Journal of Experimental Botany, Vol. 54, No. 386, pp. 1407-1414,
May 1, 2003
© 2003 Oxford University Press
Evidence for a tonoplast-associated form of sucrose synthase and its potential involvement in sucrose mobilization from the vacuole
Received 11 November 2002; Accepted 3 February 2003
Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, Fl 33850-2299, USA
1 To whom correspondence should be addressed. Fax: +1 863 956 4631. E-mail: eje{at}lal.ufl.edu
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Abstract |
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The following work presents new evidence for a tonoplast localization of sucrose synthase and its functional role during the mobilization of vacuolar sucrose. Highly purified tonoplast vesicles were associated with significant SuSy levels as determined by immuno-recognition, enzymatic activity, and by-product measurements. Total tonoplast-bound SuSy was estimated to be approximately 7% of the total tissue activity. SuSy affinity to the tonoplast was confirmed by the lack of SuSy displacement by ionic washes and also by the tonoplast ability to bind to exogenously added SuSy as compared to the cytosolic marker alcohol dehydrogenase. UL-[14C]sucrose-loaded vesicles incubated with ATP and UDP produced [14C]UDP-Glc as determined by UDP-Glc dehydrogenase and by the ability of the product to bind to DEAE-cellulose and to co-migrate with authentic UDP-Glc on TLC. ATP alone induced sucrose efflux but not the production of [14C]ADP-Glc. Kinetic analysis of [14C]UDPG formation under conditions of low sucrose availability suggests sucrose channelling between the ATP-dependent sucrose transporter and SuSy, thus corroborating the association of SuSy with the tonoplast and its involvement in sucrose mobilization from the vacuole.
Key words: Red beet, sucrose efflux, sucrose mobilization, sucrose synthase, tonoplast transport.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Since its discovery by Leloir and coworkers in 1955 (Cardini et al., 1955), sucrose synthase (SuSy, EC 2.4.1.13 [EC] ) has been assigned pivotal roles in a variety of plant metabolic processes. The enzyme has been ascribed a central function in the determination of sink strength in both storage (Zrenner et al., 1995) and developing vegetative tissues (Pak et al., 1995; Pfeiffer and Kutschera, 1995). In addition, SuSy is believed to participate in the conversion of sucrose to starch in numerous instances (Su, 1995; Pozueta-Romero et al., 1999), in the synthesis of the cellulose precursor UDPG (Chourey and Miller, 1995; Delmer and Amor, 1995), in nitrogen fixation by legume nodules (Gordon et al., 1999), and is directly involved in the metabolic changes brought about by cold stress (Maraña et al., 1990; Crespi et al., 1991) and anaerobiosis (Maraña et al., 1990; Ricard et al., 1991). Its functional plasticity is probably due to the enzyme’s capacity to utilize several nucleosides, its catalytic reversibility, and its diverse cellular compartmentation. Although initially believed to be a soluble cytosolic enzyme (Keller et al., 1988), recent evidence has shown a portion of the activity in close association with plasmalemma (Amor et al., 1995; Carlson and Chourey, 1996) and actin filaments (Winter et al., 1998).
Early studies on sucrose mobilization from the vacuole of germinating maize scutellum cells alluded to the likely possibility of SuSy being tonoplast associated (Humphreys, 1973; Echeverria and Humphreys, 1984). This conclusion was based on evidence demonstrating that total invertase activity was insufficient to account for the measured rates of sucrose utilization, while cytosolic sucrose remained unmetabolized. The fact that vacuolar sucrose was catabolized by SuSy, while cytosolic sucrose remained untouched, strongly suggested an association of SuSy with the tonoplast and its participation in sucrose transport. If vacuolar sucrose were to be released into the cytosol, a soluble SuSy could not distinguish between cytosolic sucrose and that of vacuolar origin. Additional evidence for an association between SuSy and the tonoplast has been advanced by Goldschmidt and Branton (1977), Calderón and Pontis (1985) and Fiew and Willenbrink (1987).
