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Plant fructokinases: a sweet family get-together
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Plant fructokinases: a sweet family get-together Jónatas V. Pego and Sjef C.M. Smeekens Plant fructokinases are the gateway to fructose metabolism. Here, we discuss the properties of published plant fructokinases and compare the available protein sequences. In addition, we speculate on the possible function of fructokinases as sugar sensors. A proposal is presented to clarify the confusing fructokinase nomenclature. Only a few plant fructokinase genes have been cloned but the recent isolations of two such genes in tomato and three in Arabidopsis have given this research an important impulse.
ructokinases (FRKs) are enzymes with a high, specific affinity for fructose. Plant and bacterial FRKs catalyze the conversion of fructose to fructose-6-phosphate, whereas mammalian FRKs [also known as ketohexokinases (KHKs)] phosphorylate fructose to fructose-1-phosphate. Halophilic archaebacteria harbor mammalian-like FRKs and also produce fructose-1-phosphate1. In most plants, sucrose is the major form of carbohydrate transported by the vascular system. It can be cleaved by either invertase or sucrose synthase (SUSY), and free fructose is a product of both reactions (Fig. 1). Fructose is probably then phosphorylated by FRKs in vivo because their affinities for fructose are much higher than those of the hexokinases2 (HXKs). Antonia Medina and Alberto Sols presented the first report of a plant fructosespecific kinase as a short communication in 1956 from work in pea3. However, the enzyme was not isolated and characterized until 20 years later4. Since then, plant FRKs have been purified and analyzed from a range of species (Table 1).
F
Plant FRK proteins and genes
FRK activities have been purified by ion-exchange chromatography from a variety of plant species and tissues. Between one and three peaks of FRK activity have been reported. This number varies not only among species but also among different tissues within a given species. The designation attributed to each individual activity peak was generally dictated by the order of elution from the column. This led to a diversity of designations given to the different plant FRK proteins. Three activity peaks were found in potato, maize and barley, and they were designated FRK1, FRK2 and FRK3 (Refs 2,5), FRK0, FRK1 and FRK2 (Ref. 6), and FRK1a, FRK1b and FRK2 (Ref. 7), respectively. However, because the characteristics of each peak are similar in the three species, they are likely to represent homologous FRKs. Thus FRK2 in potato is probably equivalent to FRK1 in maize and FRK1b in barley. In other cases, only one or two FRK peaks were observed, adding to the complexity of FRK nomenclature. Recently, two tomato FRK cDNAs were cloned8. The authors named them FRK1 and FRK2, in the order in which they were cloned. They speculate that the FRK1 homolog in potato is FRK3 and that the potato FRK1 and FRK2 activity peaks might be the products of a single gene, homologous to tomato FRK2 (Ref. 9). This confusion in FRK terminology, which is due to the absence of nomenclature standards, has led us to attempt to unify plant FRK nomenclature. We have based our system on plant and bacterial DNA sequence data, the characteristics of the individual enzymes, the order of elution of the FRK activity peaks, and
results obtained with a novel FRK2-deficient Arabidopsis mutant. We believe that all the published FRK activity peaks can be attributed to two different enzymes (Table 1), and these new designations will be used throughout this manuscript. In the future, we propose that the names of FRK-like genes should be based on deduced amino acid sequence similarity to their Arabidopsis and tomato counterparts. In accordance, the potato and sugar beet FRK genes should be redesignated FRK2 (Table 1). Enzymatic properties of FRKs
The molecular weight of plant FRKs has been determined in several studies (Table 1). SDS polyacrylamide gel electrophoresis
Sucrose
Invertase Fru + Glc Hexosetransporter Apoplast Cytosol
Sucrosetransporter
Sucrose Sucrose Invertase synthase UDP-Glc + Fru Fructokinase Fru-6-P
Fru + Glc Hexokinase Glc-6-P
Glycolysis Trends in Plant Science
Fig. 1. Sucrose metabolism in plant cells. Sucrose can be cleaved by invertase in the apoplast or intracellularly. The resulting hexoses will be converted to hexose phosphates by fructokinase and hexokinase in the cytosol. Alternatively, upon cell entry, sucrose can be cleaved by sucrose synthase, yielding UDP–glucose and fructose. Following phosphorylation, the resulting hexose phosphates can be further metabolized through glycolysis. Abbreviations: Fru, fructose; Fru-6-P, fructose-6-phosphate; Glc, glucose; Glc-6-P, glucose-6-phosphate.
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01783-0
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Table 1. Plant fructokinases Organism
Tissue
Arabidopsis thaliana
Name given
Name proposed
Substrate inhibition
Km Fru (m)
FRK1 FRK2 FRK3
FRK1 FRK2 FRK3
NDa ND ND
ND ND ND
Molecular weight (KDa)
GenBank Accession no.
37.1c 35.3c 34.7c
Refs
g g g
37.3c 34.8c 34.8c/72.4d 35.0e/73.0d 35.0e/73.0d
U64817 U64818 U62329
9 9 13 10 10
35.4c/70.0 5d
U37838
12
Z12823
11 18 18 18 19 5 5 5
Tomato (Lycopersicon esculentum)
See Ref. See Ref. See Ref. Fruit Fruit
FRK1 FRK2 FRK FK1 FK2
FRK1 FRK2 FRK2 FRK2 FRK2
No Yes Yes Yes Yes
1300 54 220 131 201
Sugar Beet (Beta vulgaris)
Taproot
FK
FRK2
Yes
68
Potato (Solanum tuberosum)
See Ref. Tuber Tuber Tuber See Ref. See Ref. See Ref. See Ref.
