Biomedical subjects
Publications and source records attributed to S P Mayfield.
Chlorophyll precursors, photosynthetic electron transport, and sugars have all been shown to be involved in signaling from the chloroplast to the nucleus, suggesting the presence of multiple signaling pathways of coordination between these two cellular compartments.
Binding of the chloroplast poly(A)-binding protein, RB47, to the psbA mRNA is regulated in response to light and is required for translation of this mRNA in chloroplasts. The RNA binding activity of RB47 can be modulated in vitro by oxidation and reduction. Site-directed mutations to individual cysteine residues in each of the four RNA binding domains of RB47 showed that changing single cysteines to serines in domains 2 or 3 reduced, but did not eliminate, the ability of RB47 to be redox-regulated. Simultaneously changing cysteines to serines in both domains 2 and 3 resulted in the production of RB47 protein that was insensitive to redox regulation but retained the ability to bind the psbA mRNA at high affinity. The poly(A)-binding protein from Saccharomyces cerevisiae lacks cysteine residues in RNA binding domains 2 and 3, and this poly(A)-binding protein lacks the ability to be regulated by oxidation or reduction. These data show that disulfide bond formation between RNA binding domains in a poly(A)-binding protein can be used to regulate the ability of this protein to bind mRNA and suggest that redox regulation of RNA binding activity may be used to regulate translation in organisms whose poly(A)-binding proteins contain these critical cysteine residues.
Chloroplast development and function relies both on structural and on regulatory factors encoded within the nucleus. Recent work has lead to the identification of several nuclear encoded genes that participate in a wide array of chloroplast functions. Characterization of these genes has increased our understanding of the signalling between these two compartments. Accumulating evidence shows that a variety of molecular mechanisms are used for intercompartmental communication and for regulating co-ordinated chloroplast protein expression.
The 5' untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5' terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5' untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.
A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5' untranslated region of the psbA mRNA. The results presented here confirm RB47's role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.
High-affinity binding of a set of proteins with specificity for the 5' untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation. In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from Escherichia coli binds to the psbA 5' UTR with similar specificity and affinity as RB47 isolated from C. reinhardtii chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.
Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5'-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomerase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP through redox potential or adenosine 5'-diphosphate-dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloroplast.
Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.
Translation of key proteins in the chloroplast is regulated by light. Genetic and biochemical studies in the unicellular alga Chlamydomonas reinhardtii suggest that light may regulate translation by modulating the binding of activator proteins to the 5' untranslated region of chloroplast messenger RNAs. In vitro binding of the activator proteins to psbA messenger RNA and in vivo translation of psbA messenger RNA is regulated by the redox state of these proteins, suggesting that the light stimulus is transduced by the photosynthesis-generated redox potential.
Light-regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5'-untranslated region (5'-UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5'-UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark- and light-grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA-binding complex, that utilizes the beta-phosphate of ADP to phosphorylate and inactivate psbA mRNA-binding in vitro. The inactivation of mRNA-binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark-grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA-binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.
Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine-Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message-specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.
Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.
Explore the source record for details and available documents.
We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.
We have isolated a 5.7-kbp dispersed moderately repeated DNA sequence (TOC1) from the mutant OEE1 gene of the Chlamydomonas reinhardtii strain FUD44. The copy number (2 to over 30) and genomic locations of TOC1 elements vary widely in different C. reinhardtii strains. Our standard laboratory photosynthetic strain exhibits a high degree of TOC1 instability during short periods of mitotic growth. TOC1 appears to be a retrotransposon: it contains LTRs and an oligonucleotide stretch that corresponds to a conserved pentapeptide of reverse transcriptase. TOC1 is an unusual retrotransposon: it is not flanked by a target site duplication in the OEE1 gene, the left end of TOC1 only contains a fraction of the LTR the remainder of which is present at its right end and TOC1 does not start with a 5' TG and end with a 3' CA. In most cases, TOC1 excision leaves behind a complete solo LTR sequence (577 bp) and in one case a deleted solo LTR sequence (191 bp). Solo LTR sequences form a separate family of repeated sequences in most of the strains tested.
Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.
In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem II (PS II), is coded for by a single nuclear gene (psb1). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psb1 gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.