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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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SDS-PAGE analysis of HvBT1 protein.Escherichia coli C43 and rosetta2 strains harboring the original (org) or the optimized (opc) ORF of HvBT1 were grown in 2xYT liquid media supplied with IPTG (0.5 mM final concentration). His-tagged HvBT1 membrane protein was purified and subjected to 12% SDS-PAGE. Lane 1 and 2 represent Rosetta 2 harboring opc and org ORF of HvBT1, respectively. Lane 3 and 4 represent C43 harboring opc and org ORF of HvBT1, respectively. Black arrows point to a band size of 45 KDa. Protein standard molecular weight is shown.
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pone-0098524-g006: SDS-PAGE analysis of HvBT1 protein.Escherichia coli C43 and rosetta2 strains harboring the original (org) or the optimized (opc) ORF of HvBT1 were grown in 2xYT liquid media supplied with IPTG (0.5 mM final concentration). His-tagged HvBT1 membrane protein was purified and subjected to 12% SDS-PAGE. Lane 1 and 2 represent Rosetta 2 harboring opc and org ORF of HvBT1, respectively. Lane 3 and 4 represent C43 harboring opc and org ORF of HvBT1, respectively. Black arrows point to a band size of 45 KDa. Protein standard molecular weight is shown.

Mentions: SDS-PAGE analysis of the purified HvBT1 membrane protein showed that both E. coli strains C43 and Rosetta2 harboring the optimized ORF of HvBT1 are able to express the HvBT1 protein with expected mass of ∼45 KDa. Rosetta2, harboring the original ORF was also able to express the HvBT1 at very low level (Figure 6). The Rosetta 2 strain cells harboring either org or opc ORFs of HvBT1 showed growth inhibition, while the cell growth appeared to be normal in the case of C43 strain (Figure S1). The use of optimized ORF of HvBT1 along with the C43 strain provided an ideal expression system to sustain cell viability and perform the transport assay procedures.


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

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

SDS-PAGE analysis of HvBT1 protein.Escherichia coli C43 and rosetta2 strains harboring the original (org) or the optimized (opc) ORF of HvBT1 were grown in 2xYT liquid media supplied with IPTG (0.5 mM final concentration). His-tagged HvBT1 membrane protein was purified and subjected to 12% SDS-PAGE. Lane 1 and 2 represent Rosetta 2 harboring opc and org ORF of HvBT1, respectively. Lane 3 and 4 represent C43 harboring opc and org ORF of HvBT1, respectively. Black arrows point to a band size of 45 KDa. Protein standard molecular weight is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098524-g006: SDS-PAGE analysis of HvBT1 protein.Escherichia coli C43 and rosetta2 strains harboring the original (org) or the optimized (opc) ORF of HvBT1 were grown in 2xYT liquid media supplied with IPTG (0.5 mM final concentration). His-tagged HvBT1 membrane protein was purified and subjected to 12% SDS-PAGE. Lane 1 and 2 represent Rosetta 2 harboring opc and org ORF of HvBT1, respectively. Lane 3 and 4 represent C43 harboring opc and org ORF of HvBT1, respectively. Black arrows point to a band size of 45 KDa. Protein standard molecular weight is shown.
Mentions: SDS-PAGE analysis of the purified HvBT1 membrane protein showed that both E. coli strains C43 and Rosetta2 harboring the optimized ORF of HvBT1 are able to express the HvBT1 protein with expected mass of ∼45 KDa. Rosetta2, harboring the original ORF was also able to express the HvBT1 at very low level (Figure 6). The Rosetta 2 strain cells harboring either org or opc ORFs of HvBT1 showed growth inhibition, while the cell growth appeared to be normal in the case of C43 strain (Figure S1). The use of optimized ORF of HvBT1 along with the C43 strain provided an ideal expression system to sustain cell viability and perform the transport assay procedures.

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.

Show MeSH