FRK FK1 FK2 FK3 FRK FK1 FK2 FK3
FRK2 FRK2 FRK2 FRK1 FRK2 FRK2 FRK2 FRK1
ND Yes Yes No Yes Yes Yes Yes
ND 41 116 128 1140 64 90 100
Soybean (Glycine max)
Nodule
FRK
FRK1
Yes
77
Maize (Zea mays)
Seed Seed Seed Leaf
FRK0 FRK1 FRK2 FRK1
FRK2 FRK2 FRK1 FRK2
ND Yes Yes ND
ND 148 121 ND
ND 59.0d 59.0d ND
6 6 6 22
Barley (Hordeum vulgare)
Leaf Leaf Leaf
FRK1a FRK1b FRK2
FRK2 FRK2 FRK1
Yes Yes Yesb
200 100 180
37.0d 37.0d 73.0d
7 7 7
Avocado (Persea americana)
Mesocarp Mesocarp
FRK1 FRK2
FRK2 FRK2
No No
140 100
85.0 3d 85.0 3d
14 14
Honey Locust (Gleditsia triacanthos)
Cotyledon
FRK
FRK1
Yes
190
ND
60
Spinach (Spinacia oleracea)
Leaf Leaf
FK1 FK2
FRK2 FRK1
ND ND
85 140
ND ND
22 22
Lily (Lilium longiflorum and L. lancifolium)
Pollen
FRK
FRK2
Yes
180/400
ND
61
Camellia (Camellia japonica)
Pollen
FRK
FRK2
Yes
300
ND
61
Pea (Pisum sativum)
Seed Seed Leaf
FRK FRK FRK1
FRK2 FRK1 FRK2
Yes Yesb ND
ND 57 ND
79.0 2e/72.0 4d ND ND
17 24 22
34.4c 36.0e/70.0df 36.0e/70.0df 36.0e/70.0df ND 102.0d 105.0d 118.0d 84.0 5d
20
a
ND not determined. Only slight substrate inhibition found. Calculated. d Native. e Determined by SDS-PAGE. f It is unclear to which of the three potato FRKs these values refers to. g J.V. Pego and S.C.M Smeekens, unpublished. b c
(SDS-PAGE) analysis showed a FRK2 protein of 35 kDa in developing tomato fruit10, one of 36 kDa in potato tubers11 and one of 37 kDa in sugar beet taproots12. This is in close agreement with values later obtained based on deduced amino acid sequences in 532
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tomato8, sugar beet12 and potato11. Gel filtration assays in tomato, potato, barley, soybean, avocado and sugar beet revealed a native molecular weight of 70–105 kDa (Refs 7,13,14), showing that, in these species, the protein is present as a dimer. A native molecular
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trends in plant science Reviews weight of 59 kDa has been reported15 but a mixture of the monomeric and dimeric forms of the enzyme might have caused FRK2 Tomato this. A similar case has been reported for Lactococcus lactis FRK, FRK2 Potato in which a 44 kDa value was obtained by gel filtration, proving later to be a 80:20% mixture of the 66 kDa dimer and the 33 kDa FRK2 Arabidopsis monomer16. Although most plant FRKs are present as dimers, pea FRK2 Sugar beet seed and barley leaf FRK2 might be present in the monomeric form7,17. The pea isoform seems to be an exception, giving a molFRK3 Arabidopsis ecular weight of 72 kDa on SDS-PAGE (Ref. 17), approximately twice the size of all other plant FRKs. The general picture for FRK1 Arabidopsis FRK1 is similar to that of FRK2. The native molecular weight of FRK1 Tomato FRK1 is reported to be 118 kDa or 70 kDa in potato tubers5,18, 7 19 73 kDa in barley leaves and 84 kDa in soybean nodules . The calculated weight for the tomato enzyme is 37 kDa (Ref. 8), sug300 200 100 0 gesting that FRK1, like FRK2, is dimeric. Trends in Plant Science No. of substitution events The pH optima for both FRK1 and FRK2 are reported to be Fig. 2. Phylogenetic tree of the deduced amino acid sequences of around pH 8 (Refs 10,12), and the Km for fructose is between seven plant fructokinases using the J. Hein method62 with PAM250 41 M and 220 M (Table 1). Two isolated reports of a higher Km residue weight table. of ~1.2 mM (Refs 9,20) were obtained from yeast cell extracts expressing a plant FRK and might reflect unphysiological modifications. FRKs are widely reported to have a higher affinity for FRK1 Arabidopsis 74 ATP than for other nucleotides and, unless 78 FRK1 Tomato they are in a region or cellular compartment 60 FRK2 Arabidopsis with high GTP or UTP concentrations, ATP FRK2 Tomato 62 FRK2 Sugar beet 64 will be the principal substrate for fructose FRK2 Potato 63 phosphorylation in vivo12,13. Mg2 is necesFRK3 Arabidopsis 57 sary for FRK activity2,13 and, in some cases, A1 activity can be further stimulated by the 182 addition of K (Ref. 12). Several FRK 186 purification studies show that most FRKs 168 are associated with the cytosol fraction and 170 the enzyme is therefore presumably also 172 171 cytosolic21. In spinach, organelle fractiona165 tion by sucrose-gradient centrifugation or differential centrifugation suggested that, B although FRK2 was presumably present in 210 214 the cytosol, FRK1 was associated with the 196 chloroplast22. 198 An interesting feature of plant FRKs is 200 their reported fructose substrate inhibition 191 193 in vitro (Table 1). At approximately pH 8, FRK2 is highly sensitive to inhibition by B fructose, with Ki values of 1–6 mM in 219 254 potato, tomato, pea and maize2,10,15. Sub223 258 205 238 strate inhibition has also been reported for 207 240 FRK2 at pH 6.6 (Ref. 23) but an earlier 209 242 report from the same group24 showed FRK1 199 231 inhibition at pH 8 but not at pH 6.6. Barley 202 235 FRK1 displayed only a slight substrate inhiB A2 bition at 20 mM fructose7 and, in tomato, 288 FRK1 is reported not to be inhibited by up 292 to 50 mM fructose9. Results obtained from 272 potato tuber FRK1 are conflicting because 274 276 both a Ki of 21 mM and the absence of inhi265 bition (up to 16 mM fructose) have been 269 2,18 reported . The picture that emerges is that, A3 Trends in Plant Science although fructose is a potent inhibitor of FRK2, it seems to be less effective against Fig. 3. Alignment of conserved regions of the deduced amino acid sequences of seven plant FRK1. fructokinases. This shows three signature patterns for the pfkB family of carbohydrate kinases (A1, A2, A3) and a region specific to fructokinases (B). The A1 motif is involved in FRK2 has been suggested to play a role ATP binding and the B motif contains two sugar-binding domains. Black boxes indicate in starch production in sink tissues25, where identical residues between proteins. SUSY cleaves incoming sucrose into UDP–glucose and fructose (Fig. 1). SUSY, December 2000, Vol. 5, No. 12
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Hepatocyte nucleus
GAL1/GAL3 GAL1/GAL3 Galactose
GAL4
HXK
GAL80 GAL4
F1P
mRNA Trends in Plant Science
Fig. 4. Representation of the galactose-mediated pathway for regulation of gene expression in yeast. In the presence of galactose, a complex is formed consisting of GAL4, GAL80 and one of the proteins GAL3 or GAL1 (galactokinase). This complex activates gene transcription. When galactose is absent, the GAL80–GAL1 (or GAL80–GAL3) interaction is disrupted and GAL80 inhibits GAL4 transcription activity.
