TARGETING OF AUXIN CARRIERS TO THE PLASMA-MEMBRANE - DIFFERENTIAL-EFFECTS OF BREFELDIN-A ON THE TRAFFIC OF AUXIN UPTAKE AND EFFLUX CARRIERS

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein s ecretion, 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 respons es result solely from perturbation of the efflux carrier or whether sp ecific auxin uptake carrier function is also affected, the in influenc e of BFA on the cellular transport of a range of auxins with contrasti ng affinities for specific auxin uptake and efflux carriers was invest igated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight add ition of BFA (3.10(-5) mol dm(-3)) caused a rapid (lag < 10 min) and s ubstantial (fourfold) increase in the rate of [1-C-14]IAA net uptake b y zucchini hypocotyl tissue. In the presence of the specific auxin eff lux carrier inhibitor N-l-naphthylphthalamic acid (NPA; 3 10(-6) mol d m(-3)), BFA slightly reduced the rate of [1-C-14]IAA net uptake. Stimu lation of [1-C-14]IAA net uptake by BFA was concentration-dependent. I n the absence of BFA, net uptake of [1-C-14]IAA exhibited the characte ristic biphasic response to increasing concentrations of competing col d IAA but in the presence of BFA, [1-C-14]IAA uptake decreased smoothl y with increase in concentration of competing unlabelled IAA, indicati ng a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-C-14]IAA fro m 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, zu cchini tissue of [1-C-14]2,4-dichlorophenoxyacetic acid ([1-C-14]2,4-D ), a substrate for the auxin uptake carrier but not the auxin efflux c arrier. Uptake of [1-C-14]2,4-D declined smoothly with increasing conc entrations 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-H-3]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 zucchin i cells.