Rolf Zettl

An Auxin Binding Protein is Localized in the Symbiosome Membrane of Soybean Nodules

Binding of tritiated indole-3-acetic acid ([3H]IAA) to symbiosome membranes of soybean nodules occurred in a protein-dependent manner and was competitively inhibited by unlabeled indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (1-NAA) and dithiothreitol (DTT), but not by tryptophan and benzoic acid. The symbiosome membranes bound IAA with a KD of 1 × 10-6 m. Photoaffinity labeling identified an auxin-binding protein (ABP) in the symbiosome membrane with an apparent molecular mass of 23 kDa. This 23 kDa protein was labeled either with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA) or with 5′-azido-[3,6-3H2]-1-naphthylphthalamic acid ([3H2]N3NPA). Labeling of the 23 kDa protein with [3H]N3IAA was competitively inhibited by unlabeled IAA and 1-NAA. NPA and quercetin, inhibitors of polar auxin transport, as well as rutin, a glycosylated derivative of quercetin, competed with IAA for binding. Conversely, [3H2]N3NPA labeling was inhibited by unlabeled IAA and NPA. The 23 kDa symbiosome membrane protein was partially solubilized with Triton X-100 and nearly completely using Triton X-114. The observation that auxin transport inhibitors compete with IAA for binding suggests that the symbiosome membrane ABP could be part of an auxin efflux carrier system required to control the auxin concentration in infected soybean nodule cells.

Auxin binding proteins are located in the ER and in the plasma membrane: Identification by photoaffinity-labelling and characterization

Plants modify gene expression and metabolism in response to a large variety of exogenous and endogenous signals. Among the various signals sensed by plants the phytohormones e.g. auxins, cytokinins, ethylene, abscisic acid and gibberellins, have received particular attention. It has been argued that, similar to hormone action in `vertebrates’ the first step in phytohormone action is the interaction of a ligand with binding sites, most likely proteins, located either at the plasma membrane or at various other intracellular locations. It is thought that binding of phytohormones to such proteins should be specific, reversible, of high affinity and saturable and result in a defined biological response.

Photoaffinity labeling and strategies for plasma membrane protein purification.

Publisher Summary This chapter discusses the photoaffinity labeling and strategies for plasma-membrane-protein purification. For the purification of membrane proteins solubilized with detergents, the same methods can be applied as those used to purify soluble proteins; these include gel filtration, ion-exchange, reversed-phase, and affinity chromatography. Auxins play a major role in the regulation of plant cell elongation, cell division, morphogenesis, and differentiation. The molecular sequence of events responsible for the mode of action of auxins is not well understood. Receptor-like proteins that bind auxin and transmit the auxin signal have been postulated. Because of the low concentration of most of these proteins, conventional approaches to identify and characterize them for molecular studies have been fraught with technical difficulties. To overcome problems associated with traditional auxin-binding studies, photolabile synthetic auxins may provide a valuable tool with which to tag auxin-binding proteins. Photoaffinity probes have contributed greatly to the identification of receptor proteins and have aided in structural studies.