Genetic engineering of the unsaturation of fatty acids in membrane lipids alters the tolerance of Synechocystis to salt stress. (73/15260)

The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing the desA-/desD- mutant of Synechocystis sp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid in desA-/desD- cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than in desA-/desD- cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+ antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.  (+info)

BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize. (74/15260)

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize.  (+info)

Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. (75/15260)

Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant. Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between +info)

Molecular chaperone-like properties of an unfolded protein, alpha(s)-casein. (76/15260)

All molecular chaperones known to date are well organized, folded protein molecules whose three-dimensional structure are believed to play a key role in the mechanism of substrate recognition and subsequent assistance to folding. A common feature of all protein and nonprotein molecular chaperones is the propensity to form aggregates very similar to the micellar aggregates. In this paper we show that alpha(s)-casein, abundant in mammalian milk, which has no well defined secondary and tertiary structure but exits in nature as a micellar aggregate, can prevent a variety of unrelated proteins/enzymes against thermal-, chemical-, or light-induced aggregation. It also prevents aggregation of its natural substrates, the whey proteins. alpha(s)-Casein interacts with partially unfolded proteins through its solvent-exposed hydrophobic surfaces. The absence of disulfide bridge or free thiol groups in its sequence plays important role in preventing thermal aggregation of whey proteins caused by thiol-disulfide interchange reactions. Our results indicate that alpha(s)-casein not only prevents the formation of huge insoluble aggregates but it can also inhibit accumulation of soluble aggregates of appreciable size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. This protein seems to have some characteristics of cold shock protein, and its chaperone-like activity increases with decrease of temperature.  (+info)

Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light. (77/15260)

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.  (+info)

A role for the light-dependent phosphorylation of visual arrestin. (78/15260)

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cell-specific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.  (+info)

How vertebrate and invertebrate visual pigments differ in their mechanism of photoactivation. (79/15260)

In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the "counterion" is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation-that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.  (+info)

Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. (80/15260)

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H2O2. In this report, we show that violet-blue light, as well as UVA, stimulated H2O2 production in cultured mouse, monkey, and human cells. We found that H2O2 originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H2O2 in cells. Because H2O2 and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H2O2 production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.  (+info)