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Characterization of auxin-binding proteins from zucchini plasma membrane

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Abstract

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948–4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or mutimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

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Abbreviations

HPLC:

high-pressure liquid chromatography

IAA:

indole-3-acetic acid

azido-IAA:

5-N3-7-3H-IAA

pI:

isoelectric point

PM:

plasma membrane

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. (1989) Short protocols in molecular biology. Wiley, New York

  2. Bayley, H., Knowles, J.R. (1977) Photoaffinity labeling. Methods Enzymol. 46, 69–114

  3. Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607

  4. Chowdhry, V., Westheimer, F.H. (1979) Photoaffmity labeling of biological systems. Annu. Rev. Biochem. 48, 293–325

  5. Cross, J.W. (1985) Auxin action: the reach for the receptor. Plant Cell Environ. 8, 352–359

  6. Davies, P.J. (1987) Plant hormones and their role in plant growth and development. Nijhof, Dordrecht

  7. Dunbar, B.S. (1987) Two-dimensional electrophoresis and immunological techniques. Plenum, New York

  8. Feldwisch, J., Zettl, R., Hesse, F., Schell, J., Palme, K. (1992) An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: Identification by photoaffinity labeling and purification of a 23 kDa polypeptide. Proc. Natl. Acad. Sci., USA 89, 475–479

  9. Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Plant Physiol. 28, 439–478

  10. Hames, B.D. (1981) Gel electrophoresis of proteins. IRL Press, Oxford

  11. Hertel, R., Lomax, T.L., Briggs, W.R. (1983) Auxin transport in membrane vesicles from Cucurbita pepo L. Planta 157, 193–201

  12. Hesse, T., Feldwisch, J., Balshusemann, D., Bauw, G., Puype, M., Vandekerckhove, J., Löbler, M., Klämbt, D., Schell, J., Palme, K. (1989) Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin. EMBO J. 8, 2453–2461

  13. Heukeshoven, J., Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103–112

  14. Hicks, G.R., Rayle, D.L., Jones, A.M., Lomax, T.L. (1989a) Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin. Proc. Natl. Acad. Sci., USA 86, 4948–4952

  15. Hicks, G.R., Rayle, D.L., Lomax, T.L. (1989b) The diageotropica mutant of tomato lacks high specific activity auxin binding sites. Science 245, 52–54

  16. Inohara, N., Shimomura, S., Fukui, T., Futai, M. (1989) Auxinbinding protein located in the endoplasmic reticulum of maize shoots: Molecular cloning and complete primary sequence. Proc. Natl. Acad. Sci. USA 86, 3564–3568

  17. Jones, A.M., Venis, M.A. (1989) Photoaffinity labeling of an indole-3-acetic acid-binding protein in maize. Proc. Natl. Acad. Sci. USA 86, 6153–6156

  18. Jones, A.M., Melhado, L.L., Ho, T-H.D., Leonard, N.J. (1984a) Azido auxins. Quantitative binding data in maize. Plant Physiol. 74, 295–301

  19. Jones, A.M., Melhado, L.L., Ho, T-H.D., Pearce, J., Leonard, N.J. (1984b) Azido auxins. Photoaffinity labeling of auxin-binding proteins in maize coleoptile with tritiated 5-azidoindole-3-acetic acid. Plant Physiol. 75, 1111–1116

  20. Kehry, M.R., Dahlquist, F.W. (1982) The methyl-accepting chemotaxis proteins of Escherichia coli: Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I. J. Biol. Chem. 257, 10378–10386

  21. Klekamp, M.S., Weil, P.A. (1987) Properties of yeast class III gene transcription factor TFIIIB. J. Biol. Chem. 262, 7878–7883

  22. Libbenga, K.R., Mennes, A.M. (1987) Hormone binding and its role in hormone action. In: Plant hormones and their role in plant growth and development, pp. 194–221, Davies, P.J., ed., Nijhoff, Dordrecht

  23. Lomax, T.L., Conley, P.B., Schilling, J., Grossman, A.R. (1987) Isolation and characterization of light regulated phycobilisome linker polypeptide genes and their transcription as a polycistronic mRNA. J. Bacteriol. 169, 2675–2684

  24. Lomax, T.L., Hicks, G.R. (1992) Specific auxin binding proteins in the plasma membrane: Receptors or transporters? Biochem. Soc. Trans. 20, 64–69

  25. Lomax, T.L., Melhorn, R.J., Briggs, W.R. (1985) Active auxin uptake by zucchini membrane vesicles: Quantitation using ESR volume and ΔpH determinations. Proc. Natl. Acad. Sci. USA 82, 6541–6545

  26. Maher, P.A., Singer, S.J. (1985) Anomalous interaction of the acetylcholine receptor protein with the nonionic detergent Triton X-114. Proc. Natl. Acad. Sci. USA 82, 958–962

  27. Mcdonald, H., Jones, A.M., King, P. (1991) Photoaffinity labeling of soluble auxin-binding proteins. J. Biol. Chem. 266, 7393–7399

  28. Melhado, L.L., Jones, A.M., Leonard, N.J., Vanderhoef, L.N. (1981) Azido auxins: Synthesis and biological activity of fluorescent photoaffinity labeling agents. Plant Physiol. 68, 469–475

  29. Napier, R.M., Venis, M.A. (1990) Receptors for plant growth regulators: Recent advances. J. Plant Growth Regul. 9, 113–126

  30. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021

  31. Prasad, P.V., Jones, A.M. (1991) Putative receptor for the plant hormone auxin identified and characterized by anti-idiotypic antibodies. Proc. Natl. Acad. Sci. USA 88, 5479–5483

  32. Pryde, J.G., Phillips, J.H. (1986) Fractionation of membrane protein by temperature-induced phase separation in Triton X-114. Biochem. J. 233, 525–533

  33. Rubery, P.H., Sheldrake, R.A. (1974) Carrier mediated auxin transport. Planta 118, 101–121

  34. Rubery, P.H. (1981) Auxin receptors. Annu. Rev. Plant Physiol. 32, 569–596

  35. Ruoho, A., Rashidbaigi, A., Roeder, P. (1984) Approaches to the identification of receptors utilizing photoaffinity labeling. In: Receptor biochemistry and methodology, 1, pp. 119–160, Venter, C.J., Harrison, L.C., ed. Alan R. Liss, New York

  36. Spector, T. (1987) Refinement of Coomassie blue method of protein quantitation. Anal. Biochem. 86, 142–146

  37. Tillmann, U., Viola, G., Kayser, B., Siemeister, G., Hesse, T., Palme, K., Löbler, M., Klämbt, D. (1989) cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): Isolation and characterization by immunological methods. EMBO J. 8, 2463–2467

  38. Zettl, R., Feldwisch, J., Boland, W., Schell, J., Palme, K. (1992) 5′-azido-[3,6-3H2]-1-naphthylphthalamic acid, a photoactivatable probe for napthylphthalamic acid receptor proteins from higher plants: Identification of a 23 kDa protein from maize coleoptile plasma membranes. Proc. Natl. Acad. Sci. USA 89, 480–484

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Author information

Correspondence to Terri L. Lomax.

Additional information

We thank R. Hopkins and I. Gelford for excellent technical work and our colleagues, especially T. Wolpert and D.L. Rayle, for many helpful discussions. This work was supported by grants to T.L.L. from National Science Foundation (DCB 8904114), National Aeronautics and Space Administration (NAGW 1253) and by a grant to D.L. Rayle and T.L.L. from U.S. Department of Agriculture (90-37261-5779). G.R.H. is supported by a National Aeronautics and Space Administration Predoctoral Fellowship (NGT 50455).

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Hicks, G.R., Rice, M.S. & Lomax, T.L. Characterization of auxin-binding proteins from zucchini plasma membrane. Planta 189, 83–90 (1993). https://doi.org/10.1007/BF00201348

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Key words

  • Auxin
  • Azido-indole-3-acetic acid
  • Cucurbita (auxin-binding protein)
  • Plasma membrane
  • Protein (auxin-binding)