Journal of Experimental Botany, Vol. 52, No. 359, pp. 1191-1201,
June 1, 2001
© 2001 Oxford University Press
Original Papers |
Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, RJ, Brasil
Received 20 June 2000; Accepted 8 February 2001
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Abstract |
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Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (Ki=30 µM) and mannoheptulose (Ki
300 µM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The Ki for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases. Key words: Non-cytosolic hexokinase, fructokinase, hexose-phosphorylation, glucose analogues, Zea mays L.
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Introduction |
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Hexokinase [EC 2.7.1.1] catalyses the ATP-dependent conversion of hexoses to hexose-6-phosphate and ADP. Several hexokinases with tissue specificity or developmentally regulated expression have been described in plants (Renz et al., 1993
; Galina et al., 1995). Different genes encoding hexokinases (Dai et al., 1995; Jang et al., 1997) and a fructokinase (Smith et al., 1993) have been cloned. From the viewpoint of metabolic flux, the hexokinase reaction plays a key role in the removal of free hexoses from the cytosol, channelling the carbons to either glycolysis or biosynthesis, depending on cellular demands.
In previous studies, two hexokinases were distinguished by different sensitivities to ADP and different subcellular locations in maize roots (Galina et al., 1995, 1999). The mitochondria-bound hexokinase has a higher affinity for glucose than for fructose and is strongly inhibited by ADP (Ki=20–40 µM). The inhibition is non-competitive with respect to both ATP and glucose. High cytoplasmic ADP concentrations (
200 µM) are found in maize roots during hypoxia (Hooks et al., 1994), indicating a low mitochondrial hexokinase activity under this condition. In contrast, the soluble hexokinase is not inhibited by ADP concentrations up to 1 mM (Galina et al., 1995), a kinetic property that is compatible with its glycolytic role. Recently, using [U-14C]-glucose as an isotopic tracer, it was found that non-cytosolic hexokinase forms the precursors that are essential for conversion of glucose to NDP-5'-sugar in maize root homogenates (Galina and da-Silva, 2000). A surprising finding was that ADP and mannoheptulose block NDP-5'-sugar formation, although they do not impair the formation of cytosolic glucose-6-phosphate. These results indicate that the non-cytosolic hexokinase is not engaged in glycolysis. Mannoheptulose, a specific hexokinase inhibitor, has little effect on the cytosolic glucose phosphorylation (Galina and da-Silva, 2000).
Several sugar hexokinase inhibitors such as mannoheptulose, N-acetylglucosamine and glucosamine have been widely used as experimental tools, for example, to determine catalytic site structure of glucokinases and hexokinases or to inhibit carbohydrate metabolism in many cell types (Jang and Sheen, 1994; Xu et al., 1995; Board et al., 1995; Malaisse, 1998). In plants these investigations are scarce, in part due to a small number of isolated hexokinase isoforms. Only in the past few years has the existence of sugar-regulated gene expression in plants become apparent, based on a molecular genetic approach (Dai et al., 1999; Graham et al., 1994; Jang et al., 1997; Koch, 1996). Of crucial importance in these studies was the observation that specific hexokinase inhibitors, such as mannoheptulose and glucosamine, block glucose repression in plant cells (Jang and Sheen, 1994; Umemura et al., 1998).
Although hexokinase is commonly known as a glycolytic enzyme, it has been implicated as a glucose sensor that mediates the repression of genes involved in photosynthesis, the glyoxylate cycle and synthesis of 
-amylase (Jang and Sheen, 1994; Graham et al., 1994; Umemura et al., 1998). This property may have potential interest as a target for manipulation and modification of hexokinases for crop improvement (Dunwell, 2000). As pointed out by Taylor (Taylor, 1997; see also Umemura et al., 1998), knowledge of the subcellular distribution of hexokinases in plants may help to elucidate the components in the signal transduction pathways triggered by hexokinase phosphorylation of fructose and glucose.
