The α-d-glucosyl C-2 hydroxyl is required for binding to the H+-sucrose transporter in phloem

Griffin, S. D. J.; Buxton, Karen D.; Donaldson, Iain A.

doi:10.1016/0005-2736(93)90231-n

Cited by 8 publications

(9 citation statements)

References 33 publications

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“…For LacY, the 2, 3, 4, and 6 hydroxyl groups –OH in the galactose ring of lactose, especially 3-OH and 4-OH, are important for the substrate binding and transport [37,38]. Similarly, the 2, 3, 4, and 6 hydroxyl groups in the glucose moiety of sucrose, particularly 3-OH and 4-OH, are essential for the substrate recognition and transport in SUTs [21-24]. Further work will be required to determine whether one of the NH 2 groups of Arg188 interacts with another amino acid in the N-terminal half of protein, which triggers a major conformational change (“rocker-switch”) analogous to the mechanism in LacY [33].…”

Section: Discussionmentioning

confidence: 99%

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Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

Sun

Ward

2012

BMC Biochemistry

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BackgroundPlant sucrose uptake transporters (SUTs) are H+/sucrose symporters related to the major facilitator superfamily (MFS). SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS) in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT.ResultsThe OsSUT1(R188K) mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by β-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K) and LjSUT4(R169K), respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of α-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant.ConclusionOsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation.

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Section: Discussionmentioning

confidence: 99%

“…Replacement of the glucosyl 4-OH or 3-OH with hydrogen or fluorine showed the most dramatic decrease in substrate recognition [21-23], indicating that the two hydroxyls interact with the SUT protein via hydrogen bonding [22]. Hydrogen substitution or fluorine substitution of the 2-OH [21,24] or 6-OH [22] also inhibited substrate transport.…”

Section: Introductionmentioning

confidence: 99%

Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

Sun

Ward

2012

BMC Biochemistry

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“…Several studies postulated a second linear component in addition to active uptake resulting in biphasic kinetics (e.g., van Bel and Koops, 1985; Daie, 1987). This linear uptake was assigned to facilitated diffusion, but could be equally well the linear section of a low-affinity MM system.…”

Section: Discussionmentioning

confidence: 99%

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

Hafke¹,

Höll²,

Kühn³

et al. 2013

Front. Plant Sci.

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Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.

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“…These positions are more likely to determine whether substrate binding leads to translocation. We know that the glucosyl moiety of the sugar substrate is required for binding to SUTs (10) and that glucosyl hydroxyls at positions 2, 3, 4, and 6 are important for binding (33,34). An Arg residue (Arg-188 in TMS4 of OsSUT1) that is fully conserved in all SUTs has been hypothesized to interact with glucosyl hydroxyls (35), similar to the situation in LacY in which Arg-144 interacts with galactosyl hydroxyls (36).…”

Section: Discussionmentioning

confidence: 99%

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

Reinders

Sun

Karvonen

et al. 2012

Journal of Biological Chemistry

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Background: Type II sucrose transporters (SUTs) are more selective than type I SUTs. Results: OsSUT1 was modified using gene shuffling. Variants were selected for the ability to transport the fluorescent ␤-glucoside esculin. Conclusion: Replacement of five amino acids conferred substrate specificity typical of type I SUTs. Significance: The first loop and top of TMS2 and TMS5 are involved in controlling substrate specificity in SUTs.

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The α-d-glucosyl C-2 hydroxyl is required for binding to the H+-sucrose transporter in phloem

Cited by 8 publications

References 33 publications

Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling

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