(Received for publication, July 27, 1995; and in revised form, August 23, 1995)
From the
The Schizosaccharomyces pombe gene tnr3 has been genetically defined as a negative regulator of genes involved in thiamine metabolism (Schweingruber, A. M., Fankhauser, H., Dlugonski, J., Steinmann-Loss, C., and Schweingruber, M. E.(1992) Genetics 130, 445-449). We have isolated and sequenced the gene and show that it codes for a putative protein of 569 amino acids which exhibits, in its carboxyl-terminal half, good homology to Saccharomyces cerevisiae thiamine pyrophosphokinase (TPK). tnr3 mutants have reduced levels of intracellular thiamine diphosphate, show impaired TPK activity, which is enhanced by introducing the tnr3 wild type gene on a plasmid, and can be complemented by the S. cerevisiae TPK-encoding gene THI80. These data strongly suggest that tnr3 encodes S. pombe TPK. We present evidence that TPK also acts as a negative regulator for gene pho1, which is derepressed when cells are starved for phosphate and show that in contrast to wild type cells, tnr3 mutants mate constitutively in response to thiamine, indicating that TPK is also involved in regulation of mating. Disruption of the tnr3 gene is lethal, and a tnr3 mutant expressing only residual TPK activity grows slowly and shows aberrant morphology.
Thiamine (vitamin B) acts in the form of thiamine
diphosphate (TDP) (
)as coenzyme of several enzymes including
pyruvate dehydrogenase, pyruvate decarboxylase, transketolase,
-keto-glutaraldehyde dehydrogenase, and others (for reviews see (1) and (2) ). Its metabolism is still poorly
understood, and we have, therefore, chosen to study it in the fission
yeast Schizosaccharomyces pombe. In this organism thiamine is
not only essential for growth but is also involved in the regulation of
mating of cells of opposite mating type(3) . Expression of
genes involved in thiamine metabolism is strongly regulated in S.
pombe. The first thiamine-repressible gene we identified was pho4, which encodes an acid
phosphatase(4, 5) . This phosphatase is an N-glycosylated cell wall protein and is believed to
dephosphorylate thiamine phosphates, which may occur as natural
substrates in growth media(6) . Further studies revealed that
expression of the three structural genes thi2, thi3,
and thi4, which encode enzymes of the thiamine biosynthetic
pathway, are also repressed by the vitamin (6, 7, 8) . thi3 is involved in the
biosynthesis of the pyrimidine moiety of the thiamine molecule and
corresponds to nmt1(9) . thi2 is responsible
for the thiazole part of thiamine and is the same gene as nmt2(10) . thi4 defines a gene that is
involved in the phosphorylation of the pyrimidine or thiazole
precursors and/or in the coupling of the two phosphorylated precursors
to thiamine monophosphate(8) . Recent results indicate that the thi4 gene product is a bifunctional enzyme exhibiting
hydroxyethylthiazole kinase and thiamine phosphate pyrophosphorylase
activity. (
)
As an approach to define regulatory genes of
thiamine metabolism, we searched for mutants defective in regulation of
thiamine-repressible acid phosphatase(11) . Two classes of
mutants were isolated: mutants with thiamine non-repressible and
mutants with thiamine non-derepressible pho4-encoded acid
phosphatase. The mutants exhibiting thiamine non-repressible acid
phosphatase map in three genes, tnr1, tnr2, and tnr3. Mutations causing non-derepressible pho4-encoded acid phosphatase activity map in gene thi1. We showed that all tnr mutants are not only derepressed
for pho4 but also thi2, thi3, thi4,
and thiamine transport(7, 8, 11) . Similarly, thi1 mutants are repressed for pho4 as well as for
the other known thiamine-repressible genes. We cloned and sequenced the
gene thi1(12) . It codes for a Cys zinc-finger motif-containing protein, which may bind to upstream
activator sequences of thiamine-repressible genes.
In this study, we report cloning and characterization of the tnr3 gene. We show that it codes for TPK and that this enzyme is not only involved in the regulation of thiamine metabolism but also acts as a regulator of phosphate metabolism, mating, and cell growth.
Strains were grown in liquid or on solid yeast extract and minimal medium (MM, MMA), which was supplemented as indicated in the text(3) . To derepress phosphate-repressible acid phosphatase(13) , cells were cultivated in MM containing 0.1 mM phosphate (low phosphate MM). To achieve good repression and derepression of this enzyme, phosphate has to be autoclaved separately.
