Limits...
Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

Show MeSH

Related in: MedlinePlus

Influence of boron on glycosylinositol phosphorylceramide (GIPC) extraction. (a) A cloudy layer was observed during butanol/water phase-partitioning of a GIPC-enriched lipid sample extracted with neutral ethanol from Rosa cell cultures that had been grown in the usual B concentration (i, iv). This cloudy layer disappeared in the presence of 0.1 m HCl (iii, vii), 10 mm βMCD (ii), or 6 mm borate buffer, pH 9.2 (vi). The horizontal arrow indicates the slight cloudy layer left in the presence of βMCD (butanol above). In contrast, 6 mm ammonium buffer, pH 9.2 (v), only led to a partial disappearance. (b) TLC of the different phases after butanol/water phase-partitioning of a GIPC-rich lipid extract from Rosa cell cultures grown in media with (B+) or without boron (B−). The lipids had been extracted in 70% ethanol that contained 0.1 m HCl (H+) or lacking acid (H−). BP, butanol phase; CL, cloudy layer; AP, aqueous phase; Suc, sucrose (marker). In lanes 9 and 10, 10 mm βMCD was present during the partitioning step. Lipids labelled on lane 10: bands 1–3, as in Figure1; band 4, (Pent)2-(Hex)2-HexA-Ins-P-Cer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230332&req=5

fig02: Influence of boron on glycosylinositol phosphorylceramide (GIPC) extraction. (a) A cloudy layer was observed during butanol/water phase-partitioning of a GIPC-enriched lipid sample extracted with neutral ethanol from Rosa cell cultures that had been grown in the usual B concentration (i, iv). This cloudy layer disappeared in the presence of 0.1 m HCl (iii, vii), 10 mm βMCD (ii), or 6 mm borate buffer, pH 9.2 (vi). The horizontal arrow indicates the slight cloudy layer left in the presence of βMCD (butanol above). In contrast, 6 mm ammonium buffer, pH 9.2 (v), only led to a partial disappearance. (b) TLC of the different phases after butanol/water phase-partitioning of a GIPC-rich lipid extract from Rosa cell cultures grown in media with (B+) or without boron (B−). The lipids had been extracted in 70% ethanol that contained 0.1 m HCl (H+) or lacking acid (H−). BP, butanol phase; CL, cloudy layer; AP, aqueous phase; Suc, sucrose (marker). In lanes 9 and 10, 10 mm βMCD was present during the partitioning step. Lipids labelled on lane 10: bands 1–3, as in Figure1; band 4, (Pent)2-(Hex)2-HexA-Ins-P-Cer.

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.


Glycosylinositol phosphorylceramides from Rosa cell cultures are boron-bridged in the plasma membrane and form complexes with rhamnogalacturonan II.

Voxeur A, Fry SC - Plant J. (2014)

Influence of boron on glycosylinositol phosphorylceramide (GIPC) extraction. (a) A cloudy layer was observed during butanol/water phase-partitioning of a GIPC-enriched lipid sample extracted with neutral ethanol from Rosa cell cultures that had been grown in the usual B concentration (i, iv). This cloudy layer disappeared in the presence of 0.1 m HCl (iii, vii), 10 mm βMCD (ii), or 6 mm borate buffer, pH 9.2 (vi). The horizontal arrow indicates the slight cloudy layer left in the presence of βMCD (butanol above). In contrast, 6 mm ammonium buffer, pH 9.2 (v), only led to a partial disappearance. (b) TLC of the different phases after butanol/water phase-partitioning of a GIPC-rich lipid extract from Rosa cell cultures grown in media with (B+) or without boron (B−). The lipids had been extracted in 70% ethanol that contained 0.1 m HCl (H+) or lacking acid (H−). BP, butanol phase; CL, cloudy layer; AP, aqueous phase; Suc, sucrose (marker). In lanes 9 and 10, 10 mm βMCD was present during the partitioning step. Lipids labelled on lane 10: bands 1–3, as in Figure1; band 4, (Pent)2-(Hex)2-HexA-Ins-P-Cer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230332&req=5

fig02: Influence of boron on glycosylinositol phosphorylceramide (GIPC) extraction. (a) A cloudy layer was observed during butanol/water phase-partitioning of a GIPC-enriched lipid sample extracted with neutral ethanol from Rosa cell cultures that had been grown in the usual B concentration (i, iv). This cloudy layer disappeared in the presence of 0.1 m HCl (iii, vii), 10 mm βMCD (ii), or 6 mm borate buffer, pH 9.2 (vi). The horizontal arrow indicates the slight cloudy layer left in the presence of βMCD (butanol above). In contrast, 6 mm ammonium buffer, pH 9.2 (v), only led to a partial disappearance. (b) TLC of the different phases after butanol/water phase-partitioning of a GIPC-rich lipid extract from Rosa cell cultures grown in media with (B+) or without boron (B−). The lipids had been extracted in 70% ethanol that contained 0.1 m HCl (H+) or lacking acid (H−). BP, butanol phase; CL, cloudy layer; AP, aqueous phase; Suc, sucrose (marker). In lanes 9 and 10, 10 mm βMCD was present during the partitioning step. Lipids labelled on lane 10: bands 1–3, as in Figure1; band 4, (Pent)2-(Hex)2-HexA-Ins-P-Cer.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Edinburgh, EH9 3JH, UK.

Show MeSH
Related in: MedlinePlus