TAILIEUCHUNG - Báo cáo khoa học: A sucrose binding protein homologue from soybean exhibits GTP-binding activity that functions independently of sucrose transport activity

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 ki-netic features and theprotein isnot similar toother 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 pre-cipitation byGTP-agarose demonstrated that S64/SBP2 is a membrane-associated protein that exhibits GTP binding activity. . | Eur. J. Biochem. 269 3998-4008 2002 FEBS 2002 doi A sucrose binding protein homologue from soybean exhibits GTP-binding activity that functions independently of sucrose transport activity Carlos P. Pirovani1 Joci Neuby A. Macedo2 Luis Antonio S Contim2 Fabiana S. V. Matrangolo2 Marcelo E. Loureiro1 and Elizabeth P. B. Fontes2 Departments of 1Biologia Vegetal and 2Bioquimica e Biologia MolecularỊBIOAGRO Universidade Federal de Viposa Brazil 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 con taining 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 .

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