ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

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).

In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization.

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.

In vitro fluorescent analysis of preprotein import into chloroplasts.

Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al.

Evaluating the energy-dependent "binding" in the early stage of protein import into chloroplasts.

During protein import into chloroplasts, precursor proteins are synthesized in the cytosol with an amino-terminal extension signal and irreversibly bind to chloroplasts under stringent energy conditions, such as low levels of GTP/ATP and low temperature, to form the early translocation intermediates. Whether the states of the early-intermediates that are formed under different energy conditions are similar has not been well studied. To evaluate the early intermediate states, we analyzed how precursor proteins within the early intermediates behave by employing two different approaches, limited proteolysis and site-specific cross-linking.

Alternative processing of Arabidopsis Hsp70 precursors during protein import into chloroplasts.

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms.

The transition of early translocation intermediates in chloroplasts is accompanied by the movement of the targeting signal on the precursor protein.

During protein import into chloroplasts, precursor proteins are docked to these organelles under stringent energy conditions to form early translocation intermediates. Depending on the temperature and the requirement for ATP, different types of early-intermediates are present, for which the extent of precursor protein translocation differs [H. Inoue, M.

Development and optimization of an in vitro chloroplastic protein import assay using recombinant proteins.

The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins.

Three sets of translocation intermediates are formed during the early stage of protein import into chloroplasts.

During the early stage of protein import into chloroplasts, precursor proteins synthesized in the cytosol irreversibly bind to chloroplasts to form the early translocation intermediate under stringent energy conditions. Many efforts have been made to identify the components involved in protein import by analyzing the early intermediate. However, the state of the precursor within the intermediate has not been well investigated so far.

Stimulation of transit-peptide release and ATP hydrolysis by a cochaperone during protein import into chloroplasts.

Three components of the chloroplast protein translocon, Tic110, Hsp93 (ClpC), and Tic40, have been shown to be important for protein translocation across the inner envelope membrane into the stroma. We show the molecular interactions among these three components that facilitate processing and translocation of precursor proteins. Transit-peptide binding by Tic110 recruits Tic40 binding to Tic110, which in turn causes the release of transit peptides from Tic110, freeing the transit peptides for processing.

Early steps in the biosynthesis of NAD in Arabidopsis start with aspartate and occur in the plastid.

NAD is a ubiquitous coenzyme involved in oxidation-reduction reactions and is synthesized by way of quinolinate. Animals and some bacteria synthesize quinolinate from tryptophan, whereas other bacteria synthesize quinolinate from aspartate (Asp) using L-Asp oxidase and quinolinate synthase. We show here that Arabidopsis (Arabidopsis thaliana) uses the Asp-to-quinolinate pathway.

Identification of three shikimate kinase genes in rice: characterization of their differential expression during panicle development and of the enzymatic activities of the encoded proteins.

The shikimate pathway is common to the biosynthesis of the three aromatic amino acids and that of various secondary metabolites in land plants. Shikimate kinase (SK; EC 2.7.

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon.

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal.