Redox state regulates HIF-1alpha and its DNA binding and phosphorylation in salmonid cells.

Rainbow trout (Oncorhynchus mykiss) hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor structurally similar to mammalian HIF-1. It consists of HIF-1alpha and HIF-1beta subunits, of which the HIF-1alpha subunit confers the hypoxia sensitivity. HIF-1alpha is rapidly degraded by a proteasome under normal oxygen (21% O2) conditions, mainly as a result of prolyl hydroxylation needed for protein destabilization. Although prolyl hydroxylation at conserved proline residues is a major factor controlling HIF-1alpha stability, the redox state of the cells may, in addition, influence the function of HIF-1alpha like proteins by influencing their stability, DNA binding and phosphorylation. Sensitivity of the protein to oxidation/reduction may be due to cysteine residues at critical positions. The predicted amino acid sequence of rainbow trout HIF-1alpha contains several unique cysteine residues, notably in the DNA-binding area at position 28 and in the transactivation domain of the molecule in the vicinity of the conserved proline residue at position 564 of mammalian HIF-1alpha. In the present studies we have investigated if the redox state influences HIF-1alpha stability, DNA binding and phosphorylation in two established salmonid cell lines RTG-2 and CHSE-214. The results indicate that reducing conditions, achieved using N-propylgallate (nPG) or N-acetylcysteine (NAC), stabilize HIF-1alpha, facilitate its DNA binding, and increase its phosphorylation even under normal oxygen conditions. On the other hand, oxidizing conditions, achieved using L-buthionine sulfoximine (BSO) dampen the hypoxia response. Furthermore, the hypoxia-like effect of cobalt is increased in the presence of the reducing agent. On the basis of these results, we suggest that redox state influences the accessibility of the conserved prolyl residues to oxygen-dependent hydroxylation and the accessibility of the residues involved in the phosphorylation of HIF-1alpha.

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes.

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.

Multiple effects of antibiotics on chloroplast and nuclear gene expression.

Antibiotics are widely used to monitor signalling cascades within a plant cell, for example between the nucleus and chloroplasts, and to study the function of the photosynthetic machinery. In the present study, we attempted to test various antibiotics with respect to their expected modes of function and also to monitor their possible side effects on metabolic processes in mature leaves of pea (Pisum sativum L.). Streptomycin, despite its reported prokaryotic nature, prevented translation not only in the chloroplast, but also in the cytosol. Application of puromycin, an inhibitor of protein synthesis in both the pro- and eukaryotes, resulted in severe photoinhibition of photosystem II upon illumination, yet had no effect on plastid translation, thus implying a severe side effect on plastid metabolism. Prokaryotic-type translation inhibitors lincomycin, spectinomycin and erythromycin blocked translation in the chloroplast without any direct effects on cytoplasmic protein synthesis. More detailed studies with lincomycin, however, revealed a strong modulation of the expression of nuclear-encoded genes by slowing down the transcription rate of photosynthesis-related Lhcb and RbcS genes, and furthermore, lincomycin clearly decreased the phosphorylation level of the LHCII proteins.