The properties of ADP regulation are different between soluble and particulate hexokinase fractions (Galina et al., 1995). The aim of this study was to investigate the localization of hexokinases by differential centrifugation and to correlate their subcellular distribution with their sensitivity to glucose analogue inhibitors and their steady-state kinetic properties. All hexokinase activities measured in 10000 and 100000 g pellet fractions are defined as non-cytosolic hexokinases (NC-HK) in order to distinguish them from the cytosolic soluble fractions FK1, FK2 and HK1. Most of the NC-HK activity is bound to mitochondria (Galina et al., 1995, 1999), and resides in submitochondrial particle membranes (SMP). Although, in pea roots, it was found that 16% of HK activity was associated with plastids (Borchert et al., 1993), in maize roots, this activity corresponds to only 5% of the total, as estimated by the plastidic markers ADP-Glc pyrophosphorylase and triose phosphate isomerase (Galina et al., 1995; Galina and da-Silva, 2000). Non-cytosolic hexokinase activity is also not detectable in amyloplast preparations derived from developing corn endosperm (Echeverria et al., 1988). The results of this study show that ADP and hexokinase inhibitors preferentially affect the phosphorylation of fructose and glucose in mitochondria and Golgi-vesicle preparations, with little or no effect on cytosolic hexokinase activities.
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Materials and methods |
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Preparation of plant material and isolation of cell fractions
Maize seeds (Zea mays L.) were surface-sterilized with sodium hypochlorite (
10 min in a 3% solution) and then washed with sterile water. Radicles were harvested from seeds allowed to germinate for 3–4 d on wet filter paper in the dark at 28 °C. Root homogenates and submitochondrial particles were obtained as previously described (Galina et al., 1995) using a cold extraction buffer (buffer A) containing: 5 mM HEPES/TRIS pH 7.4, 0.3 M sucrose, 7 mM cysteine, 1 mM EGTA, and 1 mM PMSF. The crude mitochondrial pellet was obtained from the first centrifugation (10000 g for 12 min). The supernatant of the first 10000 g centrifugation (cytosolic supernatant) was used for cytosolic HK assays. The cytosolic fraction was then centrifuged for 100000 g for 50 min, the pellet (microsomes) was stored and the supernatant (soluble proteins) was used to obtain soluble FK and HK.
Discontinuous sucrose gradient
Washed mitochondria (1.2 ml samples; 50–60 mg of protein) were resuspended in buffer A, added to a discontinuous sucrose gradient and centrifuged in a swinging bucket rotor (Beckman SW 27) for 45 min at 40 000 g (Douce et al., 1972). The gradients were prepared by layering sucrose solutions into a centrifuge tube in the following sequence: 1.5 M (2 ml), 1.2 M (2 ml), 0.9 M (2 ml), and 0.6 M (2 ml). Following centrifugation six main bands were obtained: B1 ( 2 ml of 0.6 M), B2 (interface of 0.6 and 0.9 M), B3 ( 2 ml of 0.9 M), B4 (interface of 0.9 and 1.2 M), B5 ( 2 ml of 1.2 M) and B6 (interface of 1.2 and 1.5 M). The sucrose gradient separates the Golgi vesicles and mitochondria. For some experiments, Golgi vesicles (bands B1 to B2) were further diluted to 0.5 M sucrose and purified by flotation centrifugation as described earlier (Morré, 1971).
