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Results for “Cytochrome b6f Complex”

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PubMed1994

Cyclic GMP and calcium mediate phytochrome phototransduction.

We have previously used single-cell assays in a phytochrome-deficient tomato mutant to demonstrate that phytochrome signaling involves heterotrimeric G proteins, calcium, and calmodulin. While G protein activation could stimulate full chloroplast development and anthocyanin pigment biosynthesis, calcium and calmodulin could not induce anthocyanins and were only able to stimulate the development of immature chloroplasts lacking cytochrome b6f and photosystem I core components. We now report that cyclic GMP is able to trigger the production of anthocyanins, and that a combination of cyclic GMP with calcium can induce the development of fully mature chloroplasts containing all the photosynthetic machinery. Furthermore, using reporter genes for these different pathways (cab-gus, chs-gus, and fnr-gus) we demonstrate that cGMP and calcium act primarily by modulating gene expression.

Anthocyanins↗
PubMed1990

MacPROT: a set of BASIC programs for protein structure analysis.

MacPROT is a set of seven interactive application programs and a coordinating menu for storing, editing, and analysing protein sequences. Analyses for hydropathy, amphilicity, helix wheel, and dot matrix comparison are plotted as graphs on the screen and can be printed immediately or transferred to a picture accepting program. Programs are written in BASIC for the Apple 512 kbyte Macintosh. The source code is accessible and can be adapted to other machines.

Amino Acid Sequence↗
PubMed2006

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions.

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.

3' Untranslated Regions↗
PubMed2001

Quinolones and their N-oxides as inhibitors of photosystem II and the cytochrome b(6)/f-complex.

4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C(5) to C(17) have been synthesized and tested for biological activity in photosystem II and the cytochrome b(6)/f-complex. In photosystem II, quinolones 1 and 2 showed only moderate activity, whereas 3<5<4 (increasing activity) were potent inhibitors. Displacement experiments with [(14)C]atrazine indicated that the quinolones share an identical binding site with other photosystem II commercial herbicides. In the cytochrome b(6)/f-complex, only 3<4 showed enhanced activity. Maximal inhibitory potency was achieved at a carbon chain length of 12-14 A. Further increase of the chain length decreased activity. In a quantitative structure-activity relationship inhibitory activity in photosystem II and the cytochrome b(6)/f-complex could be correlated to the physicochemical parameters lipophilicity pi and/or to STERIMOL L.

Binding Sites↗
PubMed2002

Synthesis and preliminary biochemical assessment of ethyl-terminated perfluoroalkylamine oxide surfactants.

The synthesis and usefulness in membrane biochemistry of a new class of surfactants have been investigated. 1-Ethyl-2-dimethylamine oxide polar heads were grafted onto a hydrocarbon, a fluorocarbon or an ethyl-capped fluorocarbon hydrophobic tail. The ability of the resulting surfactants to extract and/or to stabilize in aqueous solution a test membrane protein, cytochrome b(6)f, was evaluated. While it is not a detergent, the hemifluorinated derivative efficiently kept purified cytochrome b(6)f soluble, native and functional. The data suggest that alkyl-capped fluorocarbon surfactants provide an interesting alternative to classical detergents for handling membrane proteins in aqueous solutions under non-dissociating conditions.

Cytochrome b Group↗
PubMed1999

Synthesis and preliminary assessments of ethyl-terminated perfluoroalkyl nonionic surfactants derived from tris(hydroxymethyl)acrylamidomethane.

We describe the synthesis and preliminary physicochemical and biological assessments of a new class of nonionic hybrid hydrofluoro amphiphiles derived from tris(hydroxymethyl)aminomethane (THAM). The synthesis of the hydrophobic tail of these amphiphiles is based on the preparation of an asymmetrical hydrofluorocarbon derivative containing an ethyl segment, a fluorocarbon core, and an ethyl thiol moiety. This molecule led to either THAM galactosylated monoadducts or telomers. These amphiphiles exhibit neither detergency toward cell membranes nor membrane protein denaturation.

