A sucrose binding protein homologue from soybean exhibits GTP‐binding activity that functions independently of sucrose transport activity

Abstract

The sucrose binding protein (SBP) has been implicated as an important component of the sucrose uptake system in plants. SBP-mediated sucrose transport displays unique kinetic features and the protein is not similar to other transport proteins. Here, we report the characterization of a member of the SBP family from soybean [Glycine max (L) Merrill] designated S64 or SBP2. Subcellular fractionation and precipitation by GTP-agarose demonstrated that S64/SBP2 is a membrane-associated protein that exhibits GTP binding activity. Purified recombinant S64/SBP2 protein, expressed as a histidine-tagged protein in Escherichia coli, exhibited nucleotide-binding specificity to guanine nucleotides. The GTP binding site was mapped to an imperfect Walker A type-sequence, Ala279-Leu-Ala-Pro-Thr-Lys-Lys-Ser286, by site-directed mutagenesis. Escherichia coli-produced wildtype protein and a truncated version of the protein containing the putative binding-sequence-bound GTP, although not with the same efficiency. In contrast, replacement of Thr283 and Lys284 residues to Leu and Glu residues prevented GTP binding. The site directed mutant failed to bind GTP but retained the ability to undergo oligomerization and to promote growth of the susy7 yeast strain, deficient in utilizing extracellular sucrose, on medium containing sucrose as the sole carbon source. Our results indicate that GTP binding and sucrose transport by SBP are separable and function independently. The implications of our findings with respect to the function and membrane topology of SBP are discussed.Keywords: sucrose transporter; soybean; yeast complementation assay; Glycine max.In many higher plants, sucrose is the predominant form of photoassimilate that is transported from mature leaves (source tissues) to sink tissues, such as seeds, stems, reproductive organs and roots, via the vascular system [1]. Biochemical studies have demonstrated that sucrose uptake kinetics in leaves is complex and consists of multiple components; for example, in Vicia faba, two saturable (high-and low-affinity) components and one linear, low-affinity component have been described [2]. Our understanding of sucrose translocation has advanced considerably over the last decade with the molecular and biochemical characterization of the sucrose transporter (SUT) family of low-and high-affinity sucrose transporters [1]. The SUT1 protein has been described as the protonmotive-force-driven sucrose symporter that mediates phloem loading and long-distance transport, the key transport step in assimilate partitioning for many plants [3][4][5]. SUT1 serves as a high-affinity transporter, whereas SUT4, a second member of this sucrose transporter family, corresponds to the low-affinity/high capacity saturable component of sucrose uptake found in leaves [6]. A third structurally related-member of the family has been identified and designated SUT2 [7]. The SUT2 protein has been proposed to act as a sugar sensor that controls sucrose fluxes across the plasma membrane of sieve elements by regulating express...

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