Cycling of auxin-binding protein through the plant cell: pathways in auxin signal transduction.

The auxin receptor literature contains a glaring discrepancy that invites explanation. While some physiological experiments suggest that active auxin receptors are sited inside the cell, others point to action at the cell surface. Furthermore, although the major auxin-binding protein (ABP) of maize (Zea mays) coleoptiles is found in the lumen of the endoplasmic reticulum (ER), exogenous ABP can mediate auxin-dependent changes in the plasma membrane potential of protoplasts. How can an ER protein mediate changes in cell potential? To resolve this dilemma, I propose that ABP cycles through the cell. In response to auxin, ABP is released from the ER and follows a secretory pathway to the cell surface. After secretion, ABP would bind sites on the cell surface and become subject to endocytosis, cycling back to the ER. Elevated auxin would accelerate the cycling of ABP between the ER and the cell surface. If cell wall precursors interacted with ABP during their progression through the secretory pathway, this would provide a mechanism for regulating cell wall synthesis. At the cell surface ABP would regulate an enzyme responsible for maintaining membrane potential. Both of these responses are components of auxin-regulated growth. This hypothesis does not exclude other mechanisms of signal transduction, particularly in gene regulation.

Solubilized auxin-binding protein : Subcellular localization and regulation by a soluble factor from homogenates of corn shoots.

Discontinuous sucrose gradient fractionations indicate that the high-affinity auxin binding protein which can be solubilized from the microsomes of coleoptiles and primary leaves of Zea mays L. seedlings is probably located in the endoplasmic reticulum (ER). Since aromatic hydroxylations are enzymatic activities typical of the ER of plant cells, we have examined the effects of several electron-transport inhibitors on the binding of 1-naphthylacetic acid (NAA). NaN3 strongly inhibits this binding, but KCN and CO do not. Trans-cinnamic acid and trans-p-coumaric acid, which are the substrates of ER hydroxylase activities in plants (but which are themselves not auxins), also inhibit this binding. Supernatant fractions from corn shoots contain factors inhibitory to the binding of NAA to the intact membranes and solubilized Site I auxin-binding protein. Here we show that these factors are competitive inhibitors of the binding of [(14)C]NAA but do not change the apparent affinity of the protein for indoleacetic acid, 2,4-dichlorophenoxyacetic acid or naphthoxyacetic acid. Several tissues were assayed for factors inhibitory to auxin binding to the solubilized protein, but only supernants from corn shoots were markedly inhibitory at low concentrations.

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays.

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-(14)C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 x 10(-8) molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-(14)C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.

Auxin receptors of maize coleoptile membranes do not have ATPase activity.

Membrane-localized auxin-binding sites from coleoptiles and primary leaves of Zea mays L. which may be auxin receptors can be fully solubilized by 1 to 1.5 mg of Triton X-100 per mg of membrane protein (about 1 mg per gram of original tissue fresh weight), while 70% of the basal (Mg(2+))-ATPase and 85% of the K(+)-stimulated (Mg(2+))-ATPase (pH 6) remain pelletable. Gel exclusion chromatography on Bio-Gel A-1.5m indicates that the solubilized receptors occur as detergent-protein micelles of about 90,000 daltons equivalent molecular weight. Solubilized ATPase activities occur (a) as very large particles excluded from the gel, and (b) as particles of a size substantially smaller than the particles that exhibit auxin binding. The auxin-binding receptor therefore appears not to be an ATPase.

Labeling of membranes from erythrocytes and corn with fluorescamine.

Fluorescamine was used as a fluorescent label for intact human erythrocytes and slices of corn coleoptile tissue. This reagent has a greater affinity for membranous than for soluble proteins, and also labels membrane lipids which contain primary amine groups. In addition, some membrane fractions from labeled coleoptiles have a higher affinity for fluorescamine than do others. The relative labeling of the various fractions can be altered by changing the pH of the external labeling medium. Because the pH of the medium determines the rate of hydrolysis of fluorescamine to an unreactive form, this result suggests that the specificity of this reagent towards different cellular structures is determined by the lifetime of the active reagent. Fluorescamine was not found to be a specific reagent for the cell surface.