Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.
Bottom Line: Using high-voltage paper electrophoresis, we showed that addition of GIPCs decreased the electrophoretic mobility of radiolabelled RG-II, suggesting formation of a GIPC-B-RG-II complex.We conclude that B plays a structural role in the plasma membrane.Finally, our results suggest a role for GIPCs in the RG-II dimerization process.
Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.Show MeSH
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Mentions: To explore the putative existence of borate-bridged GIPC in vivo, we took advantage of aqueous solubility of GIPCs (Markham et al., 2006) and investigated the effect of boric acid (H3BO3) and HCl treatment on GIPC extractability. We extracted GIPCs from rose cell cultures grown with (B+) or without (B−) the routine concentration (3.3 μm) of H3BO3. Also, as cold 0.1 m HCl is able to hydrolyse the borate diester linkage in RG-II, we used acidic aqueous ethanol (H+) or non-acidic aqueous ethanol (H−). When B+ cultures were investigated, the use of H− as extractant resulted in the appearance of a prominent cloudy layer during the subsequent butan-1-ol/water phase-partitioning, interpreted as B-bridged lipid-rich material (Figure2a,d). This layer was less abundant in B−H−than in B+H−, and nearly absent in B−H+ and B+H+ (data not shown). Adding 0.1 m HCl to the B+H−sample during the butanol/water phase-partition step made this cloudy layer disappear (Figure2a-iii,vii). In the B−H+ and B+H+ samples (lacking a cloudy layer, as mentioned) no cloudiness subsequently formed after removal of the acidity by neutral acetone washes, suggesting that the disappearance of the cloudy layer was not simply pH dependent. As a consequence, a chemical modification such as the disruption of borate bridging must be responsible for the non-formation or the disappearance of the cloudy layer. Likewise, excess borate buffer, which could potentially disrupt B bridges (figure 7 of Bassil et al., 2004), solubilised all the cloudy layer, whereas ammonium buffer at the same concentration and pH did not (Figure2a-v,vi). Finally, 10 mm methyl β-cyclodextrin (βMCD), a cholesterol- and phytosterol-complexing agent that is capable of disrupting detergent-insoluble glycolipid-enriched complexes (potentially lipid rafts; Roche et al., 2008), solubilised most of, but not all, the cloudy layer (Figure2a-ii). The cloudy layer left was solubilised by addition of 0.1 m HCl.
Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.