Mobilization of sucrose from the vacuole of plant cells is indispensable for the maintenance of numerous metabolic processes, especially during dormancy, sprouting, low photosynthetic activity, and post-harvest life of agricultural commodities. For example, during the second year of their biennial life cycle, sprouting red beet plants require the mobilization of vacuolar sucrose from the underground hypocotyl. Mobilized sucrose is used to support the increased intracellular metabolic demands, as well as for long-distance transport to developing vegetative and reproductive organs. Mobilization of sucrose from the vacuole occurs by means of two distinct mechanisms. For extracellular long-distance transport, a vesicle-mediated system carries sucrose from the vacuole of storage cells into the apoplast (Echeverria, 2000). By contrast, to sustain internal metabolic demands, sucrose is transported across the tonoplast by an ATP-dependent primary active transport system (Echeverria and Gonzalez, 2000). The utilization of sucrose transported across the tonoplast for internal metabolism requires the participation of SuSy, given that invertase activity is absent from mobilizing storage parenchyma cells and there are virtually no hexoses present in the storage vacuoles (Echeverria, 1998).
During a previous investigation on the mechanisms of sucrose efflux from the vacuole of sprouting red beet hypocotyl cells, SuSy activity was consistently detected in highly purified native tonoplast vesicles. Upon realizing the tight regulation that is likely to exist in maintaining a controlled sucrose concentration in the cytosol (Koch, 1996), it became pertinent to investigate in further detail the likelihood of some SuSy activity being tonoplast associated and its potential involvement in the process of sucrose mobilization from the vacuole. The results of this investigation demonstrate that a portion of SuSy activity is associated with the tonoplast. In addition, the data also demonstrate that under appropriate conditions, SuSy participates in the in vitro mobilization of sucrose from tonoplast vesicles.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Red beet (Beta vulgaris L.) mature plants were purchased at a local market. Existing leaves were detached and hypocotyls were individually planted in 0.5 l pots for at least 3 weeks at room temperature. Newly formed leaves were excised once they attained full size.
Tonoplast isolation
Tonoplast vesicles from red-beet hypocotyls were obtained in a discontinuous sucrose gradient as described by Bennett et al. (1985). Membrane fractions containing purified tonoplast vesicles were rapidly diluted with a 10x volume of 10 mM TRIS-MES (pH 7.0) and 2 mM DTT inducing an osmotic shock. The solution containing the tonoplast vesicles was centrifuged at 80 000 g for 40 min. The final tonoplast pellet was resuspended in 1 ml of storage buffer containing 10 mM TRIS-MES and 2 mM DTT at pH 5.5. Tonoplast vesicles were then stored at –80 °C until needed. Isolation procedures were conducted at 4 °C.
Enzyme assays
ATPase activity was measured colorimetrically following the production of free Pi by the method of Chifflett et al. (1988). The reaction contained 50 mM HEPES (pH 7.5), 2 mM DTT, 4 mM ATP, 4 mM MgSO4, 50 mM KCl, 10 µM gramicidin, and vesicles in a total volume of 1 ml. For the determination of bafilomycin-sensitive ATPase, bafilomycin-A (Sigma B-1793) was added at a final concentration of 30 nM. V-PPase reaction was similar to that of ATPase except PPi was added instead of ATP.
SuSy activity was measured following the production of sucrose in a solution containing 100 mM HEPES (pH 8.0), 2 mM UDPG, 10 mM NaF, 10 mM fructose, 2 mM MgCl2, and vesicles in a total volume of 500 µl. One hundred microlitre aliquots were taken at determined times and the reaction stopped by combining with 100 µl of 30% KOH. Sucrose was analysed using the modified anthrone method of Van Handel (1968).
Alcohol dehydrogenase (ADH) was assayed following the increase in absorbance at 340 nm in a reaction containing 50 mM glycylglycine (pH 8.7), 2 mM NAD, 100 mM ethanol, and vesicles in 1 ml reaction volume as described by MacDonald and ap Rees (1983).
Immunological detection of SuSy
Immunoblot analyses were performed after electrophoresis according to Laemmli (1970). Proteins were transferred to a nitrocellulose membrane and immunostained with antisera to SuSy from maize endosperm. Antibodies were a gift from Dr Karen Koch (University of Florida) (Nolte et al., 1995). As the standard, commercially available SuSy from Sigma (Sigma S-6379) was used. However, during the course of the experimentation, it was possible to purify SuSy from red beet hypocotyls which co-migrated and reacted similarly to the wheat germ enzyme. Figure 2A was re-done using purified red beet SuSy to show the authenticity of all figures. Antibodies against subunit E of barley leaf V-ATPase were a gift from Dr Karl-Joseph Dietz and used as specified by Betz and Dietz (1991).
