order
to clarify this point, we constructed the S. pombe double-
tagged strain C694. This strain expresses Ntp1p and Tps1p
fused at their C-terminus to Ha6H and GST epitopes,
respectively, and in both cases the synthesis is regulated by
their own genomic promoters. The Tps1p fusion protein is
active because strain C694 synthesizes trehalose at normal

enzyme activity of Ntp1p bounded to Tps1p in exponen-
tially growing cells from double-tagged strain C694 and in
cultures subjected to heat or osmotic stress. As shown in
Fig. 2B, affinity-purified Ntp1p–Ha6H from growing cells
displayed a typical pattern of neutral trehalase activity,
with low level of enzyme activity in unstressed cells that
increases strongly upon stress. Notably, we were unable to
detect in situ any neutral trehalase activity associated to
native Tps1–GST protein purified with glutathione–
Sepharose beads in samples from either unstressed,
osmotic- or heat-shocked cells. A quantitative estimation
of neutral trehalase activity gave similar results (Fig. 2B,
lower panel). Because a significant fraction of Ntp1p
protein is bounded in vitro toTps1p(seeFigs1and2A),
these results demonstrate that in S. pombe the active form
of neutral trehalase exists in a free, non Tps1p-associated
state, independently of the environmental condition used
for stress.
We focussed then our attention on the likelihood that
Tps1p or Ntp1p undergo self-association. This has been
previously reported for the Tps1p homologue in
S. cerevisiae [6]. Using the same experimental procedure
used in Fig. 1, we observed that self-association does
indeed take place for both proteins. As can be seen in
Fig. 3A (lane 4) and Fig. 3B (lane 4), both Ntp1p–
Ha6H and Tps1p–Ha6H were detected employing anti-
Ha Ig after Ntp1p–GST and Tps1p–GST purification,
respectively. These and other results (see below) support
Fig. 2. Ntp1p and Tps1p coimmunoprecipitate and associate to form
complexes devoid of neutral trehalase activity. (A) The double-tagged

Ntp1–Tps1 complex
Recently a third member of the trehalose metabolism
pathway in S. pombe,thetpp1
+
gene, which codes for
trehalose-6-P phosphatase, has been isolated and charac-
terized [11]. The tpp1
+
gene has considerable sequence
homology to S. cerevisiae TPS2, which encodes trehalose-
6-P phosphatase. S. cerevisiae Tps2 has been shown to
interact, among others, with Tps1, and form part of the
trehalose synthase complex in this yeast [6,7]. Based on these
precedents, we examined if Tpp1p interacts with both Tps1p
and Ntp1p in S. pombe. The strain MMPI-3a, that
expresses a Ha6H-tagged version of Tpp1p [11], was
transformed separately with plasmids pTGST and pNGST
(expressing Tps1p and Ntp1p fused to GST, respectively),
and the GST fusions were purified with glutathione–
Sepharose beads. In either case (Fig. 4A,B), the 100 kDa
Tpp1p–HA6H protein coprecipitated with the purified GST
fusion proteins, whereas it was absent in control experi-
ments (with plasmids expressing unfused GST). These
results clearly suggest that Tpp1p may also participate
in vivo to form Ntp1p–Tps1p complexes. However, as for
Tps1p–Ntp1p complexes (see Fig. 2B), trehalase activity
wasabsentinTpp1p–Ntp1passemblieswhenassayedongel
slabs (data not shown).
Analysis of the Ntp1p–Tps1p–Tpp1p complex
by HPLC-gel filtration

containing significant amounts of all three proteins.
One corresponds to the high molecular mass complex
(700–800 kDa) and the other to a more diffusely spread
population corresponding to lower molecular mass range
(80–250 kDa, fractions 12–17). To clarify which of these
protein complexes exhibit neutral trehalase activity, we
prepared log-phase cultures from strain C335 and subjected
them to thermal (40 °C, 1 h) or osmotic (0.75
M
NaCl, 2 h)
stress. The corresponding cell extracts were then fraction-
ated by gel filtration under the conditions described above.
No significant differences in the elution profile of Ntp1p,
Tps1p and Tpp1p were found under these circumstances,
despite an increase in the overall signal (as the three
proteins behave as heat shock proteins). Surprisingly, when
the eluted fractions were assayed for neutral trehalase, no
enzyme activity was found associated with Ntp1p mono-
mers or in fractions corresponding to the high molecular
mass trehalose synthase–trehalase complex (fractions 4–5).
Instead, the heat-activated neutral trehalase was mainly
detected in complexes of about 250 kDa, whereas neutral
Fig. 4. Tpp1p associates with Ntp1p and Tps1p in vivo . (A) Tpp1p–Tps1p interaction. The Tpp1p–Ha6H epitope-tagged strain MMPI-3a was
transformed with plasmids pDS472a (unfused GST; lanes 1 and 3) or pTGST (Tps1p–GST fusion; lanes 2 and 4). GST and Tps1p–GST fusions
were expressed using the medium-strength thiamin-regulated promoter for 24 h. Yeast lysates were then adsorbed with glutathione–Sepharose
beads, and after extensive washing the proteins bound to the beads were analyzed by Western blot using anti-GST Ig (lanes 1 and 2) and anti-Ha Ig
(lanes 3 and 4). (B) Ntp1p–Tpp1p interaction. Strain MMPI-3a was transformed with plasmids pDS472a (unfused GST; lanes 1 and 3) or pNGST
(Ntp1p–GST fusion; lanes 2 and 4). GST and Tps1p–GST fusions were expressed using the medium-strength thiamin-regulated promoter for 24 h,
purified as described above, and analyzed using anti-GST (lanes 1 and 2) and anti-Ha Ig (lanes 3 and 4).
3852 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002