like FRK, is also inhibited by fructose. In tomato fruit, the Ki for fructose inhibition of FRK and SUSY are 2 mM and 2.4 mM, respectively, well below the concentration of ~40 mM fructose found in these tissues25. SUSY is also associated with other sinkspecific processes such as callose and cellulose synthesis26. Because developmental patterns of FRK and SUSY activity are expressed in a coordinated fashion in several plant species25, the two enzymes might, in concert, control sucrose use owing to elevated fructose. High SUSY activity, generating elevated fructose levels, would lead to the inhibition of both SUSY and FRK in what could be described as a ‘double brake’ mechanism. However, it remains unclear why such a mechanism would be necessary. Plant genes for FRKs and their expression
A few plant FRK genes have now been cloned, with one to three genes isolated per species. FRK2 was isolated from potato tubers27 and sugar beet taproots12, FRK1 and FRK2 from tomato fruit, and, recently, FRK1, FRK2 and FRK3 from Arabidopsis (J.V. Pego and S.C.M. Smeekens, unpublished). The isolation of FRK3 in Arabidopsis is significant as it marks the first time that three FRK genes have been found in any one organism. However, it is still uncertain whether the FRK3 gene is transcribed. The FRK3 protein would represent a novel higher-plant FRK (Fig. 2). For bacteria, 19 FRK genes from 18 different species have been reported. Alignment of the deduced amino acid sequences revealed significant homology among all seven plant FRK proteins. The three signatures of the FRK-containing pfkB family of carbohydrate kinases and a large FRK-specific domain are highly conserved in these proteins. These regions are involved in ATP and sugar binding28 (Fig. 3). The calculated molecular weights are also similar, varying from 34.4–35.4 kDa for FRK2 and FRK3, and ~37 kDa for FRK1. The higher molecular weight of FRK1 is mainly because of a longer N-terminal region present in tomato and Arabidopsis FRK1, which is absent in FRK2 and FRK3. An additional feature of all seven proteins is the low degree of homology at their C-termini. The only gene expression data available were obtained in tomato. It was recently reported that, although FRK1 is constitutively expressed throughout the entire tomato plant, FRK2 seems to be sink specific and present in source leaves only at low levels8,9. In accordance with this, potato FRK1 activity was high in leaves and low in tubers, whereas FRK2 accounted for 95% of FRK activity in tubers but was barely detectable in leaves5. These 534
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P F6
GAL80
GCKR
Cytoplasm
FRK
GCKR
Fru
Signal HXK Trends in Plant Science
Fig. 5. Sugar sensing in mammalian hepatocytes. In the presence of fructose-6-phosphate (F6P), the hexokinase IV (HXK) regulatory protein GCKR, represses HXK activity by recruiting it to the hepatocyte nuclear matrix. The binding by GCKR of fructose-1phosphate (F1P), the product of the reaction catalyzed by fructokinase (FRK), disrupts the HXK–GCKR interaction and allows HXK-mediated signaling. FRK is proposed to modulate HXK activity indirectly by controlling the F6P:F1P ratio. Abbreviation: Fru, fructose.
results were supported by studies in sugar beet taproots, where FRK2 was the only FRK found12. The situation in Arabidopsis is similar to that of potato, tomato and sugar beet because FRK2 accounts for the majority of root FRK activity and only a small fraction of activity in the shoots (A. Krapp and M. Stitt, pers. commun.). Taken together these results seem to point to a sinkspecific function for FRK2, whereas FRK1 is thought to be primarily source specific. In spite of their distinct gene expression patterns, transcription of both FRK1 and FRK2 is induced by the exogenous application of glucose, fructose or sucrose in tomato plants9. Induction was rapid (1.7 h) after application of a low concentration (4 mM) of glucose. Maximal mRNA levels were obtained at 20 mM glucose, a concentration of sugar shown to be physiologically relevant25. This is the first evidence that the FRK gene belongs to the growing class of sugar-regulated genes. Hexose kinases as sugar sensors
FRKs are members of the larger functional family of hexose kinases. These enzymes, capable of converting hexoses (e.g. glucose, fructose, mannose) to hexose phosphates, have long been recognized as essential components of metabolism. The best known family member, HXK, catalyzes the first step in glycolysis and is a key regulator of this pathway at the enzyme level. An additional, more global, regulatory role was discovered when it was linked to the process of sugar-mediated regulation of gene expression. Sugars are now known to affect the expression of an impressive number of genes in yeast29,30, mammals31,32 and plants33,34. Here, sugar-mediated regulation of gene expression seems to be a central and basic process that is probably common to all higher plants33,35,36. In Saccharomyces cerevisiae, in which the underlying mechanism is best understood, HXK acts in the signaling cascade as a sugar sensor that can relay the initial signal to downstream cascade components37,38. Although it is questioned39, a significant body of evidence points to an analogous function for HXKs in both plants35,40 and mammals41,42, suggesting that the sugar-sensing
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trends in plant science Reviews mechanism might be partly conserved in these systems. In plants, the signaling cascade(s) itself is understood poorly 36, and both the component directly downstream of HXK and the mechanism by which HXK transmits the sugar signal remains unknown. Separation of phosphorylating and signaling functions in yeast HXK mutants has contributed to the understanding of how HXK operates as a sensor43,44 and a similar approach would be of value in plants. Yeast galactokinase represents a second class of bifunctional hexose kinases. Not only is it involved in galactose metabolism but it is also, like HXK, the sensor in a sugar-signaling cascade45. The triggering of this cascade by galactokinase leads to the activation of the GAL genes, encoding the enzymes necessary for further galactose metabolism. In S. cerevisiae, transmission of the galactose signal requires either GAL1 (galactokinase) or GAL3, a GAL1 homolog lacking galactokinase activity46. GAL4, a transcription activator and the key regulator of galactose metabolism in yeast47, binds upstream of the GAL genes. Its transcriptional activity is controlled by a negative regulator, GAL80 (Ref. 48), which inhibits GAL4 activity in the absence of galactose49 (Fig. 4). When both galactose and ATP are available, galactokinase binds to GAL80, relieving its inhibitory effect on GAL4 (Ref. 50) and transcription of the GAL genes is thereby activated. In spite of the functional similarities between the galactokinase-dependent and HXK-dependent mechanisms described above, the signal transduction pathways involved are clearly distinct. However, in yeast, the two pathways interact because the presence of glucose triggers the HXK-dependent signaling pathway, ultimately inhibiting expression of the GAL1 (galactokinase) and GAL4 genes51. By doing so, glucose is established as the preferred substrate over galactose. Galactokinase activity in plants has been demonstrated in purified extracts from several species52. An Arabidopsis galactokinase gene was isolated by functional expression in yeast53 and, recently, a second member of the Arabidopsis galactokinase gene family (ARA1) was identified54. Although the possible involvement of plant galactokinases in sugar-mediated regulation of gene expression has not yet been addressed, the cloning of these two genes should facilitate the isolation of galactokinase knockout mutants. Analysis of such mutants should provide valuable insight into the putative galactokinase sugar-sensing role in plants. FRKs and sugar sensing