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[14C]sucrose loading of tonoplast vesicles
Tonoplast vesicles were centrifuged at 80 000 g for 40 min and the pellet resuspended in a solution containing 5 mM UL-[14C]Suc (51.8x103 Bq µmol–1), 10 mM TRIS-MES (pH 5.5) and 2 mM DTT. The solution was sonicated for 5 s and subsequently subjected to three freeze/thaw cycles. Vesicles were then centrifuged at 100 000 g for 1 h, the supernatant discharged, and the pellet washed with a buffer solution containing 5 mM sorbitol, 10 mM TRIS-MES (pH 5.5) and 2 mM DTT.
Treatment of vesicles with different agents
Vesicle samples (200 µl) were incubated with 0.5 M KCl, 10 mM CAPS (pH 10.4), and 3 M urea, respectively, in buffer containing 10 mM HEPES (pH 7.5) and 2 mM DTT. After incubation for 15 min, the vesicles were separated at 100 000 g for 1 h, washed with a buffer solution of 10 mM TRIS-MES pH 5.5 and 2 mM DTT, and finally resuspended in the same buffer for enzyme activity determination and immunodetection analysis.
Separation of membrane proteins with Triton X-114 was performed using the method of Bordier (1981). The resulting pellet and supernatant were used for the immunodetection of SuSy.
Sucrose efflux experiments and product determination
Sucrose efflux experiments and sucrose determination were carried out as described by Echeverria and Gonzalez (2000). Efflux of sucrose from [14C]sucrose-loaded vesicles was started by the addition of Mg/ATP. Aliquots were taken at given times, filtered, and the remaining [14C]sucrose determined by scintillation spectroscopy. For UDP-Glc formation, UL-[14C]Suc loaded vesicles (100 µg protein) were resuspended in a solution containing 5 mM sorbitol, 10 mM HEPES (pH 7.5), 2 mM DTT, 2.5 mM ATP/MgSO4, 30 nM bafilomycin, and 2.5 mM UDP in a total volume of 250 µl and incubated at 30 °C. Samples of 100 µl were taken at time zero and 30 min, boiled for 2 min, centrifuged at 13 000 g for 10 min, and the supernatant filtered through DEAE-cellulose (DE-81, 2.5 cm) paper (Whatman Science, Maidstone, UK). The ion binding paper was rinsed with water and radioactivity in the filter paper measured by liquid scintillation spectroscopy. Thin-layer chromatographic (TLC) identification of newly formed UDP-[14C]Glc was carried out on cellulose plates (Sigma, St Louis, MO) with a mixture of tert- amyl alcohol:formic acid:water (3:2:1 by vol.) as solvent. Chromatography was run for 2 h according to Randerath (1964). The UDP-[14C]Glc formed was visualized using UV light following its co-migration with authentic UDP-Glc (Sigma, U-4625) and the spots counted using scintillation spectroscopy. The authenticity of UDP-Glc was verified using UDPG dehydrogenase (Sigma U-7251, St Louis, MO).
Larger reactions were needed to produce sufficient product for spectrophotometric and colorimetric measurements. The reactions contained 10 mM HEPES (pH 7.5), 100 mM sorbitol, 2 mM DTT, 2.5 mM ATP/MgSO4, 30 nM bafilomycin, 2.5 mM UDP, and tonoplast vesicles (400 µg protein) loaded with non-radiolabelled sucrose at 100 mM in a total volume of 1.0 ml. At given times, 200 µl aliquots were sampled, boiled for 2 min, centrifuged at 13 000 g for 5 min, and the supernatant analysed for UDP-Glc formed following the increase in absorbance at 340 nm in the presence of 2.5 mM NAD, 5 mM MgSO4, 100 mM glycine buffer (pH 8.7), and 0.2 units UDP-Glc dehydrogenase. Fructose was determined by the increase in reducing power measured by the method of Nelson (1944).
Protease treatment
Tonoplast samples were incubated for 2 h at 4 °C in a solution containing 20 mM HEPES buffer (pH 7.5), 10 mM MgCl2, 250 mM sorbitol, and 0.1 U protease (P-4630, Sigma-Aldrich, St Louis, MO). The vesicles were then centrifuged at 100 000 g for 40 min and washed twice with similar buffer (without protease). The final suspension was used for SuSy analysis. Control samples were treated similarly but without protease.
