©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Schizosaccharomyces pombe Thiamine Pyrophosphokinase Is Encoded by Gene tnr3 and Is a Regulator of Thiamine Metabolism, Phosphate Metabolism, Mating, and Growth (*)

(Received for publication, July 27, 1995; and in revised form, August 23, 1995)

Hans Fankhauser Andreas Zurlinden Anne-Marie Schweingruber Eleonore Edenharter M. Ernst Schweingruber (§)

From the Institute of General Microbiology, University of Bern, Baltzer-Strasse 4, CH-3012 Bern, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Thiamine (vitamin B(1)) acts in the form of thiamine diphosphate (TDP) (^1)as coenzyme of several enzymes including pyruvate dehydrogenase, pyruvate decarboxylase, transketolase, alpha-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. (^2)

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(6) 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.


MATERIALS AND METHODS

Strains and Media

S. pombe wild type strains and the mutants ura4-D18, ade6-M216, and ade6-149 are from our collection. Strains pho1-44, pho1-44 tnr3-10, pho1-44 tnr3-5, and pho1-44 tnr3-8 have been described previously(11) . Mutant tnr3-1 has been selected by the same procedure as tnr3-10 and tnr3-5(11) . Strains pho1-44 tnr3-10pUR19tnr3 and pho1-44 tnr3-10pUR19THI80 are transformants carrying the S. pombe tnr3 and the Saccharomyces cerevisiae THI80 gene, respectively, on plasmid pUR19.

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.

Molecular Cloning and Disruption of Gene tnr3

The gene tnr3 was cloned with the partial Sau3A genomic library ligated in the shuttle vector pUR19 as described previously(14) . Strain tnr3-5 ura 4-D18 h was transformed using the alkali cation method (15) and plated on MMA. The growing ura colonies were stained with alpha-naphtyl phosphate and Fast Blue(16) . Plasmids recovered from transformants were propagated in Escherichia coli XL1-blue(17) . Gene tnr3 was disrupted by inserting a 1.8-kb HindIII fragment of the ura4 gene (18) into the single HindIII site in the ORF of the tnr3 gene (Fig. 1). Standard methods were used for restriction endonuclease digestion, ligation, and transformation in E. coli(19) .


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 pBSDeltatnr3::ura4 (c).



Synthesis of the S. cerevisiae THI80 Gene

This gene was synthesized by polymerase chain reaction and ligated into pUR19 via a SacI and PstI restriction site. The primers corresponding to the beginning and end of the THI80 gene were CGCGCGAGCTCAAAAAGCTGAATATCACTGC and CGCGCGCTGCAGTAGTTAAAGAACACATGC(20) .

DNA Sequencing

DNA sequences were determined from denatured, double-stranded templates by the dideoxy chain termination method (21) with modified T7 polymerase and [alpha-S]dATP using the Sequenase II kit (U. S. Biochemical Corp.) following the manufacturer's instructions. DNA and protein sequences were analyzed with the University of Wisconsin Genetics Computer Group program(22) .

Genetic Techniques and Physical Mapping

Standard genetic techniques for S. pombe have been described(23) . The tnr3 gene was physically mapped using P1 and cosmid libraries covering the complete S. pombe genome(24) . The ura4 gene was taken as a known reference probe. Nylon filters spotted with the genomic fragments were kindly provided by Dr. Elmar Maier (Imperial Cancer Research Fund, London) and hybridized with the probes as described(24) .

Northern Analyses

Cells were grown in normal or low phosphate MM supplemented with or without 1 µM thiamine to an optical density A of 0.9-1.1. Total RNA was extracted, separated by the glyoxal method, blotted, and hybridized as described (11) .

Determination of Intracellular Thiamine and Thiamine Phosphate Pools

Thiamine and its phosphates were extracted in HCl and determined by HPLC as described previously(6) .

