Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1996 Sep;112(1):421–432. doi: 10.1104/pp.112.1.421

Cytoplasmic Orientation of the Naphthylphthalamic Acid-Binding Protein in Zucchini Plasma Membrane Vesicles.

M W Dixon 1, J A Jacobson 1, C T Cady 1, G K Muday 1
PMCID: PMC157964  PMID: 12226399

Abstract

Polar transport of the plant hormone auxin is blocked by substances such as N-1-naphthylphthalamic acid (NPA), which inhibit auxin efflux and block polar auxin transport. To understand how auxin transport is regulated in vivo, it is necessary to discern whether auxin transport inhibitors act at the intra- or extracellular side of the plasma membrane. Populations of predominantly in-side-in plasma membrane vesicles were subjected to treatments that reverse the orientation. These treatments, which included osmotic shock, cycles of freezing and thawing, and incubation with 0.05% Brij-58, all increased NPA-binding activity and the accessibility of the binding protein to protease digestion. Marker activities for inside-out vesicles also increased, indicating that these treatments act by altering the membrane orientation. Finally, binding data were analyzed by multiple analyses and indicated that neither the affinity nor abundance of binding sites changed. Kinetic analyses indicated that the change in NPA-binding activity by Brij-58 treatment was due to an increase in the initial rates of both association and dissociation of this ligand. These experiments indicated that the NPA-binding site is on the cytoplasmic face of the plasma membrane in zucchini (Cucurbita pepo L. cv Burpee Fordhook).

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernasconi P., Patel B. C., Reagan J. D., Subramanian M. V. The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein. Plant Physiol. 1996 Jun;111(2):427–432. doi: 10.1104/pp.111.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloch R. J. Actin at receptor-rich domains of isolated acetylcholine receptor clusters. J Cell Biol. 1986 Apr;102(4):1447–1458. doi: 10.1083/jcb.102.4.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Jacobs M., Gilbert S. F. Basal localization of the presumptive auxin transport carrier in pea stem cells. Science. 1983 Jun 17;220(4603):1297–1300. doi: 10.1126/science.220.4603.1297. [DOI] [PubMed] [Google Scholar]
  4. Jacobs M., Rubery P. H. Naturally occurring auxin transport regulators. Science. 1988 Jul 15;241(4863):346–349. doi: 10.1126/science.241.4863.346. [DOI] [PubMed] [Google Scholar]
  5. Johansson F., Olbe M., Sommarin M., Larsson C. Brij 58, a polyoxyethylene acyl ether, creates membrane vesicles of uniform sidedness. A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles. Plant J. 1995 Jan;7(1):165–173. doi: 10.1046/j.1365-313x.1995.07010165.x. [DOI] [PubMed] [Google Scholar]
  6. Lomax T. L., Mehlhorn R. J., Briggs W. R. Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6541–6545. doi: 10.1073/pnas.82.19.6541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Muday G. K., Brunn S. A., Haworth P., Subramanian M. Evidence for a Single Naphthylphthalamic Acid Binding Site on the Zucchini Plasma Membrane. Plant Physiol. 1993 Oct;103(2):449–456. doi: 10.1104/pp.103.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Muday G. K., Haworth P. Tomato root growth, gravitropism, and lateral development: correlation with auxin transport. Plant Physiol Biochem. 1994 Mar-Apr;32(2):193–203. [PubMed] [Google Scholar]
  9. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  10. Palmgren M. G. An H-ATPase Assay: Proton Pumping and ATPase Activity Determined Simultaneously in the Same Sample. Plant Physiol. 1990 Nov;94(3):882–886. doi: 10.1104/pp.94.3.882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Palmgren M. G., Askerlund P., Fredrikson K., Widell S., Sommarin M., Larsson C. Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation. Plant Physiol. 1990 Apr;92(4):871–880. doi: 10.1104/pp.92.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Palmgren M. G., Sommarin M. Lysophosphatidylcholine stimulates ATP dependent proton accumulation in isolated oat root plasma membrane vesicles. Plant Physiol. 1989 Jul;90(3):1009–1014. doi: 10.1104/pp.90.3.1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rubery P. H. Phytotropins: receptors and endogenous ligands. Symp Soc Exp Biol. 1990;44:119–146. [PubMed] [Google Scholar]
  14. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  15. Sussman M. R., Gardner G. Solubilization of the receptor for N-1-naphthylphthalamic Acid. Plant Physiol. 1980 Dec;66(6):1074–1078. doi: 10.1104/pp.66.6.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yoshihara C. M., Hall Z. W. Increased expression of the 43-kD protein disrupts acetylcholine receptor clustering in myotubes. J Cell Biol. 1993 Jul;122(1):169–179. doi: 10.1083/jcb.122.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES