Antibodies to the tonoplast from the storage parenchyma cells of beetroot recognize a major intrinsic protein related to TIPs.
A vacuole membrane (= tonoplast) subfraction was purified by sedimentation in a discontinuous sucrose gradient (0.6 M/0.3 M) from a crude vacuole fraction isolated from red beetroot storage cells. The vacuole membrane-enriched fraction was injected into the popliteal lymph nodes of rabbits to raise polyclonal antibodies. The resultant serum was tested by indirect immunofluorescence microscopy on cryosections of shoot meristem. Antibodies specifically bound to the tonoplast but not the cytoplasm or the nucleus. By immunogold microscopy of ultrathin frozen sections, the anti-tonoplast antibodies were shown to label the luminal (exoplasmic) surface of the vacuole membrane and vesicular elements of the Golgi complex. Almost all of the antibodies were directed against a polypeptide with an M(r) of 27,000 as determined by Western blotting. A 30,000 M(r) polypeptide was occasionally detected in tonoplast-enriched fractions. It was weakly labeled by the crude immune serum, but not by the serum purified by affinity on the M(r) 27,000 band. The 27 kDa polypeptide which accounted for 10 to 15% of the total membrane proteins was partially characterized. The M(r) 27,000, but not the M(r) 30,000 polypeptide, had Triton X-114 binding properties and was extracted by chloroform:methanol, indicating its high hydrophobicity. On the basis of their partitioning in detergent/aqueous phases it is suggested that the 27 kDa and the 30 kDa polypeptides belong to the tonoplast and to the vacuolar sap, respectively. Neither polypeptide binds to Con A or is sensitive to Endo F digestion, suggesting that they are either unglycosylated polypeptides or glycopeptides with modified oligosaccharides. When separated by SDS-PAGE under non-reducing conditions, the 27 kDa antigen displays a small increase in apparent relative mobility. Its NH2-terminal amino acid sequence shares homology with plant members of the MIP channel family. As suggested by its relative molecular mass, its high hydrophobicity and its NH2-terminal amino acid sequence, the M(r) 27,000 polypeptide from the tonoplast of beetroot may be a new member of the TIP family. It appears to represent a useful specific marker for the vacuole membrane at all developmental stages of the storage parenchyma cells.
Marty-Mazars
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Laboratoire de Phyto-Biologie Cellulaire, Université de Bourgogne, Dijon/France.
Clémencet
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Dozolme
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P
Marty
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D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D010940:Plant Proteins / Q000032:analysis*; D018517:Plant Roots / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D000941:Antigens; D008565:Membrane Proteins; D010940:Plant Proteins; C077491:tonoplast intrinsic protein, plant