Partial purification of cytosolic fructokinases and hexokinase
The soluble proteins were fractionated by the addition of solid (NH4)2SO4. The precipitate formed at 50% salt saturation (31.3 g added to 100 ml of final volume) was centrifuged (30 000 g, 15 min) and discarded. The salt saturation of the supernatant was raised to 60% (adding 6.6 g to 100 ml) and the precipitate was centrifuged and dissolved in 3 ml buffer B (like buffer A, but without sucrose and with 20 mM TRIS-HCl instead of HEPES/TRIS). The supernatant concentration was raised to 70% salt saturation (adding 6.9 g to 100 ml) and the precipitate was centrifuged and dissolved in 3 ml buffer B. After (NH4)2SO4 precipitation each fraction (60% and 70% saturation) was separated from contaminating proteins with a DEAE-Toyopearl column (21.6x1.2 cm). With 1 mM fructose as substrate, only one peak of activity was detected in the 60% fraction (FK1) and there was no glucose phosphorylation activity present. The 70% fraction had both fructose and glucose phosphorylation activities (FK2, HK1). The proteins bound to the column were eluted with a linear salt gradient from 0 to 0.4 M NaCl. An attempt was made to further separate the glucose and fructose activities by affinity chromatography (Renz et al., 1993) to determine if they were due to one or two enzymes. The method involved a Cibacron-Blue 3GA (Sigma) column (4x1.5 cm) and elution with 0–3 mM ATP. The three enzymes were characterized kinetically.
Assay of hexokinase activity
Each fraction was assayed for hexokinase activity in a medium containing 20 mM TRIS-HCl pH 7.5, 6 mM MgCl2, 1 mM ATP, 2 mM phosphoenolpyruvate (PEP), 10 units ml-1 pyruvate kinase, 0.1% (v/v) Triton X-100, 5 mM NaN3, 5 mM NaF, 0.1 mM P1,P5-diadenosine-5'-pentaphosphate, and the appropriate glucose or fructose concentrations. When ADP was studied, the pyruvate kinase was omitted from the medium. Assays were initiated with ATP or by adding protein fractions (20–120 µg protein ml-1) and were quenched after 5–10 min at 30 °C by heating (1 min at 100 °C). The glucose-6-phosphate formed was measured by adding an equal volume of a solution containing 20 mM TRIS-HCl pH 7.5, 6 mM MgCl2, 0.7 units ml-1 glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroids) (Sigma Chemical Co.) and 0.3 mM ß-NAD+. For fructose-6-phosphate determination 3.5 units ml-1 phosphoglucoseisomerase were included in the assay medium. In continuous spectrophotometric assays the hexose-6-phosphate production was coupled with glucose-6-phosphate dehydrogenase and NAD reduction, by measuring the increase in A340. In all cases, activities were linear with the amount of protein added. Kinetic constants were calculated from Woolf–Augustinsson–Hofstee (v versus v/[S]) replots of kinetic data and by non-linear regression analysis applied to the Michaelis–Menten or Hill equations using the program ENZFITTER. Ki values were calculated from Dixon (1/v versus [I]) plots.
ADP quantification in homogenates and cytosolic fractions
In the experiments where the ADP inhibition was tested during hexokinase activity assay, the ADP concentration was checked in order to evaluate the degree of its consumption by potential side-reactions in the homogenate and cytosolic fractions. An aliquot of 150 µl was removed and boiled from the reaction mixture at the beginning and at the end of the assay. After centrifugation at 5000 g for 15 min, 100 µl of the supernatant was removed and the ADP was quantified by the addition of 400 µl of a medium containing: 20 mM TRIS-HCl pH 7.5, 6 mM MgCl2, 2 mM PEP, 10 units ml-1 lactate dehydrogenase, 7 units ml-1 pyruvate kinase (rabbit muscle) (Sigma Chemical Co.), and 0.5 mM ß-NADH. The decrease in the absorbance at 340 nm was proportional to the amount of ADP.
Other enzyme assays
The UDPase activity, a Golgi marker enzyme (Widell and Larsson, 1990), was measured as previously described (Nagahashi and Kane, 1982). Membranes (10–100 µl) were added to 0.9 ml of assay buffer containing 30 mM MOPS-TRIS, pH 6.5, 1 mM UDP and 2 mM MnSO4. Duplicate tubes with 0.02% (v/v) Triton X-100 were prepared. The reaction was incubated at 35 °C for 20 min. Released Pi was determined according to Fiske and Subbarow (Fiske and Subbarow, 1925). Triton-activated UDPase activity, referred to as latent UDPase activity, increased linearly with time. The mitochondrial ATPase activity was determined by measuring the release of Pi from ATP in the absence and presence of 5 mM NaN3 and 2 µg ml-1 oligomycin in a reaction medium containing 50 mM TRIS-HCl (pH 8.0), 10 mM MgCl2, 1 mM ATP, and 5 µM FCCP. The difference between these activities is referred to as the azide-sensitive ATPase activity and is related to the maize FoF1ATPase complex (Galina et al., 1995).