Given the sugar-sensing capability of HXK and galactokinase, it is tempting to speculate that this feature is shared by other hexose kinases. This suggestion is supported by the recent isolation of sugar-insensitive mig mutants in Arabidopsis34. In one of these mutants, a specific FRK activity was found to be completely absent. Further analysis revealed a null mutation in the FRK2 gene and the mutant was therefore renamed frk2; tests are under way to determine whether this mutation is responsible for the mig phenotype. We have since cloned two additional FRKs in Arabidopsis and work is in progress to isolate mutants in these genes. The question of whether sugar sensing is a general feature of hexose kinases is currently being addressed, for FRK, in the frk2 mutant. FRK has previously been associated with the process of sugarmediated gene regulation in mammals55. Pancreatic -cell HXKIV, which is highly similar to yeast HXK (Ref. 56), functions as a glucose sensor that regulates insulin secretion and gene activation. It thus plays an important role in regulating glucose homeostasis. Mutations in the HXKIV gene or in GCKR, the gene encoding the HXKIV regulatory protein, are linked to matureonset diabetes of the young, a form of non-insulin-dependent diabetes57. Mammalian FRK plays a regulatory role in this system. GCKR is bound to the hepatocyte nuclear matrix and represses
HXKIV activity by binding it (Fig. 5). This interaction is relieved by binding of fructose-1-phosphate, the product of the FRKcatalyzed reaction, to GCKR (Refs 58,59). FRK activity is therefore proposed to modulate the glucose sensor (HXKIV) activity in the islet cell. Hepatic FRK deficiency does not impair glucose tolerance but the molecular relationship between islet-cell and hepatic FRK is unknown. In plants, several putative sugar-sensing screens have been devised and many Arabidopsis mutants isolated34. However, recent findings have shown that gin1 and sun6 are mutated in interacting hormone pathway components36. This shows that more specific screening strategies are needed that can discriminate between the primary sugar-signaling cascades and secondary, interacting pathways. These interactions can be further dissected with well characterized mutants in enhancer and suppressor screens to identify pathway-specific mutations. The integration of data obtained from the different mutant screens is beginning to provide insight into the underlying plant sugar-sensing and -signaling mechanism(s), but many issues remain. For example, does FRK act as sugar sensor in plants or does it control sensing, such as in the mammalian system? The available frk2 mutant, and other frk mutants that will be identified, should be valuable tools in addressing these and other questions. Acknowledgements
Our work was financially supported by the Fundação para a Ciência e Tecnologia, Lisbon, Portugal (grant PRAXIS XXI/BD/3103/94 awarded to J.V.P.). References 1 Rangaswamy, V. and Altekar, W. (1994) Ketohexokinase (ATP:D-fructose 1phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties. J. Bacteriol. 176, 5505–5512 2 Renz, A. and Stitt, M. (1993) Substrate specificity and product inhibition of different forms of fructokinases and hexokinases developing potato tubers. Planta 190, 166–175 3 Medina, A. and Sols, A. (1956) A specific fructokinase in peas. Biochim. Biophys. Acta 19, 378–379 4 Frankart, W.A. and Pontis, H.G. (1976) Fructose metabolism in plants. Isolation and properties of pea seed fructokinase. Acta Physiol. Latinoamericana. 26, 319–329 5 Renz, A. et al. (1993) Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity. Planta 190, 156–165 6 Doehlert, D.C. (1990) Fructokinases from developing maize kernels differ in their specificity for nucleoside triphosphates. Plant Physiol. 93, 353–355 7 Baysdorfer, C. et al. (1989) Partial purification and characterization of fructokinase activity from barley leaves. J. Plant Physiol. 134, 156–161 8 Kanayama, Y. et al. (1997) Divergent fructokinase genes are differentially expressed in tomato. Plant Physiol. 113, 1379–1384 9 Kanayama, Y. et al. (1998) Tomato fructokinases exhibit differential expression and substrate regulation. Plant Physiol. 117, 85–90 10 Martinez-Barajas, E. and Randall, D.D. (1996) Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits. Planta 199, 451–458 11 Taylor, M.A. et al. (1995) Characterisation of a cDNA encoding fructokinase from potato (Solanum tuberosum L.). J. Plant Physiol. 145, 253–256 12 Chaubron, F. et al. (1995) Partial purification and characterization of fructokinase from developing taproots of sugar beet (Beta vulgaris). Plant Sci. 110, 181–186 13 Martinez-Barajas, E. et al. (1997) Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli. Protein Expres. Purif. 11, 41–46 14 Copeland, L. and Tanner, G.J. (1988) Hexose kinases of avocado. Physiol. Plant. 74, 531–536
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41 Matschinsky, F. et al. (1993) Glucokinase as pancreatic cell glucose sensor and diabetes gene. J. Clin. Invest. 92, 2092–2098 42 Grupe, A. et al. (1995) Transgenic knockouts reveal a critical requirement for pancreatic cell glucokinase in maintaining glucose homeostasis. Cell 83, 69–78 43 Hohmann, S. et al. (1999) Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2. Microbiology 145, 703–714 44 Kraakman, L.S. et al. (1999) Structure-function analysis of yeast hexokinase: structural requirements for triggering cAMP signalling and catabolite repression. Biochem. J. 343, 159–168 45 Zenke, F.T. et al. (1996) Activation of gal4p by galactose-dependent interaction of galactokinase and gal80p. Science 272, 1662–1665 46 Bhat, P.J. et al. (1990) Analysis of the GAL3 signal transduction pathway activating GAL4 protein-dependent transcription in Saccharomyces cerevisiae. Genetics 125, 281–291 47 Hashimoto, H. et al. (1983) Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae. Isolation and characterization of the regulatory gene GAL4. Mol. Gen. Genet. 191, 31–38 48 Nogi, Y. and Fukasawa, T. (1984) Nucleotide sequence of the yeast regulatory gene GAL80. Nucleic Acids Res. 12, 9287–9298 49 Wu, Y. et al. (1996) Quantitation of putative activator–target affinities predicts transcriptional activating potentials. EMBO J. 15, 3951–3963 50 Platt, A. and Reece, R.J. (1998) The yeast galactose genetic switch is mediated by the formation of a Gal4p–Gal80p–Gal3p complex. EMBO J. 17, 4086–4091 51 Frolova, E. et al. (1999) Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters in vivo: regulation by glucose and chromatin structure. Nucleic Acids Res. 27, 1350–1358 52 Dey, P.M. (1983) Galactokinase of Vicia faba seeds. Eur. J. Biochem. 136, 155–159 53 Kaplan, C.P. et al. (1997) Isolation of a cDNA encoding an Arabidopsis galactokinase by functional expression in yeast. Plant Mol. Biol. 34, 497–506 54 Sherson, S. et al. (1999) The arabinose kinase, ARA1, gene of Arabidopsis is a novel member of the galactose kinase gene family. Plant Mol. Biol. 39, 1003–1012 55 Hayward, B.E. and Bonthron, D.T. (1998) Structure and alternative splicing of the ketohexokinase gene. Eur. J. Biochem. 257, 85–91 56 Gidh-Jain, M. et al. (1993) Glucokinase mutations associated with non-insulindependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships. Proc. Natl. Acad. Sci. U. S. A. 90, 1932–1936 57 Pillay, T.S. et al. (1995) The genetics of non-insulin-dependent diabetes mellitus. Adv. Genet. 32, 51–98 58 Malaisse, W.J. et al. (1990) Regulation of glucokinase by a fructose-1phosphate-sensitive protein in pancreatic islets. Eur. J. Biochem. 190, 539–545 59 Van Schaftingen, E. et al. (1997) The regulatory protein of glucokinase. Biochem. Soc. Trans. 25, 136–140 60 Myers, D.K. and Matheson, N.K. (1994) Hexose-6-kinases in germinating honey locust cotyledons: substrate specificity of D-fructo-6-kinase. Phytochemistry 37, 957–969 61 Nakamura, N. et al. (1991) Characterization of hexose kinases from camellia and lily pollen grains. Physiol. Plant. 81, 215–220 62 Hein, J.J. (1990) Unified approach to alignment and phylogenies. Methods Enzymol. 183, 626–645
Jónatas V. Pego is at the Dept of Biology, Minho University, Gualtar campus, 4710-057 Braga, Portugal; Sjef C.M. Smeekens* is at Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. *Author for correspondence (tel 31 30 2533431; fax 31 30 2513655; e-mail [email protected]).
