Chou YH, Pogorelko G, Zabotina O

Xyloglucan is the major hemicellulosic polysaccharide in the primary cell walls of most vascular dicotyledonous plants, and has important structural and physiological functions in plant growth and development. In Arabidopsis thaliana, the 1,4-β-glucan synthase CSLC4 and three xylosyltransferases, XXT1, XXT2, and XXT5, act in Golgi to form the xylosylated glucan ...

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backbone during xyloglucan biosynthesis. However, the functional organization of these enzymes in the Golgi membrane is currently unknown. In this study, we used Bimolecular Fluorescence Complementation (BiFC) and in vitro pull-down assays to investigate the supramolecular organization of the CSLC4, XXT1, XXT2 and XXT5 proteins in Arabidopsis protoplasts. Quantification of BiFC fluorescence by flow cytometry allowed us to perform competition assays which demonstrated the high probability of protein-protein complex formation in vivo and revealed differences in the abilities of these proteins to form multiprotein complexes. Results of in vitro pull-down assays using recombinant proteins confirmed that the physical interactions among XXTs occur through their catalytic domains. Additionally, co-immunoprecipitation of XXT2YFP and XXT5HA proteins from Arabidopsis protoplasts indicated that while the formation of the XXT2-XXT2 homo-complex involves disulfide bonds, the formation of the XXT2 -XXT5 hetero-complex does not involve covalent interactions. The combined data allow us to propose that the proteins involved in xyloglucan biosynthesis function in a multiprotein complex composed of at least two homo-complexes, CSLC4-CSLC4 and XXT2 -XXT2, and three hetero-complexes, XXT2-XXT5, XXT1-XXT2, and XXT5-CSLC4.

Date: Jun. 04, 2012

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