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Partial isolation of cytosolic fructokinases and hexokinase
The 70% (NH4)2SO4 saturation fraction that was obtained from soluble proteins was resolved by ion exchange chromotography (Fig. 1
). One peak, that was able to phosphorylate fructose but not glucose, was detected in the unbound protein fractions of the column (FK2). Another peak of activity was detected between 0.17 and 0.34 M of the NaCl gradient. This peak of activity phosphorylated both glucose and fructose (HK1). The affinity chromatography procedure was unable to separate these activities further so it is concluded that the activity resulted from a single enzyme.
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Localization of hexokinases in subcellular fractions of maize roots
Inhibition of the hexokinase activity by ADP and mannoheptulose was tested in two major fractions obtained after homogenate centrifugation (Fig. 2). Mannoheptulose (30 mM) and ADP (250 µM) inhibited the activity by 50% in crude homogenates (Fig. 2A). However, with the cytosolic fraction, ADP had no effect and mannoheptulose caused a small inhibition (about 20%) (Fig. 2B). The ADP concentration was not significantly decreased by side-reactions in the homogenate and cytosolic fractions. The ADP measured at the final of reaction time was 266±3 µM (n=3). An almost total inhibition was observed with either ADP or mannoheptulose in the pellet fraction obtained after a 10000 g centrifugation (Fig. 2C). The cytosolic supernatant (100000 g) (Fig. 2B) and pellet (10000 g) (Fig. 2C) fractions were fractionated further in Fig. 3.
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); 30 mM mannoheptulose–MH (•) or 250 µM ADP without pyruvate kinase (
). The reaction was started by the addition of 1 mM ATP (
). The final protein concentration was: In (A) total homogenate, 0.12 mg ml-1; (B) soluble fraction (100 000 g supernatant), 0.1 mg ml-1; and (C) pellet fraction (10 000 g), 0.05 mg ml-1.
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The total soluble glucose and fructose phosphorylating activities obtained after centrifugation at 100000 g (S100, Fig. 3A
) were fractionated sequentially by addition of (NH4)2SO4. It became apparent that, when 0.8 mM glucose or fructose was the substrate in the assays, more fructose than glucose was phosphorylated (70% of the total), especially when the (NH4)2SO4 saturation was between 50% and 70% (Fig. 3A, black bars). The glucose phosphorylated was about one-half of the fructose phosphorylated. Although the soluble activity measured with glucose was present in all (NH4)2SO4 fractions, it was more evident between 60% and 80% of (NH4)2SO4 saturation (Fig. 3A, grey bars). This result suggests the presence of distinct hexokinase and fructokinase activities in the cytosolic compartment.
When the pellet (10000 g) fraction (Fig. 2C) was further separated on a sucrose gradient, it revealed non-cytosolic hexokinase (NC-HK) activity in distinct organelles (Fig. 3B). Five major bands from the sucrose gradient were assayed. The Golgi marker (UDPase) was concentrated in bands 1 and 2, while mitochondrial marker (FoF1ATPase) predominated in bands 4, 5, and 6. The NC-HK activity appeared to be associated with both mitochondria and Golgi membranes. In addition to a large fraction of total NC-HK activity detected in band 6, where FoF1ATPase predominates, Golgi vesicles isolated from lighter bands by flotation centrifugation as described previously (Morré, 1971) also exhibited high specific activity of hexokinase, totally inhibited by mannoheptulose and ADP (data not shown). The hexokinase specific activities for different non-cytosolic fractions (mean ±s.e.) were: microsomes 0.014±0.006 (n=4), crude mitochondria 0.082±0.023 (n=7), washed mitochondria 0.18±0.05 (n=5), Golgi vesicles (Morré, 1971) 0.53±0.14 (n=3), and SMP preparations 1.31±0.32 (n=3) µmol min-1 mg-1. With this method of preparing Golgi vesicles, mitochondrial contamination is reduced to 13%.