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Plant fructokinases: a sweet family get-together Jónatas V. Pego and Sjef C.M. Smeekens Plant fructokinases are the gateway to fructose metabolism. Here, we discuss the properties of published plant fructokinases and compare the available protein sequences. In addition, we speculate on the possible function of fructokinases as sugar sensors. A proposal is presented to clarify the confusing fructokinase nomenclature. Only a few plant fructokinase genes have been cloned but the recent isolations of two such genes in tomato and three in Arabidopsis have given this research an important impulse.
ructokinases (FRKs) are enzymes with a high, specific affinity for fructose. Plant and bacterial FRKs catalyze the conversion of fructose to fructose-6-phosphate, whereas mammalian FRKs [also known as ketohexokinases (KHKs)] phosphorylate fructose to fructose-1-phosphate. Halophilic archaebacteria harbor mammalian-like FRKs and also produce fructose-1-phosphate1. In most plants, sucrose is the major form of carbohydrate transported by the vascular system. It can be cleaved by either invertase or sucrose synthase (SUSY), and free fructose is a product of both reactions (Fig. 1). Fructose is probably then phosphorylated by FRKs in vivo because their affinities for fructose are much higher than those of the hexokinases2 (HXKs). Antonia Medina and Alberto Sols presented the first report of a plant fructosespecific kinase as a short communication in 1956 from work in pea3. However, the enzyme was not isolated and characterized until 20 years later4. Since then, plant FRKs have been purified and analyzed from a range of species (Table 1).
F
Plant FRK proteins and genes
FRK activities have been purified by ion-exchange chromatography from a variety of plant species and tissues. Between one and three peaks of FRK activity have been reported. This number varies not only among species but also among different tissues within a given species. The designation attributed to each individual activity peak was generally dictated by the order of elution from the column. This led to a diversity of designations given to the different plant FRK proteins. Three activity peaks were found in potato, maize and barley, and they were designated FRK1, FRK2 and FRK3 (Refs 2,5), FRK0, FRK1 and FRK2 (Ref. 6), and FRK1a, FRK1b and FRK2 (Ref. 7), respectively. However, because the characteristics of each peak are similar in the three species, they are likely to represent homologous FRKs. Thus FRK2 in potato is probably equivalent to FRK1 in maize and FRK1b in barley. In other cases, only one or two FRK peaks were observed, adding to the complexity of FRK nomenclature. Recently, two tomato FRK cDNAs were cloned8. The authors named them FRK1 and FRK2, in the order in which they were cloned. They speculate that the FRK1 homolog in potato is FRK3 and that the potato FRK1 and FRK2 activity peaks might be the products of a single gene, homologous to tomato FRK2 (Ref. 9). This confusion in FRK terminology, which is due to the absence of nomenclature standards, has led us to attempt to unify plant FRK nomenclature. We have based our system on plant and bacterial DNA sequence data, the characteristics of the individual enzymes, the order of elution of the FRK activity peaks, and
results obtained with a novel FRK2-deficient Arabidopsis mutant. We believe that all the published FRK activity peaks can be attributed to two different enzymes (Table 1), and these new designations will be used throughout this manuscript. In the future, we propose that the names of FRK-like genes should be based on deduced amino acid sequence similarity to their Arabidopsis and tomato counterparts. In accordance, the potato and sugar beet FRK genes should be redesignated FRK2 (Table 1). Enzymatic properties of FRKs
The molecular weight of plant FRKs has been determined in several studies (Table 1). SDS polyacrylamide gel electrophoresis
Sucrose
Invertase Fru + Glc Hexosetransporter Apoplast Cytosol
Sucrosetransporter
Sucrose Sucrose Invertase synthase UDP-Glc + Fru Fructokinase Fru-6-P
Fru + Glc Hexokinase Glc-6-P
Glycolysis Trends in Plant Science
Fig. 1. Sucrose metabolism in plant cells. Sucrose can be cleaved by invertase in the apoplast or intracellularly. The resulting hexoses will be converted to hexose phosphates by fructokinase and hexokinase in the cytosol. Alternatively, upon cell entry, sucrose can be cleaved by sucrose synthase, yielding UDP–glucose and fructose. Following phosphorylation, the resulting hexose phosphates can be further metabolized through glycolysis. Abbreviations: Fru, fructose; Fru-6-P, fructose-6-phosphate; Glc, glucose; Glc-6-P, glucose-6-phosphate.
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01783-0
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Table 1. Plant fructokinases Organism
Tissue
Arabidopsis thaliana
Name given
Name proposed
Substrate inhibition
Km Fru (m)
FRK1 FRK2 FRK3
FRK1 FRK2 FRK3
NDa ND ND
ND ND ND
Molecular weight (KDa)
GenBank Accession no.