Beet hypocotyl protein preparation
Beet hypocotyl tissue (160 g) was homogenized in a Wareing blender for 1 min in 250 ml homogenization buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 10% ethylene glycol, 0.2% Triton X-100, 4 mM EGTA, 1 mM PMSF, and 1.5% PVPP. The solution was filtered through nylon cloth and centrifuged for 30 min at 10 000 g. The supernatant was diluted by 50% with water and brought up to 60% saturation with (NH4)2SO4 at 4 °C. The resulting precipitate, after 1 h centrifugation at 10 000 g, was resuspended in 24 ml of buffer containing 50 mM HEPES (pH 7.5) 10 mM MgCl2, 1 mM EDTA 2 mM DTT, and dialysed overnight in 4.0 l of buffer containing 10 mM HEPES (pH 7.5) 10 mM MgCl2, 1 mM EDTA, and 2 mM DTT.
Protein determination
Protein was determined as described by Bradford (1976) using ‘Coomassie Plus Protein Assay Reagent’ (Pierce Co. Rockford, IL). All experiments were performed at least three times with tonoplast preparations from different beet hypocotyls.
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Results and discussion |
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Red beet tonoplast vesicles prepared in a discontinuous sucrose gradient were extremely pure as previously demonstrated by Bennett et al. (1985) and by Echeverria and Gonzalez (2000). Contamination with other endomembrane systems, determined by the inhibition of V-ATPase towards specific membrane marker inhibitors, was minimal (Echeverria and Gonzalez, 2000). In the presence of bafilomycin-A, ATPase activity was reduced approximately 96%, whereas vanadate only inhibited total activity by 3–5%, demonstrating minimal cross contamination with plasmalemma. To eliminate the possibility of contamination by soluble cytosolic components entrapped within the vesicles during tissue homogenization, tonoplast vesicles were subjected to one osmotic shock and three 3-s sonication cycles. Measurements of the soluble cytosolic marker alcohol dehydrogenase (ADH) before and after clean-up procedures demonstrated that only traces of ADH activity remained (Table 1). Therefore, tonoplast vesicles subjected to the described purification procedure were also free of soluble cytosolic contamination by entrapment. V-PPase activity increased only by 19% when 0.05% Triton X-100 was added to the reaction medium (Table 1), indicating that tonoplast vesicles were predominantly right side out (
81%). This is in accordance with previous observations where tonoplast vesicles reform predominantly in the correct orientation (Marty, 1982) whether plasmalemma orients randomly during vesicle formation (Sze, 1985).
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When purified tonoplast vesicles were tested against antisera to SuSy from maize endosperm (Nolte et al., 1995), a distinctive immunoreaction was observed at a location corresponding to SuSy (Fig. 1, lane 3). Strong immunoreaction was also observed in crude samples from beet hypocotyl (lane 2) and in commercial SuSy samples from wheat germ used as standard (lane 1). These results suggest that significant amounts of SuSy protein remained attached to the tonoplast in the absence of cytosolic or endomembrane contamination, representing a portion of the enzyme associated with the tonoplast. In Fig. 2A, samples of membrane vesicles taken from the three distinct interfaces of the sucrose gradient showed increased immunoreactivity to SuSy antibodies proportional to tonoplast purity (Echeverria and Gonzalez, 2000). The increase in immunoreactivity to SuSy parallelled the increase in tonoplast enrichment. Tonoplast enrichment was demonstrated by reaction to antibodies against subunit E of V-ATPase from barley leaf (Fig. 2B). Therefore, the lightest membrane fractions (16/26% sucrose interface), containing the highest levels of bafilomycin-A-sensitive ATPase (Echeverria and Gonzalez, 2000) also showed strongest immunoreactivity to SuSy (Fig. 2A) and V-ATPase antibodies (Fig. 2B). As the bafilomycin-A-sensitive ATPase activity decreased and the tonoplast became contaminated with a mixture of other endomembrane components (Bennett et al., 1985; Echeverria and Gonzalez, 2000), the SuSy antibody signal subsided in a parallel manner (Fig. 2A).
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An estimation of percentage SuSy activity associated with the tonoplast was accomplished using V-ATPase as the tonoplast marker (Table 1). Based on V-ATPase and SuSy activities present in the crude preparations and in their respective activities recovered in the purified tonoplast samples, it was estimated that approximately 7.2% of SuSy remained tonoplast associated (Table 1). These values are higher than those obtained by Carlson and Chourey (1996) for SuSy associated with the plasmalemma of maize endosperm. Most of the unaccounted activities of V-ATPase and SuSy were lost during microsomal purification and to other membrane layers of hybrid origin in the sucrose gradient (Echeverria and Gonzalez, 2000). Nonetheless, in the present study, purity of tonoplast was more critical than amount recovered.
The seemingly tight association between SuSy and the tonoplast is illustrated by the experiments presented in Fig. 3. When antibody quantification was used, significant SuSy signal remained at the tonoplast after washing with 0.5 M KCl, high pH treatment (pH >10), or 3 M urea. Very similar results were obtained by Amor et al. (1995) with plasmalemma associated SuSy in cotton fibres. When enzymatic activity was tested after the above described treatments, only urea abolished SuSy activity, although the enzyme remained strongly attached to the membrane (Fig. 3A). Partition experiments of tonoplast samples using Triton X-114 revealed that SuSy is not a trans-membrane protein. After only one detergent wash, SuSy was recovered in the fraction containing hydrophilic proteins (Fig. 3B). A second wash of the hydrophobic portion of the gradient did not extract any additional SuSy. The enzyme, therefore, appears to be bound to a membrane component at the surface of the lipid bilayer and is not a transmembrane protein.
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The affinity of SuSy to the tonoplast was further substantiated by the experiments of Table 2, where tonoplast vesicles were incubated in an iso-osmotic solution containing resuspended beet protein extract. The resuspended beet protein solution contained 54.6 mU of alcohol dehydrogenase, used as cytosolic control, and 6.4 mU of SuSy activity. After incubation for 15 min at 4 °C in 10 mM HEPES (pH 7.5), 2 mM DTT and 2.5 mM MgCl2, the vesicles were separated from the protein solution in a sucrose gradient, washed twice, and activities of alcohol dehydrogenase and SuSy reassessed. Whereas there was no increase in alcohol dehydrogenase activity after treatment, SuSy activity increased by 86% (Table 2). The notable increase in SuSy activity occurred despite the fact that ADH activity was seven times higher than SuSy in the soluble resuspended protein fraction.
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Previous studies demonstrated that addition of UDP to sucrose-loaded tonoplast vesicles did not result in sucrose efflux (Echeverria and Gonzalez, 2000). The lack of sucrose efflux from sucrose-loaded vesicles in the presence of UDP alone, in addition to the peripheral location of SuSy at the tonoplast, imply that SuSy by itself is not involved in sucrose mobilization. However, when ATP was added in conjunction of UDP to [14C]Suc-loaded vesicles, substantial sucrose efflux was measured. Sucrose efflux was accompanied by the production of UDP-[14C]Glc as determined by the ability of the product to bind to DEAE-cellulose paper and to comigrate with authentic UDP-Glc on TLC (Table 3). In the absence of UDP, ATP alone induced sucrose efflux (Echeverria and Gonzalez, 2000; Table 3), but no [14C]anionic compound (ADP-[14C]Glc) was detected. Verification of the UDPG formed was accomplished spectrophotometrically using UDP-Glc dehydrogenase (Fig. 4) in reactions containing vesicles with higher sucrose concentrations (100 mM). The figure shows the increase in absorbance at 340 nm when sucrose-loaded tonoplast vesicles were incubated in the presence of ATP, UDP and 0.25 U UDPG-dehydrogenase. Control samples were carried out in the absence of ATP until commercial UDPG was added (arrow). In similar reactions, product ratio (UDP-Glc:fructose) was determined to be 1:1 as corresponds to the SuSy reaction.
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The levels of sucrose efflux and products (UDP-Glc and fructose) produced during efflux experiments could not have been the result of contamination by the small amounts of inside-out plasmalemma and/or tonoplast vesicles. First, plasmalemma contamination was too low to account for the measured rates of sucrose efflux (Echeverria and Gonzalez, 2000). Second, in inside-out tonoplast vesicles (only 19%, Table 1), the ATP added to energize sucrose efflux and the UDP for SuSy activity would be at opposite sides of the membrane which contains the active ATP-dependent sucrose transporter. In such cases, sucrose efflux would not be induced under the present conditions. Verification that tonoplast-bound SuSy activity was located at the periphery of the membrane was obtained by proteolytic treatment of tonoplast vesicles (Table 4). When tonoplast vesicles were treated with 0.1 U protease, approximately 22% of the activity remained compared to untreated controls. This value is very close to the increase in SuSy activity after detergent treatment (Tables 1, 4).
