Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Abstract.

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells.