RESUMO
Functional characterization of Arabidopsis thaliana GAT1 in heterologous expression systems, i.e. Saccharomyces cerevisiae and Xenopus laevis oocytes, revealed that AtGAT1 (At1g08230) codes for an H(+)-driven, high affinity gamma-aminobutyric acid (GABA) transporter. In addition to GABA, other omega-aminofatty acids and butylamine are recognized. In contrast to the most closely related proteins of the proline transporter family, proline and glycine betaine are not transported by AtGAT1. AtGAT1 does not share sequence similarity with any of the non-plant GABA transporters described so far, and analyses of substrate selectivity and kinetic properties showed that AtGAT1-mediated transport is similar but distinct from that of mammalian, bacterial, and S. cerevisiae GABA transporters. Consistent with a role in GABA uptake into cells, transient expression of AtGAT1/green fluorescent protein fusion proteins in tobacco protoplasts revealed localization at the plasma membrane. In planta, AtGAT1 expression was highest in flowers and under conditions of elevated GABA concentrations such as wounding or senescence.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Arabidopsis/química , Betaína/química , Ligação Competitiva , Transporte Biológico , Butilaminas/química , Membrana Celular/metabolismo , Senescência Celular , Clorofila/química , DNA/química , Eletrofisiologia , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glicina/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Modelos Químicos , Oócitos/metabolismo , Filogenia , Prolina/química , Ligação Proteica , Biossíntese de Proteínas , Protoplastos/metabolismo , RNA/química , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Nicotiana/metabolismo , Cicatrização , Xenopus/metabolismoRESUMO
The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding. Purified NAC can prevent protein aggregation in vitro and thus shows certain properties of a molecular chaperone. Interestingly, the alpha-subunit of NAC interacts with the 54 kDa subunit of SRP. Consistent with a regulatory role of NAC in protein translocation into the endoplasmic reticulum (ER), we find that deletion of NAC results in an induction of the ER stress-response pathway. These results identify L25 as a conserved interaction platform for specific cytosolic factors that guide nascent polypeptides to their proper cellular destination.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas Fúngicas/genética , Complexos Multiproteicos , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Ribossômicas/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K(m) = 0.1-0.3 mM), lower for Pro (K(m) = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K(m) = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.