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Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).

Soliman A, Ayele BT, Daayf F - PLoS ONE (2014)

Bottom Line: Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM.The study also showed that AMP is another possible exchange substrate.The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACT
In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

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Exchange of the intracellular radiolabeled substrates.A: E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α-32P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B: the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C: E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α-32P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.
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pone-0098524-g008: Exchange of the intracellular radiolabeled substrates.A: E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α-32P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B: the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C: E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α-32P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.

Mentions: Efflux of the intracellular [α-32P] ADP-Glc and [α-32P] ADP was monitored using the intact IPTG-induced E. coli cells harboring HvBT1 as described in the materials and methods. Rapid export of [α-32P] ADP-Glc was enhanced by dilution with a high concentration of ADP or AMP, which caused a 75% or 60% reduction from the initial amount, respectively. Meanwhile, dilution of [α-32P] ADP-Glc with a high concentration of non-labeled ADP-Glc or ATP led to 43% or 36% reduction from the initial amount, respectively within 8 min of incubation (Figure 8A). Increased efflux of putative [α-32P] ADP was also observed by dilution of the medium with high concentration of non-labeled ADP-Glc at different incubation periods (Figure 8B). No significant difference in efflux was found between dilution with ADP and the control.


Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).

Soliman A, Ayele BT, Daayf F - PLoS ONE (2014)

Exchange of the intracellular radiolabeled substrates.A: E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α-32P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B: the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C: E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α-32P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043945&req=5

pone-0098524-g008: Exchange of the intracellular radiolabeled substrates.A: E. coli cells harboring the vector containing HvBT1 and the control vector were incubated with 1 µM [α-32P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. B: the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. C: E. coli C43 cells harboring the vector containing HvBT1 and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α-32P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.
Mentions: Efflux of the intracellular [α-32P] ADP-Glc and [α-32P] ADP was monitored using the intact IPTG-induced E. coli cells harboring HvBT1 as described in the materials and methods. Rapid export of [α-32P] ADP-Glc was enhanced by dilution with a high concentration of ADP or AMP, which caused a 75% or 60% reduction from the initial amount, respectively. Meanwhile, dilution of [α-32P] ADP-Glc with a high concentration of non-labeled ADP-Glc or ATP led to 43% or 36% reduction from the initial amount, respectively within 8 min of incubation (Figure 8A). Increased efflux of putative [α-32P] ADP was also observed by dilution of the medium with high concentration of non-labeled ADP-Glc at different incubation periods (Figure 8B). No significant difference in efflux was found between dilution with ADP and the control.

Bottom Line: Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM.The study also showed that AMP is another possible exchange substrate.The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACT
In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

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