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 inside-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% Brii-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 1. cv Burpee Fordhook).The auxins are a class of phytohormones that control plant growth, development, and response to the environment through regulation of elongation, cell division, differentiation, and tropic responses to light and gravity. Auxins, of which IAA is the predominant naturally occurring type, are delivered to plant cells by a unique polar transport mechanism (for review, see Goldsmith, 1977;Lomax et al., 1995). Polar transport is critica1 for the growth and development of plants and their ability to respond to environmental stimuli. Auxins are transported from their source in the shoot apex at the growing tip toward the base of stems. Polar auxin transport depends on the proton gradient across the plasma membrane of plant cells and has been described as chemiosmotic (Rubery and Sheldrake, 1974). The ionization states of IAA in the extracellular space and in the cytoplasm affect the membrane permeability of this phytohormone and lead to an accumulationThe work performed at Wake Forest University was supported
421of the anionic form of the molecule within the cytoplasm. Polar transport is believed to be mediated by an uptake 2H+/IAA-symport carrier (Lomax et al., 1985;Sabater and Rubery, 1987) and an IAA-efflux carrier (Rubery and Sheldrake, 1974). Basal localization of this IAA-efflux carrier has been proposed to drive the polarity of IAA transport (Rubery and Sheldrake, 1974;Jacobs and Gilbert, 1983).Severa1 synthetic auxin transport inhibitors, or phytotropins, have been identified, but NPA is the best characterized (Rubery, 1990). These phytotropins specifically block auxin transport at the site of the efflux carrier (Rubery, 1990). Natural inhibitors, or flavonoids, have also been shown to block transport at the site of the efflux carrie...