Determination of TPK and Acid Phosphatase Activities

For measurements of TPK activity, we followed a modified procedure of Chernikevich et al.(25) . Cells were grown in MM to mid-log phase, harvested by centrifugation, and disrupted in 20 mM Tris-HCl, pH 7.4, containing 1 mM dithiothreitol. Cell debris was centrifuged, and the extracts were dialyzed in the same buffer. The total volume of the standard assays was 1 ml and contained 20 mM Tris-HCl, pH 8.6, 4 mM ATP, 10 mM MgSO(4), 40 µM thiamine, and crude extract (100-200 µg of protein). The reaction was started by adding thiamine and stopped by the addition of 100 µl of HClO(4). The incubation conditions were 30 min at 37 °C. Proteins were centrifuged, the supernatant was neutralized with 125 mg KHCO(3), and TDP was measured by HPLC as described above. Protein concentrations were determined by the method of Bradford(26) . The activity is given as pMol of TDP/µg of protein and 30 min.

Acid phosphatase activity was measured as described previously (27) .

Mating

Mating of cells was determined in liquid media as described previously(3) . In these experiments phosphate was not autoclaved separately.


RESULTS

Cloning and Physical Mapping of the tnr3 Gene

tnr3 mutants are derepressed for thiamine-repressible acid phosphatase on a medium containing high amounts of thiamine. As an approach to clone the tnr3 gene, we therefore transformed strain tnr3-5 pho1-44 ura4-D18 with a wild type gene bank and selected transformants that exhibited a repressed phenotype for thiamine-repressible acid phosphatase. Out of approximately 250,000 tested colonies one showed a reduced pho4-encoded phosphatase activity on a thiamine-containing medium. The plasmid of this strain (pUR19tnr3) was isolated, propagated in E. coli, and upon back transformation shown to complement the parental strain tnr3-5 and other tnr3 strains such as tnr3-10 and the newly isolated strain tnr3-1 (see below). Quantitative measurements of thiamine-repressible acid phosphatase in liquid media confirmed that the activity is repressed in transformed strains (data not shown).

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.



Nucleotide Sequence of Gene tnr3

Using a set of nested deletions and specific primers, virtually the whole sequence of the 5.2-kb insert was sequenced. There was only one ORF of reasonable length in the whole sequence. It codes for a putative protein with 569 amino acids with a predicted molecular weight of 64,231 (Fig. 2). The amino-terminal half of the protein exhibits no obvious sequence relationship to known proteins. As shown in Fig. 2, the 260 amino acids of the carboxyl-terminal moiety, however, show 31% amino acid sequence identity and 54% similarity with the S. cerevisiae TPK determined by Nosaka et al.(20) . This suggests that tnr3 encodes S. pombe TPK.

TPK Activity in Different S. pombe Strains

Before measuring TPK activity of tnr3 mutants and strains overexpressing the tnr3 gene, we first characterized the activity of the wild type enzyme. All measurements were done using dialyzed crude extracts. As expected, the enzyme was fully dependent on ATP (or other triphosphates, see below), Mg and thiamine and its activity was linear for at least 30 min under the standard conditions described under ``Materials and Methods'' (data not shown). Activity is only detectable if the buffer, in which cells are disrupted and extracts are dialyzed, contains dithiothreitol or similar reducing agents such as mercaptoethanol. By measuring TPK activity at different substrate concentrations, we determined the K(m) values for the different substrates (TPK activity was measured at 10 different concentrations, and the data were plotted according to Eadie and Hofstee). These are 1.9 mM for ATP, 3 mM for Mg, and 6 µM for thiamine under the standard assay conditions (data not shown). The pH optimum for the activity is around 9, and the enzyme shows a broad in vitro specificity for ribonucleoside triphosphates as pyrophosphate donors, as it has already been observed for S. cerevisiae TPK(25) .

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.



TDP Pools and Growth of tnr3 Mutants

The tnr3 mutants tnr3-8 and tnr3-10 described previously are strongly derepressed for thiamine-repressible genes but do not reveal a significant growth defect, and measured TDP pools are only slightly lower than in the parent strain(11) . Knowing that the mutants exhibit drastically decreased TPK activities in vitro and that TDP is essential for growth, this was rather surprising and suggested that TPK is either encoded by more than one gene or that the previously isolated mutants were very leaky. To test the second possibility, we screened for further tnr3 mutants and subsequently isolated mutant tnr3-1. This mutant is approximately 10-fold more derepressed for pho4-encoded acid phosphatase (data not shown) and, as shown in Table 1, exhibits a much higher intracellular thiamine pool than mutants tnr3-10 or tnr3-5, indicating that thiamine-repressible genes in tnr3-1 are more derepressed than in the tnr3 mutants published previously. TPK activity of strain tnr3-1 is only 5% compared to the wild type, and the amount of extractable TDP is only about a third (Table 1). As shown in Fig. 3, the strain shows a drastically reduced growth rate, and the morphology of cells is aberrant. Slow growth, aberrant cell morphology, and derepressed acid phosphatase activity cosegregate (data not shown). These results suggest that the low TPK activity in strain tnr3-1 is responsible for the low TDP pool, slow growth, and aberrant cell shape.


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 pBSDeltatnr3::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.

Expression of tnr3 mRNA

To test whether expression of gene tnr3 is itself also regulated by thiamine and under the control of thi1, tnr1, tnr2, and itself, we grew cells of the wild type and strains thi1-1, tnr1-18, tnr2-2, and tnr3-5 in the presence and absence of thiamine and examined tnr3 expression by Northern blotting. No significant alterations of tnr3 mRNA levels could be detected (data not shown). Furthermore, growing a thiamine auxotrophic strain (thi4-4) under extreme thiamine limitation (20 nM) did not affect tnr3 mRNA levels. From this result, we conclude that transcription of tnr3 is not regulated by thiamine.

Mutations in tnr3 Affect Expression of Gene pho1 Expression

In addition to thiamine-repressible acid phosphatase encoded by pho4, S. pombe also codes for a phosphate-repressible acid phosphatase(13, 30) . This acid phosphatase is encoded by gene pho1 and, like thiamine-repressible acid phosphatase, is a cell wall glycoprotein. It is rather substrate unspecific and is thought to scavenge phosphate from organic compounds in the growth medium. In contrast to the situation in S. cerevisiae, regulation of pho1 expression by phosphate in the MM is not strong. In attempts to improve phosphate regulation of pho1 expression, we found that not only phosphate but also thiamine reproducibly represses pho1 expression (Table 2, Fig. 4). Thiamine apparently not only regulates thiamine- but also phosphate-repressible acid phosphatase. Repression by thiamine is relatively weak and occurs mainly in MM. To test whether TPK plays a role in this regulation, we examined pho1-encoded acid phosphatase activity from two different tnr3 mutants (tnr3-10, tnr3-5) and observed that gene pho1 is clearly more derepressed in the mutants than in the wild type (Table 2). The derepressed phenotype cosegregates with the tnr3 lesion (data not shown). The derepressing effect of the tnr3 mutation is stronger in MM than in low phosphate MM. The Northern blot for the wild type and mutant tnr3-10 is shown in Fig. 4. The relative intensities of the bands roughly match the values given in Table 2. The pho4 mRNA levels are given as a control. They are regulated as described previously(4) . In summary, the results illustrate that TPK is also a regulator of phosphate-repressible acid phosphatase.




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.



TPK Is Involved in Regulation of Mating

Thiamine represses mating in S. pombe(3) . We therefore examined mating behavior of tnr3 mutants in the presence and absence of thiamine. As shown in Fig. 5, mating in the two tnr3 strains is not or is only weakly repressible by thiamine. We dissected 10 tetrads resulting from a cross of tnr3-10 with wild type and found that constitutive mating in the 10 tested combinations only occurs when both partner strains carry the tnr3 mutation (data not shown). This suggests that not only thiamine but also TPK is a regulator of mating.


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.




DISCUSSION

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.


FOOTNOTES

*
This study was supported by the Swiss National Foundation and Hoffmann-La Roche and Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84417[GenBank].

§
To whom correspondence should be addressed. Tel.: 41-31-631-46-58; Fax: 41-31-631-46-84.

(^1)
The abbreviations used are: TDP, thiamine diphosphate; TPK, thiamine pyrophosphokinase; MM, minimal medium; HPLC, high performance liquid chromatography; kb, kilobase pair(s); ORF, open reading frame.

(^2)
A. Zurlinden and M. E. Schweingruber, unpublished results.


ACKNOWLEDGEMENTS

We thank A. Carr for the partial Sau3A genomic library, E. Maier for the filters spotted with the genomic fragments and evaluations of the results, M. Rusu for dissecting some tetrads, and W. Wilson and T. Seebeck for helpful comments on the manuscript.


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