HK inhibitors impair phosphorylation of hexoses by non-cytosolic hexokinase
The activities of soluble cytosolic fructokinases and hexokinases that had been partially purified from the soluble fraction (Fig. 2B), were compared with that of the NC-HK fraction (Fig. 4). Neither mannoheptulose nor N-acetylglucosamine inhibited the cytosolic FK1 activity, even at a low fructose concentration (0.2 mM). The ability of mannoheptulose to inhibit phosphorylation of glucose by the cytosolic HK1 fraction was also very low: in the presence of 0.8 mM glucose the activity was reduced by 30% with 30 mM mannoheptulose (Fig. 5B). These results indicate that neither of the cytosolic hexokinases is greatly affected by a wide range of inhibitor concentrations. Similar results were observed with mannoheptulose using cytosolic glucokinase purified from young tomato fruit (Martinez-Barajas and Randall, 1998).
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, 0.1 mg ml-1), FK2 (
, 0.12 mg ml-1), HK1 (•, 0.12 mg ml-1) or NC-HK (
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A significant inhibition occurred when the inhibitors were added to assays containing the NC-HK fraction associated with SMPs (open symbols in Figs 4, 5A
). The inhibition was observed with either fructose or glucose as substrate. The inhibition was practically abolished when the glucose concentration was raised from 0.8 mM to 20 mM, indicating a competitive inhibition (Fig. 5A). An increase in fructose concentration from 30 to 200 mM did not totally overcome the mannoheptulose inhibition, either with SMP or Golgi-vesicle preparations (data not shown). In agreement with previous data (Galina et al., 1995), the NC-HK was effectively inhibited by ADP when the substrate was glucose (Fig. 4C, open circles). However, when fructose was the substrate, the ADP inhibition is much lower (Fig. 4C, open triangles) (Galina et al., 1999). A small ADP inhibition was detected with FK1, FK2 and HK1 at higher ADP concentrations (Fig. 4C).
Kinetic properties of cytosolic and non-cytosolic hexokinases
The kinetic constants for the nucleotide-5'-triphosphate and hexose substrates of FK1, FK2, HK1, and NC-HK are shown in Table 1. For FK1 and FK2, the Vmax values were practically the same for all NTP's. However, the NC-HK showed a marked preference for ATP, with a Vmax ranging from 2.4–5.4-fold higher with ATP than other NTPs. Thus, although all hexokinases analysed in this study utilized ATP most efficiently as a phosphoryl donor, based on the Vmax/Km ratio (Table 1, last column), the difference between ATP and the other NTPs was much greater for NC-HK. This result suggests that cytosolic fructokinases may utilize other NTPs fairly readily.
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A comparison of the kinetic constants for glucose and fructose revealed that FK1 uses only fructose as substrate (Table 1
; Fig. 6A). In addition, FK1 has a strong positive co-operativity with respect to fructose for fructose phosphorylation, with a Hill coefficient for fructose greater than 2 (Fig. 6A, closed triangles). Despite the similarity of kinetic constants for phosphorylation of fructose by FK1 and FK2, the co-operative behaviour was not observed for FK2 (Fig. 6A, open triangles). The Vmax of HK1 was 1.7 times higher with fructose (Fig. 6B, open circles) than that with glucose as substrate. Since the affinity of HK1 for both hexoses was practically the same, the HK1 has a catalytic efficiency higher when using fructose as substrate (Table 1). In contrast, NC-HK exhibited a catalytic efficiency for glucose that was 84-fold higher than for fructose as substrate (Fig. 6C; Table 1).