37.1c 35.3c 34.7c
Refs
g g g
37.3c 34.8c 34.8c/72.4d 35.0e/73.0d 35.0e/73.0d
U64817 U64818 U62329
9 9 13 10 10
35.4c/70.0 5d
U37838
12
Z12823
11 18 18 18 19 5 5 5
Tomato (Lycopersicon esculentum)
See Ref. See Ref. See Ref. Fruit Fruit
FRK1 FRK2 FRK FK1 FK2
FRK1 FRK2 FRK2 FRK2 FRK2
No Yes Yes Yes Yes
1300 54 220 131 201
Sugar Beet (Beta vulgaris)
Taproot
FK
FRK2
Yes
68
Potato (Solanum tuberosum)
See Ref. Tuber Tuber Tuber See Ref. See Ref. See Ref. See Ref.
FRK FK1 FK2 FK3 FRK FK1 FK2 FK3
FRK2 FRK2 FRK2 FRK1 FRK2 FRK2 FRK2 FRK1
ND Yes Yes No Yes Yes Yes Yes
ND 41 116 128 1140 64 90 100
Soybean (Glycine max)
Nodule
FRK
FRK1
Yes
77
Maize (Zea mays)
Seed Seed Seed Leaf
FRK0 FRK1 FRK2 FRK1
FRK2 FRK2 FRK1 FRK2
ND Yes Yes ND
ND 148 121 ND
ND 59.0d 59.0d ND
6 6 6 22
Barley (Hordeum vulgare)
Leaf Leaf Leaf
FRK1a FRK1b FRK2
FRK2 FRK2 FRK1
Yes Yes Yesb
200 100 180
37.0d 37.0d 73.0d
7 7 7
Avocado (Persea americana)
Mesocarp Mesocarp
FRK1 FRK2
FRK2 FRK2
No No
140 100
85.0 3d 85.0 3d
14 14
Honey Locust (Gleditsia triacanthos)
Cotyledon
FRK
FRK1
Yes
190
ND
60
Spinach (Spinacia oleracea)
Leaf Leaf
FK1 FK2
FRK2 FRK1
ND ND
85 140
ND ND
22 22
Lily (Lilium longiflorum and L. lancifolium)
Pollen
FRK
FRK2
Yes
180/400
ND
61
Camellia (Camellia japonica)
Pollen
FRK
FRK2
Yes
300
ND
61
Pea (Pisum sativum)
Seed Seed Leaf
FRK FRK FRK1
FRK2 FRK1 FRK2
Yes Yesb ND
ND 57 ND
79.0 2e/72.0 4d ND ND
17 24 22
34.4c 36.0e/70.0df 36.0e/70.0df 36.0e/70.0df ND 102.0d 105.0d 118.0d 84.0 5d
20
a
ND not determined. Only slight substrate inhibition found. Calculated. d Native. e Determined by SDS-PAGE. f It is unclear to which of the three potato FRKs these values refers to. g J.V. Pego and S.C.M Smeekens, unpublished. b c
(SDS-PAGE) analysis showed a FRK2 protein of 35 kDa in developing tomato fruit10, one of 36 kDa in potato tubers11 and one of 37 kDa in sugar beet taproots12. This is in close agreement with values later obtained based on deduced amino acid sequences in 532
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tomato8, sugar beet12 and potato11. Gel filtration assays in tomato, potato, barley, soybean, avocado and sugar beet revealed a native molecular weight of 70–105 kDa (Refs 7,13,14), showing that, in these species, the protein is present as a dimer. A native molecular
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trends in plant science Reviews weight of 59 kDa has been reported15 but a mixture of the monomeric and dimeric forms of the enzyme might have caused FRK2 Tomato this. A similar case has been reported for Lactococcus lactis FRK, FRK2 Potato in which a 44 kDa value was obtained by gel filtration, proving later to be a 80:20% mixture of the 66 kDa dimer and the 33 kDa FRK2 Arabidopsis monomer16. Although most plant FRKs are present as dimers, pea FRK2 Sugar beet seed and barley leaf FRK2 might be present in the monomeric form7,17. The pea isoform seems to be an exception, giving a molFRK3 Arabidopsis ecular weight of 72 kDa on SDS-PAGE (Ref. 17), approximately twice the size of all other plant FRKs. The general picture for FRK1 Arabidopsis FRK1 is similar to that of FRK2. The native molecular weight of FRK1 Tomato FRK1 is reported to be 118 kDa or 70 kDa in potato tubers5,18, 7 19 73 kDa in barley leaves and 84 kDa in soybean nodules . The calculated weight for the tomato enzyme is 37 kDa (Ref. 8), sug300 200 100 0 gesting that FRK1, like FRK2, is dimeric. Trends in Plant Science No. of substitution events The pH optima for both FRK1 and FRK2 are reported to be Fig. 2. Phylogenetic tree of the deduced amino acid sequences of around pH 8 (Refs 10,12), and the Km for fructose is between seven plant fructokinases using the J. Hein method62 with PAM250 41 M and 220 M (Table 1). Two isolated reports of a higher Km residue weight table. of ~1.2 mM (Refs 9,20) were obtained from yeast cell extracts expressing a plant FRK and might reflect unphysiological modifications. FRKs are widely reported to have a higher affinity for FRK1 Arabidopsis 74 ATP than for other nucleotides and, unless 78 FRK1 Tomato they are in a region or cellular compartment 60 FRK2 Arabidopsis with high GTP or UTP concentrations, ATP FRK2 Tomato 62 FRK2 Sugar beet 64 will be the principal substrate for fructose FRK2 Potato 63 phosphorylation in vivo12,13. Mg2 is necesFRK3 Arabidopsis 57 sary for FRK activity2,13 and, in some cases, A1 activity can be further stimulated by the 182 addition of K (Ref. 12). Several FRK 186 purification studies show that most FRKs 168 are associated with the cytosol fraction and 170 the enzyme is therefore presumably also 172 171 cytosolic21. In spinach, organelle fractiona165 tion by sucrose-gradient centrifugation or differential centrifugation suggested that, B although FRK2 was presumably present in 210 214 the cytosol, FRK1 was associated with the 196 chloroplast22. 198 An interesting feature of plant FRKs is 200 their reported fructose substrate inhibition 191 193 in vitro (Table 1). At approximately pH 8, FRK2 is highly sensitive to inhibition by B fructose, with Ki values of 1–6 mM in 219 254 potato, tomato, pea and maize2,10,15. Sub223 258 205 238 strate inhibition has also been reported for 207 240 FRK2 at pH 6.6 (Ref. 23) but an earlier 209 242 report from the same group24 showed FRK1 199 231 inhibition at pH 8 but not at pH 6.6. Barley 202 235 FRK1 displayed only a slight substrate inhiB A2 bition at 20 mM fructose7 and, in tomato, 288 FRK1 is reported not to be inhibited by up 292 to 50 mM fructose9. Results obtained from 272 potato tuber FRK1 are conflicting because 274 276 both a Ki of 21 mM and the absence of inhi265 bition (up to 16 mM fructose) have been 269 2,18 reported . The picture that emerges is that, A3 Trends in Plant Science although fructose is a potent inhibitor of FRK2, it seems to be less effective against Fig. 3. Alignment of conserved regions of the deduced amino acid sequences of seven plant FRK1. fructokinases. This shows three signature patterns for the pfkB family of carbohydrate kinases (A1, A2, A3) and a region specific to fructokinases (B). The A1 motif is involved in FRK2 has been suggested to play a role ATP binding and the B motif contains two sugar-binding domains. Black boxes indicate in starch production in sink tissues25, where identical residues between proteins. SUSY cleaves incoming sucrose into UDP–glucose and fructose (Fig. 1). SUSY, December 2000, Vol. 5, No. 12
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Hepatocyte nucleus
GAL1/GAL3 GAL1/GAL3 Galactose
GAL4
HXK
GAL80 GAL4
F1P
mRNA Trends in Plant Science
Fig. 4. Representation of the galactose-mediated pathway for regulation of gene expression in yeast. In the presence of galactose, a complex is formed consisting of GAL4, GAL80 and one of the proteins GAL3 or GAL1 (galactokinase). This complex activates gene transcription. When galactose is absent, the GAL80–GAL1 (or GAL80–GAL3) interaction is disrupted and GAL80 inhibits GAL4 transcription activity.