Figure 1:
Restriction map of plasmids carrying
the whole or part of the tnr3 gene. Plasmid pUR19tnr3 (a) was isolated as described in the text. The box denotes the ORF, and the arrow indicates the direction of
transcription. The underlined KpnI-HindIII fragment
in the ORF was used as a probe to physically map the tnr3 gene. To disrupt the gene, the KpnI-PstI
fragment was subcloned into pBluescript (b), and in the HindIII site of this fragment the ura4 gene was
ligated, resulting in plasmid pBStnr3::ura4 (c).
Acid phosphatase activity was measured as described previously (27) .
The plasmid pUR19tnr3 has an insert of 5.2 kb, and its restriction map is shown in Fig. 1. To examine whether the cloned fragment contains the tnr3 gene, the ars sequence was deleted, and the
resulting plasmid was linearized by NaeI digestion and
transformed into tnr3-5 ura4-D18 pho1-44
cells(28) . Four stable transformants were selected and crossed
by standard genetic methods with the parental strain pho1-44.
Out of 8200 colonies examined, no thiamine non-repressible recombinants
could be recovered, but 24% of the progeny were ura.
This indicates that the cloned 5.2-kb fragment had integrated at or
very close to the tnr3 locus, suggesting that we have isolated
the tnr3 gene.
Using the 0.7-kb KpnI-HindIII fragment (Fig. 1) of the insert, the tnr3 gene was physically mapped to the left side of the left arm of chromosome I adjacent to the probe 12 g11 of the map given in Fig. 2by Hoheisel et al. (24) .
Figure 2: Sequence comparison between the S. pombe tnr3 gene and the S. cerevisiae TPK encoded by gene THI80. Sequence identities are indicated by vertical bars, strong similarities by two dots, and weak similarities by one dot. The two sequences were aligned using the program GAP; gap weight was 3.0, and gap length weight was 0.1.
TPK activities from three tested tnr3 mutants and two transformed strains are shown in Table 1. The activities are clearly reduced in the mutants and a mutant carrying the pUR19tnr3 plasmid has a higher activity than strains lacking a plasmid-born tnr3 gene. When tnr3 cells are transformed with the S. cerevisiae TPK gene (THI80), pho4-encoded acid phosphatase is again thiamine repressible (data not shown) and cells reveal a high TPK activity, indicating that the S. cerevisiae gene is expressed in S. pombe and can complement the defect of tnr3 mutations. These results together with the finding that the tnr3 gene shows a significant sequence homology with S. cerevisiae TPK strongly suggests that tnr3 encodes TPK of S. pombe.
Figure 3:
Growth
of the wild type 972 h and the mutant strain tnr3-1. A, strains were grown in MM at 30 °C, and
at different time intervals the optical density A
was measured. B, microscopic examination of end-log
phase cells of the mutant and the wild type (Wt
972).
To test the effects of a disrupted tnr3 gene, a diploid
strain of the genetic constitution ade6-M216ura4-D18
h/ade6-149ura4-D18 h
was transformed with the 3.9-kb KpnI-SacI
fragment of plasmid pBS
tnr3::ura4 (Fig. 1), and stable integrants were selected. Southern
analysis of two integrants showed that integration had occurred at the
correct position (data not shown). One of these integrants was
sporulated, and eight tetrads were dissected. All tetrads showed the
same pattern: two spores were viable, uracil and adenine auxotrophic,
and two spores did not form visible colonies. Microscopic observation
showed that the spores germinated and underwent one or two rounds of
division. Cells were elongated and morphologically aberrant. This is
evidence that gene tnr3 is essential for cell growth and
normal cell shape and shows that most, if not all of TPK activity is
coded for by the gene tnr3.
Figure 4:
Northern analysis of pho1 and pho4 mRNA of wild type 972 h and mutant tnr3-5. Cells were grown at 30 °C in either low
phosphate (LP) or normal phosphate (NP) MM that
contained no thiamine (-T) or was supplemented with 1
µM of the vitamin (+T), and RNA was
extracted and blotted as described under ``Materials and
Methods.'' We confirmed earlier findings that pho1 and pho4 do not cross-hybridize under the conditions
used(29) . As a control, the RNA was probed with ura4.