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The inhibitor constants (Ki) for different inhibitors of the soluble and membrane-bound hexokinases are shown in Table 2
. As described for other plant fructokinases (Doehlert, 1990; Renz et al., 1993), FK1 and FK2 exhibited substrate inhibition when the fructose concentration exceeded 1 mM (Fig. 6A; Table 2). FK1 was 10-fold more sensitive to inhibition by fructose than FK2. In contrast, the membrane-bound HK did not exhibit substrate inhibition (Fig. 6C), and was sensitive to the HK inhibitors mannoheptulose, N-acetylglucosamine, glucosamine, and ADP. The Ki values for NC-HK were lower when the sugar substrate was fructose (Table 2). This was not the case for ADP: the Ki value was 10-fold higher with fructose as substrate (Galina et al., 1999). The FK1, FK2 and HK1 activities were partially affected by 1 mM ADP (Table 2; Fig. 4C).
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Discussion |
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Localization of fructokinases and hexokinases in subcellular fractions of seedling maize roots
At least two soluble cytosolic fructokinases (FK1, FK2) were detected in seedling maize roots (Figs 1, 6). A large portion of the total fructose phosphorylation occurred by cytosolic fructokinase activity which could be inhibited by high fructose concentration, similar to the results of others (Doehlert, 1990
; Renz et al., 1993) (Fig. 6A). However, it was not possible to inhibit fructose phosphorylation in non-cytosolic membrane fractions with the same technique (Fig. 6C). The preferential cytosolic localization of the fructokinases is possibly involved in the phosphorylation of fructose derived from sucrose by either the invertase or sucrose-synthase enzymes. In contrast, the hexokinase activity in seedling maize roots is distributed between cytosolic and non-cytosolic fractions (Figs 1, 2, 3). Here it is shown that a substantial portion (30%) of the non-cytosolic activity is associated with the Golgi apparatus and other cellular membranes of maize roots (Figs 2, 3B). In mammalian tissues it has been demonstrated that Golgi-glucokinase facilitates the biosynthesis of UDP-glucose (Berthillier et al., 1973; Berthillier and Got, 1974; Iijima and Awazi, 1972). It has also been found that NC-HK is coupled to UDP-glucose formation in maize roots (Figs 4, 6C; Galina and da-Silva, 2000). It may be that NC-HK is involved in glycosylation reactions in the Golgi apparatus, and ADP plays a critical role modulating its activity here and on the mitochondria. The location of hexokinases in Golgi and mitochondrial membranes suggests an interaction between these organelles (Morré, 1964).
The kinetic constants of fructokinases and hexokinases
The kinetic constants revealed that all hexokinase isoforms tested phosphorylate their sugar substrates more readily with ATP than with other nucleotides. A very high catalytic efficiency with ATP was observed for NC-HK (Table 1). In maize roots, no FK with a high specificity for UTP was found, such as the FK-0 which was described in maize endosperm (Doehlert, 1990) and suggested to take part in the cycling of UTP during sucrose degradation by sucrose synthase and FK (Huber and Akazawa, 1986). However, it was shown in maize root tips (Zeng et al., 1999) that anoxia favours sucrose cleavage via sucrose synthase. It would be of interest to know whether the FK-0 is present in maize roots under anoxic conditions.
Substrate inhibition of the FK1 and FK2 fractions resembles that described for several plant FK sources. However, with fructose as substrate, FK1 exhibited a strong co-operativity (Hill n=2.5) and a much lower Ki for fructose than FK2 (Fig. 6A, filled triangles; Table 2). This is the first kinetic evidence that the fructose phosphorylation rate may be controlled allosterically by variations in cytosolic fructose. At low fructose levels, the co-operative activation of FK1 would increase the phosphorylation rate sharply in response to small increases in fructose. At fructose concentrations above 1.5 mM, inhibition of FK1 could be a mechanism to prevent trapping Pi and depleting ATP due to a high rate of fructose phosphorylation uncoupled from the demand for fructose carbons through glycolysis (Teusink et al., 1998).
