RESUMEN
Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.
Asunto(s)
Cloroplastos/genética , Guanosina Tetrafosfato/metabolismo , Extensión de la Cadena Peptídica de Translación/genética , Proteínas de Plantas/genética , Antibacterianos/farmacología , Radioisótopos de Carbono , Cloroplastos/efectos de los fármacos , Cloroplastos/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Fusídico/farmacología , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacología , Guanosina Tetrafosfato/farmacología , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Leucina/genética , Leucina/metabolismo , Pisum sativum/genética , Pisum sativum/metabolismo , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Péptidos/genética , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Poli U/genética , ARN Mensajero/genética , Tioestreptona/farmacologíaRESUMEN
We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.
