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Abstract Detail

Biochemistry, metabolism, carbon flux

Liu, Fushan [1], Tetlow, Ian J. [2], Emes, Michael J. [3].

Phosphorylation of Specific Serine Residues of Maize Branching Enzymes Conditions Protein-Protein Interactions between SBEI and SBEIIb Isoforms.

Starch is widely used in food and non-food sectors. Its biosynthesis is a linear process known to involve at least five groups of committed enzymes: ADP-glucose pyrophosphorylase, starch synthases (SS), starch branching enzymes (SBE), debranching enzymes, and starch phosphorylase. Biochemical studies suggest that posttranslational modifications of starch branching enzymes by protein phosphorylation and protein-protein interactions are common processes in cereal endosperm, however, the biological significance in vivo has been unclear. Phosphorylation of Maize SBEIIb on Ser-649 has been observed in vitro by mass spectrometry and confirmed by site-directed mutagenesis of radiolabeled recombinant protein. In the present study, we observed phosphorylation of SBEIIb on Ser-649 and SBEI Ser-748 in intact amyloplasts of developing maize endosperm by using phospho-peptide specific antibodies. Phosphorylation of these sites was also observed during the transient and constitutive expression of maize SBEs in chloroplasts of Nicotiana benthamiana and Arabidopsis thaliana, respectively. In vitro affinity co-immunoprecipitationn, and in vivo bimolecular fluorescence complementation (BiFC) analysis demonstrated the pre-requisite for the phosphorylation of SBEIIb and SBEI in order to form a heteromeric complex in vivo. Protein-protein interactions between SSII and SBEs were also observed in vivo., The biological significance of phosphorylation-dependent, protein-protein interactions between branching enzymes was investigated in stable transgenic plants of Arabidopsis and maize, in which interactions were disrupted by site-directed mutagenesis. The effect of these altered interactions had a marked effect on starch structure, the results of which will be presented.

1 - University of Guelph, Molecular and Cellular Biology, Room 4466, Summerly Science Complex, 488 Gordon Street, Guelph, ON, N1G2W1, Canada
2 - University of Guelph, Molecular and Cellular Biology, Room 4471, Summerly Science Complex, 488 Gordon Street, Guelph, ON, N1G2W1, Canada
3 - University of Guelph, Molecular and Cellular Biology, MCB, Summerlee Science Complex, Guelph, ON, N1G2W1, Canada

Keywords:
Starch Biosynthesis
Phosphorylation
protein protein interactions
transgenic plants
Bimolecular fluorescence complementation (BiFC)
Co-immunoprecipitationn.

Presentation Type: Oral Paper:Papers for Topics
Session: 43
Location: Hall A/The Shaw Conference Centre
Date: Tuesday, July 28th, 2015
Time: 10:30 AM
Number: 43002
Abstract ID:480
Candidate for Awards:None