Biomedical subjects

C D Fairchild

Publications and source records attributed to C D Fairchild.

8 recordsLinked to original sources

PubMed2000

Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix-loop-helix factor PIF3 in a reaction requiring the PAS domain of PIF3.

The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix-loop-helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.

Arabidopsis↗

PubMed2000

HFR1 encodes an atypical bHLH protein that acts in phytochrome A signal transduction.

Phytochromes are informational photoreceptors through which plants adapt their growth and development to prevailing light conditions. These adaptations are effected primarily through phytochrome regulation of gene expression by mechanisms that remain unclear. We describe a new mutant, hfr1 (long hypocotyl in far-red), that exhibits a reduction in seedling responsiveness specifically to continuous far-red light (FRc), thereby suggesting a locus likely to be involved in phytochrome A (phyA) signal transduction. Using an insertionally tagged allele, we cloned the HFR1 gene and subsequently confirmed its identity with additional alleles derived from a directed genetic screen. HFR1 encodes a nuclear protein with strong similarity to the bHLH family of DNA-binding proteins but with an atypical basic region. In contrast to PIF3, a related bHLH protein previously shown to bind phyB, HFR1 did not bind either phyA or B. However, HFR1 did bind PIF3, suggesting heterodimerization, and both the HFR1/PIF3 complex and PIF3 homodimer bound preferentially to the Pfr form of both phytochromes. Thus, HFR1 may function to modulate phyA signaling via heterodimerization with PIF3. HFR1 mRNA is 30-fold more abundant in FRc than in continuous red light, suggesting a potential mechanistic basis for the specificity of HFR1 to phyA signaling.

Amino Acid Sequence↗

PubMed1998

The phytochromes: photosensory perception and signal transduction.

Phytochromes are regulatory photoreceptors which primarily absorb red (R) and far-red (FR) light. A great deal is known about the spectroscopic properties, primary structure, gene regulation and gross structure of phytochromes, and about the set of developmental changes which they control, but the early steps in signal transduction from phytochrome which result in these changes are still mysterious. In angiosperms, phytochromes are encoded by a small gene family, and as a result of recent work with mutants and transgenic overexpressors it is possible to assign distinct functions to some of the individual types of phytochrome. For two of these, phytochrome A and phytochrome B, overexpression of chimeras has revealed that the determinants for their photosensory specificity and the light-promoted degradation of phytochrome A reside on the amino-terminal halves of the molecules. The interchangeability of the C-terminal halves suggests that they may share a common signal transduction mechanism. These results also invite a reappraisal of the various models that have been proposed over the years to explain the complexity of phytochrome sensitivity to various light regimes.

Light↗

PubMed1996

Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability.

In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2-terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors.

Amino Acid Sequence↗

PubMed1994

Nonenzymatic bilin addition to the alpha subunit of an apophycoerythrin.

C-Phycoerythrin is a light-harvesting protein whose alpha and beta subunits carry thioether-linked phycoerythrobilin (PEB) at cysteine residues alpha-82, alpha-139, beta-48,59 (doubly-linked), beta-80, and beta-165. The two subunits of Calothrix sp. PCC 7601 C-phycoerythrin, overexpressed together as apopolypeptides in Escherichia coli, formed inclusion bodies. Purified apo-alpha was soluble in the absence of urea, whereas the apo-beta subunit was only soluble at high urea concentrations. Products of nonenzymatic addition of PEB and phycocyanobilin (PCB) to apo-alpha were characterized by isolation of bilin peptides and spectroscopy. Reaction of PEB with the apo-alpha subunit led primarily to 15,16-dihydrobiliverdin (Cys-82) or urobilin (Cys-139) adducts, and small amounts of the natural PEB adducts at both Cys-82 and Cys-139. PCB reacted primarily with Cys-82 to form phycocyanobilin and mesobiliverdin adducts. Both PEB and PCB also formed relatively small amounts of adducts with Cys-59, which is not a bilin attachment residue in natural phycoerythrin. Sodium azide was found to promote the addition of PEB to simple thiols but not to apo-alpha phycoerythrin.

Azides↗

PubMed1994

Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase.

Phycobiliproteins carry linear tetrapyrrole chromophores (bilins) thioether-linked to specific cysteine residues. The process of bilin attachment to apoprotein in vivo has been characterized for only one bilin attachment site on one phycobiliprotein, that on the alpha subunit of phycocyanin (alpha PC). In the cyanobacterium Synechococcus sp. PCC 7002, the addition of phycocyanobilin to apo-alpha PC is catalyzed by the protein products of the cpcE and cpcF genes. We have purified and further characterized the recombinant CpcE and CpcF proteins. CpcE and CpcF form an enzymatically active 1:1 complex (CpcEF), stable to size exclusion chromatography. CpcEF causes a reduction in alpha PC fluorescence and strongly affects its absorption spectrum but has no effect on the beta subunit. The CpcEF bilin addition activity exhibits simple Michaelis-Menten kinetics with respect to the apo-alpha PC and to bilin. CpcEF also catalyzes the addition of phycoerythrobilin to apo-alpha PC; phycoerythrobilin is thought to be on the biosynthetic pathway of phycocyanobilin. CpcEF shows a preference for phycocyanobilin relative to phycoerythrobilin, both in binding affinity and in the rate of catalysis, sufficient to account for selective attachment of phycocyanobilin to apo-alpha PC.

Bile Pigments↗

PubMed1992

Phycocyanin alpha-subunit phycocyanobilin lyase.

Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.

Bile Pigments↗

PubMed1991

Absence of glycosylation on cyanobacterial phycobilisome linker polypeptides and rhodophytan phycoerythrins.

The 27-, 30-, and 33-kDa rod linker polypeptides and the 75-kDa core linker of phycobilisomes from the cyanobacterium Synechococcus sp. strain PCC 7942 have been reported to be glycoproteins with carbohydrate contents ranging from 3.2 to 18.8% and composed of N-acetylgalactosamine and glucose (H.C. Riethman, T.P. Mawhinney, and L.A. Sherman, J. Bacteriol. 170:2433-2440, 1988). Synechococcus sp. strain PCC 7942 phycobilisomes were purified extensively, and the linker polypeptides were separated from the phycobiliproteins by precipitation in 1 M NaSCN. Upon hydrolysis, the linker fraction yielded 0.037% glucose and 0.015% galactosamine by weight and no other carbohydrate. Phycobilisome polypeptides separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were subjected to various glycoprotein-specific staining procedures. Linker polypeptides showed very weak concanavalin A binding and no staining by the Schiff-periodate method or by a much more sensitive periodate oxidation-based method. These results indicated that the linker polypeptides are not glycosylated. An earlier report (T. Fujiwara, J. Biochem. 49:361-367, 1961) contended, on the basis of the isolation of sugar-containing peptic chromopeptides from Porphyra tenera R-phycoerythrin, that this red algal phycobiliprotein is a glycoprotein. Analysis of Gastroclonium coulteri R-phycoerythrin and Porphyridium cruentum B-phycoerythrin revealed only traces of carbohydrate in these two proteins, 0.36 and 0.14%, respectively. Results of glycoprotein staining of gels suggested that the carbohydrate in the R-phycoerythrin preparation is due to a glycoprotein contaminant and that neither red algal phycoerythrin is glycosylated.

Carbohydrates↗