However, in S. cerevisiae the functional cytoplasmic neutral
trehalase (NTH1) is probably a dimer, as deduced from gel
filtration experiments with active enzyme [25]. No evidence
has been reported demonstrating that NTH1 participates in
the formation of the well-characterized trehalose synthase
complex in the budding yeast [6,7]. In S. pombe, we find that
part of Ntp1p is apparently present as free monomeric
protein that does not display trehalase activity in vitro
(Fig. 5). The elution profile of trehalase activity in gel
filtration indicated that after heat or osmotic shock only
fractions corresponding to protein sizes between 170 and
300 kDa contain active enzyme. On the other hand, Ntp1p
eluted mainly in two peaks, either as part of a 700–800 kDa
complex, together with Tps1p and Tpp1p, or, as expected,
in the fractions containing trehalase activity, which again
showed the coexistence of Tps1p and Tpp1p (Fig. 5).
Because Ntp1p molecules also associate among themselves
(Fig. 2), these results alone would not allow us to establish
whether trehalase activity correlates with self-assembly of
Ntp1p homomultimers or with the formation of hetero-
meric complexes involving Tps1p and Tpp1p in addition to
Ntp1p. However, the observation that Tps1p–(Tpp1p)–
Ntp1p complexes lack trehalase activity (Fig. 2B) strongly
favors the first interpretation. Active trehalase might thus
arise from the specific self-assembly of a discrete number of
Ntp1p molecules to form small oligomers (probably dimers
or trimers, considering the molecular mass of each putative
subunit, 84 kDa). An intriguing fact is that there is a small
but reproducible shift in the elution behavior of trehalase
depending on the nature of the activation stress (Fig. 5B).

S. cerevisiae, trehalose is synthesized by the 800 kDa
trehalose synthase complex as well as by free TPS1 [7]. It
is unknown whether Tps1p activity in S. pombe is present in
the high molecular mass trehalose synthase–trehalase com-
plex or linked to forms of lower molecular mass as for
neutral trehalase, although accumulation of trehalose by
ntp1-deleted strains seems to indicate that Tps1p function
does not require association with Ntp1p. In any case, our
data suggest the existence of different types of Ntp1p
complexes in S. pombe that might be specifically activated
as a function of the external stimulus. If so, an attractive
explanation for the above results would be that S. pombe
harbors at least two forms of Ntp1p that are capable of
being activated, one as a Ntp1p–Ntp1p homodimer
( 170 kDa), that becomes activated during osmotic shock,
and the other as a Tps1p–(Tpp1p)–Ntp1p heterocomplex
( 250 kDa) activated during heat shock. Apparently,
trehalase requires association with Tps1p while being
activated by heat shock acquires enzymatic activity only
when detached from the heat-induced activation complex.
Other possibilities may exist, but some experimental data
are consistent with this hypothesis. For instance, in contrast
to heat shock, Ntp1p activation during osmotic shock is
independent of the presence of Tps1p [18]. Also, the
activation of neutral trehalase induced by heat shock is
largely a post-translational event that exhibits considerably
faster kinetics than the osmotically induced activation
[17,23]. In the latter case, the Pka/Sck1p-mediated activa-
tion of Ntp1p appears to occur at the level of the enzyme
synthesized de novo [17]. Finally, the Tps1p–Ntp1p associ-

complex in S. cerevisiae, it is tempting to speculate that in
S. pombe these ORFs code for proteins that somehow
regulate trehalose metabolism by interacting with some
members of the enzyme proteins described here. The
data presented in this report reveal a crucial difference
between the two organisms. Unlike previously reported for
S. cerevisiae [25], both the anabolic and catabolic enzymes
might be integrated in some instances into a regulatory
complex in S. pombe.
ACKNOWLEDGEMENTS
T. S. and A. F. contributed equally to this work. We thank Prof F. J.
Murillo for generous access to the HPLC equipment and F. Garro for
expert technical assistance. We are indebted to Profs. M. Yamamoto,
S. L. Forsburg and A. Duran for kindly providing plasmids and
strains. A. F. is a predoctoral fellow of PFPI from the University of
Murcia. This work was supported in part by grant BMC2001-0135
from MCYT, Spain.
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