The cytosolic HK1 seems not to be regulated by glucose and fructose levels (Fig. 6B). The HK1 may be involved in maintaining the cytosolic fructose phosphorylations at low rates, even in the presence of high cytosolic fructose levels (see below).
HK inhibitors impair phosphorylation of hexoses by non-cytosolic hexokinase
In plants, has been shown that it is not the uptake of hexoses into the cell, but rather their entry into metabolism through phosphorylation by hexokinase that triggers a broad spectrum of gene repression, a phenomena known as sugar-sensing (Graham et al., 1994; Jang and Sheen, 1994; Jang et al., 1997; Pego et al., 1999; Smeekens, 1998; Sheen et al., 1999). One important finding establishing the role of hexokinase as sensor was the fact that addition of competitive inhibitors of hexose binding such as mannoheptulose, N-acetylglucosamine or glucosamine, relieved the gene repressed by hexoses (Jang and Sheen, 1994; Yamaguchi et al., 1997; Pego et al., 1999). The results shown in this study give the first kinetic evidence that the sugar-sensing cascade may occur through NC-HK bound to mitochondria, Golgi, plasmalemma or internal membranes in maize roots (Figs 3, 4; Table 2). All of the NC-HK bound to mitochondria (60%), Golgi vesicles (30%) and the microsomal fraction (10%) are inhibited by HK inhibitors, including ADP; but most of the cytosolic isoforms are not (Figs 3B, 4, 5; see also Galina et al., 1995; Galina and da-Silva, 2000). The very modest inhibition of cytosolic FK and HK by mannoheptulose, N-acetylglucosamine and glucosamine suggests a high degree of sugar stereo-selectivity of the catalytic sites of the cytosolic enzymes. Similar results were observed with cytosolic glucokinase purified from young tomato fruit, which was inhibited by only 26% with 100 mM mannoheptulose (with 0.4 mM glucose) (Martinez-Barajas and Randall, 1998).
In recent work with rice embryos (Guglielminetti et al., 2000), it was observed that two isoforms of HK (HK1 and HK2) and one of glucokinase (GK3) were inhibited by the glucose analogues, mannoheptulose and glucosamine. It was hypothesized that these isoforms are involved in the sugar-sensing process. Interestingly, the subcellular fraction employed in rice embryo studies was the 15 000 g supernatant fraction, which contains Golgi vesicles, microsomes and soluble enzymes. Here it is shown that this fraction in maize roots contains NC-HK that is inhibited by these glucose analogues.
Some caution is required when correlating glucose analogue inhibition to the sugar-sensing process, because the NC-HK may require the presence of some signal transduction factor anchored to the same membrane (Koch et al., 2000). Indirect evidence for a distinct subcellular site of NC-HK-mediated sugar-sensing comes from transgenic tobacco leaf cells studies (Herbers et al., 1996). The expression of yeast invertase in apoplasts or vacuoles leads to elevated concentrations of glucose and fructose which are sensed in plants expressing invertase, resulting in altered gene expression and leaf lesions. These effects were not observed when invertase was expressed in the cytosol. It was proposed that hexoses are sensed only in secretory membrane system of endoplasmic reticulum or Golgi apparatus (Herbers et al., 1996). Based on these results and the current paradigm that most of the hexokinase is a glycolytic and cytosolic enzyme, the role of hexokinase in sugar-sensing has been questioned (Halford et al., 1999). The results of this study indicate that in maize, HK inhibitors preferentially inhibit the mitochondrial, microsomal and Golgi-bound hexokinases and have very little effect on the cytosolic hexokinase and fructokinases (Figs 4, 5, 7 ; Table 2).