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Subsequent kinetic analysis of the SuSy reaction provided further stronger evidence in support of SuSy association with the tonoplast. For example, the total amount of sucrose contained within the tonoplast vesicles used for each reaction in Table 3 was approximately 2.2 nmol. Even if all [14C]Suc within the vesicles were released into the medium by the ATP-dependent sucrose transport, the final concentration in the reaction medium would not exceed 9 µM. Such sucrose concentration is over 300-fold lower than the Km estimated for red beet SuSy (Pavlinova and Prasolova, 1970). In fact, when SuSy assays were carried out separately under these conditions of substrate availability (9 µM), no UDP-[14C]Glc was produced (Table 3). These results indicated that even under the most favourable conditions, SuSy would not be active at such low sucrose concentrations. The fact that UDP-[14C]Glc was formed during ATP-induced sucrose efflux from tonoplast vesicles indicates that sucrose was not simply released into the solution (and greatly diluted), but that sucrose was catalysed by SuSy upon transport. The implicit direct transfer of sucrose between the ATP-dependent sucrose transporter and SuSy provides strong support for the association of SuSy with the tonoplast and implies its function in sucrose efflux from the vacuole during mobilization.
The data presented in this communication supports the existence of an association between SuSy and the tonoplast of mobilizing red beet hypocotyl cells. The results from in vitro sucrose efflux experiments and kinetic analysis describe a transport system where tonoplast-associated SuSy participates in the transport of sucrose from the vacuole to the cytosol. In this system, sucrose is exported from the vacuole by the ATP-dependent sucrose transporter and channelled to SuSy which is connected at the cytosolic side of the tonoplast. In the presence of UDP, SuSy catalyses sucrose breakdown releasing UDPG and fructose into the cytosol. Whether all sucrose transported out of the vacuole under in vivo conditions is catalysed by SuSy can not be determined based on the present data. However, catalysis of all mobilized sucrose seems an unlikely event, since sucrose has diverse physiological functions and may be required in the cytosol as a disaccharide. The fact that sucrose can be transported out of vesicles and released as a disaccharide in the absence of UDP (Table 3; Echeverria and Gonzalez, 2000) indicates that additional regulatory mechanisms exist that provides for this option (see Spivey and Ovadi, 1999, for discussion of different forms of metabolic channelling). The present work, demonstrating yet another intracellular location and function for SuSy, further supports the utilitarian plasticity of SuSy and its variable cellular localization depending on the cellular needs.
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Acknowledgements |
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The authors are deeply grateful to Drs Graciela Salerno, Karen Koch, and Karl-Joseph Dietz for the antibodies used in this study and for their critical review of the manuscript (GS and KK). This research was supported by the Florida Agricultural Experiment Station, and approved for publication as Journal Series No. R-08025.
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S. C. Hardin, K. A. Duncan, and S. C. Huber Determination of Structural Requirements and Probable Regulatory Effectors for Membrane Association of Maize Sucrose Synthase 1 Plant Physiology, July 1, 2006; 141(3): 1106 - 1119. [Abstract] [Full Text] [PDF]
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E. Etxeberria, E. Baroja-Fernandez, F. J. Munoz, and J. Pozueta-Romero Sucrose-inducible Endocytosis as a Mechanism for Nutrient Uptake in Heterotrophic Plant Cells Plant Cell Physiol., March 1, 2005; 46(3): 474 - 481. [Abstract] [Full Text] [PDF]
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S. C. Hardin, H. Winter, and S. C. Huber Phosphorylation of the Amino Terminus of Maize Sucrose Synthase in Relation to Membrane Association and Enzyme Activity Plant Physiology, April 1, 2004; 134(4): 1427 - 1438. [Abstract] [Full Text] [PDF]
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 S. Baud, M.-N. Vaultier, and C. Rochat Structure and expression profile of the sucrose synthase multigene family in Arabidopsis J. Exp. Bot., February 1, 2004; 55(396): 397 - 409. [Abstract] [Full Text] [PDF]
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