Suitable exposures of the blot were scanned and quantitated. The
relative intensities for the pho1 band are from left to right:
2.0, 1.0, 14.6, 12.1, 10.7, 5.0, 19.8, and 18.0. The results of the
blot have been reproduced in a second independent
experiment.
Figure 5: Mating of wild type and tnr3 mutant strains in the presence and absence of thiamine. Zygote formation and sporulation of cells of opposite mating types from wild type (1) and mutant strains tnr3-10 (2) and tnr3-5 (3) were examined in MM (white columns) and MM containing 1 µM thiamine (black columns) as described by Schweingruber and Edenharter(3) . Mating efficiency was determined by counting zygotes and asci(4) . The results are the mean values from two independent experiments.
We have cloned and characterized gene tnr3, and all available data suggest that the gene encodes TPK.
TDP is essential for cell growth. It is probable that the tnr3-encoded TPK is the only enzymatic activity in the cell that is able to synthesize TDP. We cannot yet exclude the possibility that a thiamine phosphate kinase, which synthesizes TDP from thiamine monophosphate, exists in the cell. Under the physiological conditions used here, however, insufficient TDP would be synthesized by this activity to allow cell growth.
The putative protein encoded by tnr3 has a molecular weight of 64,000. The molecular weights of the TPK monomers from Paracoccus dentrificans, S. cerevisiae, rat liver, and human red blood cells are known(30, 31, 32) . They are all in the range between 23,000 and 28,000. Assuming that the molecular weight of purified S. pombe TPK monomer is in the same range, we have to take into consideration the possibility that the enzyme is synthesized as a larger precursor, which is proteolytically processed in the cell, or that purified TPK does not correspond to in vivo TPK due to partial degradation of the enzyme during purification.
Previous results show that exogenously added thiamine increases the intracellular TDP level and represses expression of genes involved in thiamine metabolism(4, 6, 7, 8, 9) . The results of this communication indicate that TPK is involved in relaying the thiamine signal. This suggests that thiamine is converted by TPK to TDP and that TDP then acts in turn as an intracellular metabolic thiamine signal. The fact that mutants tnr3-5 and tnr3-10 are strongly derepressed for thiamine metabolic genes but that their intracellular TDP pools are only very slightly lower than the wild type pools would further imply that the TDP level is very critical and that even slight alterations of this level can trigger a gene-regulating signal. Earlier observations that the intracellular TDP level increases less than a factor of two by fully repressing amounts (10 µM) of thiamine is consistent with this notion. How the TDP signal is further transduced is unknown. The last target in the signal transduction cascade is possibly the transcription factor encoded by gene thi1.
Our results show that thiamine represses pho1 expression and that TPK, in addition to pho4, is also a regulator of pho1-encoded acid phosphatase. Derepression of pho1 is diagnostic for phosphate limitation. This implies that TPK is involved in signaling phosphate starvation. We speculate, as above, that thiamine is converted by TPK to TDP, which in turn affects phosphate metabolism. Knowing that thiamine regulates phosphate metabolism, we suggest that thiamine-repressible acid phosphatase acts in vivo not only as thiamine phosphate phosphatase but also as an unspecific phosphatase that is, like phosphate-repressible acid phosphatase, involved in scavenging phosphate from the growth medium when TDP levels are low. Indeed, we noted that the enzyme has not only a high affinity for thiamine phosphates (6) but also cleaves many other organic phosphates, albeit with lower affinity(4) .
A mutant (tnr3-1) exhibiting only 5% of the wild type TPK activity grows slowly and also exhibits aberrant cell morphology. Most cells are elongated and have a distorted form. This indicates that TPK also has some effect on cell division and morphogenesis.
Mating of S. pombe is regulated by a variety of different nutritional signals, one of which we have shown to be thiamine. In this study, we show that TPK is involved in regulation of mating. As previously discussed, the easiest explanation of regulation of mating by thiamine and TPK is that TDP is a critical metabolic signal for mating. We observed previously that starving cells for ammonium and glucose reduces intracellular TDP levels(3) . Such starvation conditions strongly favor mating, and we suggest that nutritional signaling of mating by glucose and ammonium can partially occur by mechanisms that alter intracellular TDP levels.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84417[GenBank].