ABSTRACT
The two plasma-membrane (PM) markers: 1,3-ß-glucan synthase and naphthylphthalamic-acid-binding capacity are localized in two peaks of activity on isopycnic Ficoll/D2O gradients prepared from a zucchini (Cucurbita pepo L.) hypocotyl post-microsomal fraction. The denser peak overlaps with the major distribution of clathrin and represents a region of the gradient enriched in coated vesicles (cv). Further purification of the pooled cv-fractions has shown that these PM marker activities are not borne by the cv, but are instead carried by smooth membrane fragments also present in these fractions. As judged from the results of Western blotting with polyclonal antibodies prepared against the 100-kilodalton (kDa) subunit of a PM H(+)-ATPase and the 70-kDa subunit of a tonoplastic H(+)-ATPase, these contaminants are of both PM and endomembrane origin. The PM contaminants however, differ from phase-partitioning- and free-flow-electrophoresis-purified PM prepared from microsomal fractions of zucchini hypocotyls in terms of their bouyant density in Ficoll/D2O gradients. Moreover, they do not appear to be present as sealed, outside-out, vesicles. Highly purified cv fractions from zucchini hypocotyls cross-react with subunit antibodies from both vacuolar and PM H (+)-ATPases. These results are discussed in terms of recent findings on cv ATPases from bovine brain.
ABSTRACT
The transmembrane transport of indole-3-acetic acidd (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) by 2 mm segments of dark-grown Cucurbita pepo L. hypocotyls involves nonme-diated diffusion of the neutral protonated auxin species as well as carrier-mediated components for influx and efflux. The efflux carrier is blocked by 2,3,5-triiodobenzoic acid (TIBA), a noncompetitive inhibitor of polar auxin transport which is itself transported in a polar fashion. The transmembrane transport of TIBA includes diffusion of the protonated species (common to lipophilic weak acids) and also a carrier-mediated efflux component which can be blocked by IAA, 2,4-D, 1-naphthylacetic acid (NAA), and by 1-naphthylphthalamic acid (NPA) which, like TIBA, noncompetitively inhibits polar auxin transport but is not itself polarly transported. The non-auxin benzoic acid has no effect on auxin or TIBA transport. No significant metabolism of IAA, 2,4-D, or TIBA occurred during the experimental periods. A model is proposed for the auxin efflux carrier in which auxins and TIBA have separate specific binding sites; occupation of either one of the sites allows ligand translocation whereas occupation of both sites prevents any transport. A third specific site may be necessary to account for the inhibitory effects of NPA. The consequences of cytoplasmic acidification which can accompany lipophilic weak acid uptake are discussed.