Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport inCucurbita pepoL. Hypocotyl Segments

Abstract

Pretreatment of 2·0 mm segments of etiolated zucchini (Cucurbita pepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) eliminated the stimulation by N-1-naphthylphthalamic acid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA), but had relatively little effect on the net uptake of IAA in the absence of NPA. The efflux of [1-¹⁴C]IAA from preloaded segments was not substantially affected by inhibitor pretreatment in the absence of NPA, but CH pretreatment significantly inhibited the reduction of efflux caused by NPA. Pretreatment with CH or MDMP did not affect net uptake by segments of the pH probe [2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or the net uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu), suggesting that neither inhibitor affected intracellular pH or the general function of proton symporters in the plasma membrane. Both compounds reduced the incorporation of label from [³⁵S]methionine into trichloroacetic acid (TCA)-insoluble fractions of zucchini tissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesicles prepared from zucchini hypocotyl tissue was enhanced by the inclusion of NPA in the uptake medium. The stimulation by NPA of [³H]IAA association with microsomes was substantially reduced when the tissue was pretreated with CH. However, CH pretreatment did not affect the level of high affinity NPA binding to the membranes indicating that treatments did not result in loss of NPA receptors. It is suggested that the auxin transport site on the efflux carrier system and the receptor site for NPA may reside on separate proteins linked by a third, rapidly turned-over, transducing protein.