An Optimized Screen Reduces the Number of GA Transporters and Provides Insights Into Nitrate Transporter 1/Peptide Transporter Family Substrate Determinants.

Based on recent in vitro data, a relatively large number of the plant nitrate transporter 1/peptide transporter family (NPF) proteins have been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes, wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities toward organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis suggests distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.

Origin and evolution of transporter substrate specificity within the NPF family.

Despite vast diversity in metabolites and the matching substrate specificity of their transporters, little is known about how evolution of transporter substrate specificities is linked to emergence of substrates via evolution of biosynthetic pathways. Transporter specificity towards the recently evolved glucosinolates characteristic of Brassicales is shown to evolve prior to emergence of glucosinolate biosynthesis. Furthermore, we show that glucosinolate transporters belonging to the ubiquitous NRT1/PTR FAMILY (NPF) likely evolved from transporters of the ancestral cyanogenic glucosides found across more than 2500 species outside of the Brassicales. Biochemical characterization of orthologs along the phylogenetic lineage from cassava to A. thaliana, suggests that alterations in the electrogenicity of the transporters accompanied changes in substrate specificity. Linking the evolutionary path of transporter substrate specificities to that of the biosynthetic pathways, exemplify how transporter substrate specificities originate and evolve as new biosynthesis pathways emerge.

Characterization of different crystal forms of the alpha-glucosidase MalA from Sulfolobus solfataricus.

MalA is an alpha-glucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus. It belongs to glycoside hydrolase family 31, which includes several medically interesting alpha-glucosidases. MalA and its selenomethionine derivative have been overproduced in Escherichia coli and crystallized in four different crystal forms. Microseeding was essential for the formation of good-quality crystals of forms 2 and 4. For three of the crystal forms (2, 3 and 4) full data sets could be collected. The most suitable crystals for structure determination are the monoclinic form 4 crystals, belonging to space group P2(1), from which data sets extending to 2.5 A resolution have been collected. Self-rotation functions calculated for this form and for the orthorhombic (P2(1)2(1)2(1)) form 2 indicate the presence of six molecules in the asymmetric unit related by 32 symmetry.

Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family.

The NAC domain (residues 1-168) of ANAC, encoded by the abscisic acid-responsive NAC gene from Arabidopsis thaliana, was recombinantly produced in Escherichia coli and crystallized in hanging drops. Three morphologically different crystal forms were obtained within a relatively narrow range of conditions: 10-15% PEG 4000 and 0.1 M imidazole/malic acid buffer pH 7.0 in the reservoir, 3.2-7.7 mg ml(-1) protein stock and a 1:1 ratio of reservoir to protein solution in the hanging drop. One of the crystal forms, designated crystal form III, was found to be suitable for further X-ray analysis. Form III crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.0, b = 75.2, c = 80.8 A at 100 K. The unit-cell volume is consistent with two molecules in the asymmetric unit and a peak in the native Patterson map suggests the presence of a non-crystallographic twofold axis parallel to a crystallographic axis. Size-exclusion chromatography of the NAC domain showed that the dimeric state is also the preferred state in solution and probably represents the biologically active form. Data sets were collected from four potential heavy-atom derivatives of the form III crystals. The derivatized crystals are reasonably isomorphous with the non-derivatized crystals and the four data sets are being evaluated for use in structure determination by multiple isomorphous replacement.