The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein–protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein–protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.
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Research Article|
November 21 2012
Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules
Fushan Liu;
Fushan Liu
*Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Nadya Romanova;
Nadya Romanova
*Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Elizabeth A. Lee;
Elizabeth A. Lee
†Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Regina Ahmed;
Regina Ahmed
‡Food Futures National Research Flagship and Division of Plant Industry, CSIRO, Canberra, ACT 2601, Australia
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Martin Evans;
Martin Evans
§Bragg Institute, ANSTO, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia
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Elliot P. Gilbert;
Elliot P. Gilbert
§Bragg Institute, ANSTO, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia
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Matthew K. Morell;
Matthew K. Morell
‡Food Futures National Research Flagship and Division of Plant Industry, CSIRO, Canberra, ACT 2601, Australia
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Michael J. Emes;
Michael J. Emes
*Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON, Canada, N1G 2W1
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Ian J. Tetlow
Ian J. Tetlow
1
*Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON, Canada, N1G 2W1
1To whom correspondence should be addressed (email itetlow@uoguelph.ca).
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Publisher: Portland Press Ltd
Received:
April 04 2012
Revision Received:
August 27 2012
Accepted:
September 10 2012
Accepted Manuscript online:
September 10 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 448 (3): 373–387.
Article history
Received:
April 04 2012
Revision Received:
August 27 2012
Accepted:
September 10 2012
Accepted Manuscript online:
September 10 2012
Citation
Fushan Liu, Nadya Romanova, Elizabeth A. Lee, Regina Ahmed, Martin Evans, Elliot P. Gilbert, Matthew K. Morell, Michael J. Emes, Ian J. Tetlow; Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules. Biochem J 15 December 2012; 448 (3): 373–387. doi: https://doi.org/10.1042/BJ20120573
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