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It was shown that Arabidopsis over-expressing the product of Athxk 1 gene (HxK I) presents high glucose sensitivity (Jang et al., 1997
). Recently, the association of a HxK I with chloroplast outer envelope membrane has been demonstrated in spinach leaves (Wiese et al., 1999). Spinach HxK I and corresponding cDNA from tobacco and potato are highly homologous to Arabidopsis HxK I and II, which also possess a hydrophobic N-terminal membrane anchor. This N-terminal seems to be important for association of HxK I with chloroplast envelope membranes (Wiese et al., 1999). A different hydrophobic N-terminal segment is known to be critical for binding of the mammalian Type I hexokinase to porin located in outer membrane of mitochondria (Wilson, 1997). However, the molecular characteristics of the HK association with mitochondria and other internal membrane systems have not yet been established. The extent to which the Arabidopsis hexokinase resembles the hexokinases has been shown to be associated with mitochondria in maize roots (Galina et al., 1995, 1999) also remains to be determined. However, the affinity of glucose and fructose for AtHxK I over-expressed in tomato plants (Dai et al., 1999) showed a very similar profile to those described for NC-HK in this paper (Fig. 6C).
Based on the HK inhibitors as markers of the sugar-sensing process, the data presented in this report suggest that the sugar-sensing hexokinase is not cytosolic in maize roots. Recently, evidence was given for an interface between transduction pathways of hexose and energy charge signals (Koch et al., 2000). It would be of interest to evaluate which signal transduction elements are associated with mitochondrial/Golgi-apparatus membranes and how ADP (a natural HK regulator) modulates the hexose sensor activity of maize non-cytosolic hexokinases.
Subcellular organization of the hexose-phosphorylation diversity in maize seedlings
Based on the data presented in this report and the current knowledge of plant carbohydrate translocation, an integrative model is proposed for subcellular organization and the short-term (kinetic) adaptive responses of the hexose-phosphorylating potential to variations in hexose and adenylate energy-charge levels in maize root cells (Fig. 7). In maize root axis, the sugar concentration varies from 500 mM in the phloem to less than 5 mM in the root tips (Bret-Harte and Silk, 1994; Dieuaide-Noubhani et al., 1995). At extremely low hexose levels (up to 1 mM), either FK1, FK2 or HK1 would phosphorylate glucose and fructose at their maximal rates (Figs 6A, B, 7B, D). Under this condition, NC-HK would function as a ‘glucokinase-like’ enzyme with a tight regulation by ADP (Figs 4C, 6C, 7B, D). At high hexose levels (>20 mM) the cytosolic FK2 activity would be reduced to 20% and FK1 completely blocked (Figs 6A, 7A, C). The fructose phosphorylations would be carried out only by the NC-HK and, at low rate, by the HK1 (Fig. 6B, C), with very little control from ADP (Figs 4C, 7A, C; Galina et al., 1999). The glucose phosphorylation catalysed by HK1 is unaffected by the adenylate charge (Figs 4C, 7A, C). In agreement with recent data (Galina and da-Silva, 2000), neither ADP nor HK inhibitors have much effect on the cytosolic FK and HK activities. The lack of ADP inhibition suggests that cytosolic FK and HK are involved in the glycolysis (Givan, 1974; Farrar and Williams, 1991). A negative regulation by ADP was found with NC-HK mediating the UDP-glucose formation (Galina and da-Silva, 2000) suggesting that these isoforms do not take part in the glycolytic pathway.
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Acknowledgments |
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WSS is a recipient of a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil. GLR is a recipient of a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. We are grateful to Martha Sorenson and Roger Seymour critically reading the manuscript. This work was supported by grants from the Fundação de Amparo a Pesquisa do Rio de Janeiro (FAPERJ) proc. no. E-26/170.713/99; Programa de Apoio ao Desenvolvimento Científico e Tecnológico, PADCT-CNPQ no. 620433/91-4; (FINEP/PRONEX/FUJB) no. 76.97.1000.000.
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Notes |
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1 To whom correspondence should be addressed. Fax: +55 21 270 8647. E-mail: galina{at}bioqmed.ufrj.br
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