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 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 wild-type 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 andto promote growth of the susy7 yeast strain, deficient inutilizing 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.
Pirovani
Carlos P
CP
Departments of Biologia Vegetal and Bioquímica e Biologia Molecular/BIOAGRO, Universidade Federal de Viçosa, Brazil.
Macêdo
Joci Neuby A
JN
Contim
Luís Antônio S
LA
Matrangolo
Fabiana S V
FS
Loureiro
Marcelo E
ME
Fontes
Elizabeth P B
EP
D000595:Amino Acid Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013395:Sucrose / Q000378:metabolism*
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C078729:sucrose transport protein, plant; D013395:Sucrose; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins