Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana |
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Authors: | Fettke Joerg Nunes-Nesi Adriano Fernie Alisdair R Steup Martin |
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Institution: | a Mass Spectrometry of Biopolymers, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, 14476 Potsdam-Golm, Germany b Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, 14476 Potsdam-Golm, Germany c Max-Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14469 Potsdam-Golm, Germany |
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Abstract: | Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants. |
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Keywords: | DMSO dimethylsulfoxide FITC fluorescein isothiocyanate HIP1 3 heteroglycan interacting protein No 1 3 PMSF phenylmethylsulfonyl fluoride HPAEC-PAD high performance anion exchange chromatography coupled to pulsed amperomeric detection SHG water soluble heteroglycan SHGS water soluble heteroglycan with an apparent size below 10 kDa SHGL water soluble heteroglycan with an apparent size of more than 10 kDa |
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