Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, γ-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Dual functioning of plant arginases provides a third route for putrescine synthesis.

Two biosynthetic routes are known for putrescine, an essential plant metabolite. Ornithine decarboxylase (ODC) converts ornithine directly to putrescine, while a second route for putrescine biosynthesis utilizes arginine decarboxylase (ADC) to convert arginine to agmatine, and two additional enzymes, agmatine iminohydrolase (AIH) and N-carbamoyl putrescine aminohydrolase (NLP1) to complete this pathway. Here we show that plants can use ADC and arginase/agmatinase (ARGAH) as a third route for putrescine synthesis. Transformation of Arabidopsis thaliana ADC2, and any of the arginases from A. thaliana (ARGAH1, or ARGHA2) or the soybean gene Glyma.03g028000 (GmARGAH) into a yeast strain deficient in ODC, fully complemented the mutant phenotype. In vitro assays using purified recombinant enzymes of AtADC1 and AtARGAH2 were used to show that these enzymes can function in concert to convert arginine to agmatine and putrescine. Transient expression analysis of the soybean genes (Glyma.06g007500, ADC; Glyma.03g028000 GmARGAH) and the A. thaliana ADC2 and ARGAH genes in leaves of Nicotiana benthamiana, showed that these proteins are localized to the chloroplast. Experimental support for this pathway also comes from the fact that expression of AtARGAH, but not AtAIH or AtNLP1, is co-regulated with AtADC2 in response to drought, oxidative stress, wounding, and methyl jasmonate treatments. Based on the high affinity of ARGAH2 for agmatine, its co-localization with ADC2, and typically low arginine levels in many plant tissues, we propose that these two enzymes can be major contributors to putrescine synthesis in many A. thaliana stress responses.