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Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus

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Sucrose transporters in the SUT family are important for phloem loading and sucrose uptake into sink tissues. The recent localization of type III SUTs AtSUT4 and HvSUT2 to the vacuole membrane suggests that SUTs also function in vacuolar sucrose transport. The transport mechanism of type III SUTs has not been analyzed in detail. LjSUT4, a type III sucrose transporter homolog from Lotus japonicus, is expressed in nodules and its transport activity has not been previously investigated. In this report, LjSUT4 was expressed in Xenopus oocytes and its transport activity assayed by two-electrode voltage clamping. LjSUT4 transported a range of glucosides including sucrose, salicin, helicin, maltose, sucralose and both α- and β-linked synthetic phenyl glucosides. In contrast to other sucrose transporters, LjSUT4 did not transport the plant glucosides arbutin, fraxin and esculin. LjSUT4 showed a low affinity for sucrose (K 0.5 = 16 mM at pH 5.3). In addition to inward currents induced by sucrose, other evidence also indicated that LjSUT4 is a proton-coupled symporter: 14C-sucrose uptake into LjSUT4-expressing oocytes was inhibited by CCCP and sucrose induced membrane depolarization in LjSUT4-expressing oocytes. A GFP-fusion of LjSUT4 localized to the vacuole membrane in Arabidopsis thaliana and in the roots and nodules of Medicago truncatula. Based on these results we propose that LjSUT4 functions in the proton-coupled uptake of sucrose and possibly other glucosides into the cytoplasm from the vacuole.

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The LjSUT4 cDNA clone in vector pSPORT was provided by Panagiotis Katinakis and Emmanouil Flemetakis, Agricultural University of Athens, Greece. We are grateful to Mark A. Sanders and Tracy E. Anderson of the College of Biological Sciences Imaging Center at the University of Minnesota for their help with confocal microscopy. We thank Elison Blancaflor (Noble Foundation) and Michael Knoblauch (Washington State University) for their helpful suggestions on microscopy. This project was supported by Department of Energy Grant No. DE-FG02-07ER15886 (to JMW) and NSF Grant No. DBI-0421676 (to JSG). ABS gratefully acknowledges funding from the Doctoral Dissertation Fellowship (DDF) program of the University of Minnesota.

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Correspondence to John M. Ward.

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Reinders, A., Sivitz, A.B., Starker, C.G. et al. Functional analysis of LjSUT4, a vacuolar sucrose transporter from Lotus japonicus . Plant Mol Biol 68, 289–299 (2008).

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