like FRK, is also inhibited by fructose. In tomato fruit, the Ki for fructose inhibition of FRK and SUSY are 2 mM and 2.4 mM, respectively, well below the concentration of ~40 mM fructose found in these tissues25. SUSY is also associated with other sinkspecific processes such as callose and cellulose synthesis26. Because developmental patterns of FRK and SUSY activity are expressed in a coordinated fashion in several plant species25, the two enzymes might, in concert, control sucrose use owing to elevated fructose. High SUSY activity, generating elevated fructose levels, would lead to the inhibition of both SUSY and FRK in what could be described as a ‘double brake’ mechanism. However, it remains unclear why such a mechanism would be necessary. Plant genes for FRKs and their expression
A few plant FRK genes have now been cloned, with one to three genes isolated per species. FRK2 was isolated from potato tubers27 and sugar beet taproots12, FRK1 and FRK2 from tomato fruit, and, recently, FRK1, FRK2 and FRK3 from Arabidopsis (J.V. Pego and S.C.M. Smeekens, unpublished). The isolation of FRK3 in Arabidopsis is significant as it marks the first time that three FRK genes have been found in any one organism. However, it is still uncertain whether the FRK3 gene is transcribed. The FRK3 protein would represent a novel higher-plant FRK (Fig. 2). For bacteria, 19 FRK genes from 18 different species have been reported. Alignment of the deduced amino acid sequences revealed significant homology among all seven plant FRK proteins. The three signatures of the FRK-containing pfkB family of carbohydrate kinases and a large FRK-specific domain are highly conserved in these proteins. These regions are involved in ATP and sugar binding28 (Fig. 3). The calculated molecular weights are also similar, varying from 34.4–35.4 kDa for FRK2 and FRK3, and ~37 kDa for FRK1. The higher molecular weight of FRK1 is mainly because of a longer N-terminal region present in tomato and Arabidopsis FRK1, which is absent in FRK2 and FRK3. An additional feature of all seven proteins is the low degree of homology at their C-termini. The only gene expression data available were obtained in tomato. It was recently reported that, although FRK1 is constitutively expressed throughout the entire tomato plant, FRK2 seems to be sink specific and present in source leaves only at low levels8,9. In accordance with this, potato FRK1 activity was high in leaves and low in tubers, whereas FRK2 accounted for 95% of FRK activity in tubers but was barely detectable in leaves5. These 534
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P F6
GAL80
GCKR
Cytoplasm
FRK
GCKR
Fru
Signal HXK Trends in Plant Science
Fig. 5. Sugar sensing in mammalian hepatocytes. In the presence of fructose-6-phosphate (F6P), the hexokinase IV (HXK) regulatory protein GCKR, represses HXK activity by recruiting it to the hepatocyte nuclear matrix. The binding by GCKR of fructose-1phosphate (F1P), the product of the reaction catalyzed by fructokinase (FRK), disrupts the HXK–GCKR interaction and allows HXK-mediated signaling. FRK is proposed to modulate HXK activity indirectly by controlling the F6P:F1P ratio. Abbreviation: Fru, fructose.
results were supported by studies in sugar beet taproots, where FRK2 was the only FRK found12. The situation in Arabidopsis is similar to that of potato, tomato and sugar beet because FRK2 accounts for the majority of root FRK activity and only a small fraction of activity in the shoots (A. Krapp and M. Stitt, pers. commun.). Taken together these results seem to point to a sinkspecific function for FRK2, whereas FRK1 is thought to be primarily source specific. In spite of their distinct gene expression patterns, transcription of both FRK1 and FRK2 is induced by the exogenous application of glucose, fructose or sucrose in tomato plants9. Induction was rapid (1.7 h) after application of a low concentration (4 mM) of glucose. Maximal mRNA levels were obtained at 20 mM glucose, a concentration of sugar shown to be physiologically relevant25. This is the first evidence that the FRK gene belongs to the growing class of sugar-regulated genes. Hexose kinases as sugar sensors
FRKs are members of the larger functional family of hexose kinases. These enzymes, capable of converting hexoses (e.g. glucose, fructose, mannose) to hexose phosphates, have long been recognized as essential components of metabolism. The best known family member, HXK, catalyzes the first step in glycolysis and is a key regulator of this pathway at the enzyme level. An additional, more global, regulatory role was discovered when it was linked to the process of sugar-mediated regulation of gene expression. Sugars are now known to affect the expression of an impressive number of genes in yeast29,30, mammals31,32 and plants33,34. Here, sugar-mediated regulation of gene expression seems to be a central and basic process that is probably common to all higher plants33,35,36. In Saccharomyces cerevisiae, in which the underlying mechanism is best understood, HXK acts in the signaling cascade as a sugar sensor that can relay the initial signal to downstream cascade components37,38. Although it is questioned39, a significant body of evidence points to an analogous function for HXKs in both plants35,40 and mammals41,42, suggesting that the sugar-sensing
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trends in plant science Reviews mechanism might be partly conserved in these systems. In plants, the signaling cascade(s) itself is understood poorly 36, and both the component directly downstream of HXK and the mechanism by which HXK transmits the sugar signal remains unknown. Separation of phosphorylating and signaling functions in yeast HXK mutants has contributed to the understanding of how HXK operates as a sensor43,44 and a similar approach would be of value in plants. Yeast galactokinase represents a second class of bifunctional hexose kinases. Not only is it involved in galactose metabolism but it is also, like HXK, the sensor in a sugar-signaling cascade45. The triggering of this cascade by galactokinase leads to the activation of the GAL genes, encoding the enzymes necessary for further galactose metabolism. In S. cerevisiae, transmission of the galactose signal requires either GAL1 (galactokinase) or GAL3, a GAL1 homolog lacking galactokinase activity46. GAL4, a transcription activator and the key regulator of galactose metabolism in yeast47, binds upstream of the GAL genes. Its transcriptional activity is controlled by a negative regulator, GAL80 (Ref. 48), which inhibits GAL4 activity in the absence of galactose49 (Fig. 4). When both galactose and ATP are available, galactokinase binds to GAL80, relieving its inhibitory effect on GAL4 (Ref. 50) and transcription of the GAL genes is thereby activated. In spite of the functional similarities between the galactokinase-dependent and HXK-dependent mechanisms described above, the signal transduction pathways involved are clearly distinct. However, in yeast, the two pathways interact because the presence of glucose triggers the HXK-dependent signaling pathway, ultimately inhibiting expression of the GAL1 (galactokinase) and GAL4 genes51. By doing so, glucose is established as the preferred substrate over galactose. Galactokinase activity in plants has been demonstrated in purified extracts from several species52. An Arabidopsis galactokinase gene was isolated by functional expression in yeast53 and, recently, a second member of the Arabidopsis galactokinase gene family (ARA1) was identified54. Although the possible involvement of plant galactokinases in sugar-mediated regulation of gene expression has not yet been addressed, the cloning of these two genes should facilitate the isolation of galactokinase knockout mutants. Analysis of such mutants should provide valuable insight into the putative galactokinase sugar-sensing role in plants. FRKs and sugar sensing
Given the sugar-sensing capability of HXK and galactokinase, it is tempting to speculate that this feature is shared by other hexose kinases. This suggestion is supported by the recent isolation of sugar-insensitive mig mutants in Arabidopsis34. In one of these mutants, a specific FRK activity was found to be completely absent. Further analysis revealed a null mutation in the FRK2 gene and the mutant was therefore renamed frk2; tests are under way to determine whether this mutation is responsible for the mig phenotype. We have since cloned two additional FRKs in Arabidopsis and work is in progress to isolate mutants in these genes. The question of whether sugar sensing is a general feature of hexose kinases is currently being addressed, for FRK, in the frk2 mutant. FRK has previously been associated with the process of sugarmediated gene regulation in mammals55. Pancreatic -cell HXKIV, which is highly similar to yeast HXK (Ref. 56), functions as a glucose sensor that regulates insulin secretion and gene activation. It thus plays an important role in regulating glucose homeostasis. Mutations in the HXKIV gene or in GCKR, the gene encoding the HXKIV regulatory protein, are linked to matureonset diabetes of the young, a form of non-insulin-dependent diabetes57. Mammalian FRK plays a regulatory role in this system. GCKR is bound to the hepatocyte nuclear matrix and represses
HXKIV activity by binding it (Fig. 5). This interaction is relieved by binding of fructose-1-phosphate, the product of the FRKcatalyzed reaction, to GCKR (Refs 58,59). FRK activity is therefore proposed to modulate the glucose sensor (HXKIV) activity in the islet cell. Hepatic FRK deficiency does not impair glucose tolerance but the molecular relationship between islet-cell and hepatic FRK is unknown. In plants, several putative sugar-sensing screens have been devised and many Arabidopsis mutants isolated34. However, recent findings have shown that gin1 and sun6 are mutated in interacting hormone pathway components36. This shows that more specific screening strategies are needed that can discriminate between the primary sugar-signaling cascades and secondary, interacting pathways. These interactions can be further dissected with well characterized mutants in enhancer and suppressor screens to identify pathway-specific mutations. The integration of data obtained from the different mutant screens is beginning to provide insight into the underlying plant sugar-sensing and -signaling mechanism(s), but many issues remain. For example, does FRK act as sugar sensor in plants or does it control sensing, such as in the mammalian system? The available frk2 mutant, and other frk mutants that will be identified, should be valuable tools in addressing these and other questions. Acknowledgements
Our work was financially supported by the Fundação para a Ciência e Tecnologia, Lisbon, Portugal (grant PRAXIS XXI/BD/3103/94 awarded to J.V.P.). References 1 Rangaswamy, V. and Altekar, W. (1994) Ketohexokinase (ATP:D-fructose 1phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties. J. Bacteriol. 176, 5505–5512 2 Renz, A. and Stitt, M. (1993) Substrate specificity and product inhibition of different forms of fructokinases and hexokinases developing potato tubers. Planta 190, 166–175 3 Medina, A. and Sols, A. (1956) A specific fructokinase in peas. Biochim. Biophys. Acta 19, 378–379 4 Frankart, W.A. and Pontis, H.G. (1976) Fructose metabolism in plants. Isolation and properties of pea seed fructokinase. Acta Physiol. Latinoamericana. 26, 319–329 5 Renz, A. et al. (1993) Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity. Planta 190, 156–165 6 Doehlert, D.C. (1990) Fructokinases from developing maize kernels differ in their specificity for nucleoside triphosphates. Plant Physiol. 93, 353–355 7 Baysdorfer, C. et al. (1989) Partial purification and characterization of fructokinase activity from barley leaves. J. Plant Physiol. 134, 156–161 8 Kanayama, Y. et al. (1997) Divergent fructokinase genes are differentially expressed in tomato. Plant Physiol. 113, 1379–1384 9 Kanayama, Y. et al. (1998) Tomato fructokinases exhibit differential expression and substrate regulation. Plant Physiol. 117, 85–90 10 Martinez-Barajas, E. and Randall, D.D. (1996) Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits. Planta 199, 451–458 11 Taylor, M.A. et al. (1995) Characterisation of a cDNA encoding fructokinase from potato (Solanum tuberosum L.). J. Plant Physiol. 145, 253–256 12 Chaubron, F. et al. (1995) Partial purification and characterization of fructokinase from developing taproots of sugar beet (Beta vulgaris). Plant Sci. 110, 181–186 13 Martinez-Barajas, E. et al. (1997) Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli. Protein Expres. Purif. 11, 41–46 14 Copeland, L. and Tanner, G.J. (1988) Hexose kinases of avocado. Physiol. Plant. 74, 531–536
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Jónatas V. Pego is at the Dept of Biology, Minho University, Gualtar campus, 4710-057 Braga, Portugal; Sjef C.M. Smeekens* is at Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. *Author for correspondence (tel 31 30 2533431; fax 31 30 2513655; e-mail [email protected]).