Acrylamides↗
PubMed1993

RNA editing in maize chloroplasts is a processing step independent of splicing and cleavage to monocistronic mRNAs.

The psbB operon contained in the plastomes of higher plants consists of the genes psbB, psbH, petB and petD. The primary transcript of this operon is subject to a series of processing steps which include cleavages resulting in four monocistronic mRNAs and splicing of the petB and petD transcripts. A search for editing sites within the two latter transcripts from maize led us to the detection of one editing site within the petB coding region which is conserved at the DNA level in other graminean species and in tobacco. This shows that editing must be considered as an additional processing step of the psbB operon encoded primary transcript. As is evident from cDNA sequences derived from the dicistronic and/or unspliced petB/D transcripts which are completely edited, editing is an early step of mRNA processing which precedes both splicing and cleavage to the monocistronic mRNAs and which must, therefore, be independent of the latter two steps. This conclusion is confirmed by a similar observation with the editing site of the rpl2 transcript which is contained in the polycistronic transcript of the rpoA operon, although here only partial editing is observed for the unspliced dicistronic rpl23/rpl2 transcript.

Animals↗
PubMed1993

In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions.

We have used the Escherichia coli beta-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function of the 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene. Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene. In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated. However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of beta-glucuronidase activity was detected. When approximately 70% of the petD 5' UTR was deleted from the translational fusion, uidA mRNA accumulation and beta-glucuronidase activity decreased 4- to 6-fold and 8-fold, respectively. Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates. Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability. The expression of beta-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.

Animals↗
PubMed1996

Amphipols: polymers that keep membrane proteins soluble in aqueous solutions.

Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

Animals↗
PubMed1997

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.

Cyclohexanecarboxylic Acids↗
PubMed2003

Secondary structures common to chloroplast mRNA 3'-untranslated regions direct cleavage by CSP41, an endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily.

CSP41 (chloroplast stem-loop-binding protein of 41 kDa), a chloroplast endonuclease belonging to the SDR superfamily, preferentially cleaves stem-loop-containing RNAs in vitro. This potentially directs it to the 3'-ends of mature chloroplast mRNAs, which generally possess such structures. To understand the basis for this discrimination, the RNA elements directing CSP41 cleavage of petD RNA in vitro were dissected. Substrates containing fully base-paired stem-loops were optimal substrates, whereas deletion of part of the stem-loop decreased activity by 100-fold, and deletion of the distal arm of the stem-loop abolished cleavage, even in substrates containing the primary CSP41 cleavage site. Competition assays showed that the decrease in activity resulted from decreased affinity for the RNA by CSP41. Mutations of the residues at the scissile bond and mutations and deletions at the terminal loop of the stem had a moderate effect on activity but no effect on cleavage site specificity, suggesting that CSP41 has no sequence specificity. Titration of ethidium bromide into the assay decreased activity to a basal level of approximately 18%, and introduction of a single base bulge into either arm of the stem-loop decreased cleavage at the primary cleavage site by up to 70%. This suggests that changing the structure of the helical stem has a mild effect on activity. Deletion analysis of CSP41 suggests that the specificity domain lies in the first 73 amino acids of the protein, a domain that also contains a putative dehydrogenaselike mononucleotide binding motif. These results are consistent with a broad role for CSP41 in the degradation of stem-loop-containing mRNAs.

3' Untranslated Regions↗
PubMed2003

Thylakoid targeting of Tat passenger proteins shows no delta pH dependence in vivo.

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane delta pH. In this paper, we assess the delta pH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the delta pH in vivo. Using experimental conditions in which the trans-thylakoid delta pH was almost zero, we found no evidence for a delta pH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a delta pH under physiological conditions, but becomes delta pH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors.

Animals↗
PubMed1997

A dominant mutation in the Chlamydomonas reinhardtii nuclear gene SIM30 suppresses translational defects caused by initiation codon mutations in chloroplast genes.

A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30. A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA. sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene. We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor. The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.

Animals↗