期刊
BIOCHEMICAL JOURNAL
卷 448, 期 -, 页码 373-387出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20120573
关键词
amyloplast; protein phosphorylation; protein-protein interaction; starch synthase; starch-branching enzyme; starch synthesis; sugary-2
资金
- Ontario Ministry of Agriculture, Food and Rural Affairs Bio-Products Research Grants [026262, 200172]
- Natural Sciences and Engineering Research Council (NSERC) Discovery Grants [262209, 341722]
- NSERC Strategic Grant [048237]
- Commonwealth Scientific and Industrial Research Organization (CSIRO) Sir Frederick McMaster Fellowship
The sugary-2 mutation in maize (Zen mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) Ha 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 (Gly(522) -> 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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