scholarly journals Enzymatic product formation impairs both the chloroplast receptor-binding function as well as translocation competence of the NADPH: protochlorophyllide oxidoreductase, a nuclear-encoded plastid precursor protein.

1995 ◽  
Vol 129 (2) ◽  
pp. 299-308 ◽  
Author(s):  
S Reinbothe ◽  
C Reinbothe ◽  
S Runge ◽  
K Apel
Keyword(s):  
Aminolevulinic Acid ◽  
Transit Peptide ◽  
Precursor Protein ◽  
Receptor Protein ◽  
Chloroplast Envelope ◽  
Dependent Manner ◽  
Envelope Membrane ◽  
Chloroplast Transit Peptide ◽  
Plastid Envelope

The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein. Its posttranslational transport into plastids of barley depends on the intraplastidic availability of one of its substrates, protochlorophyllide (PChlide). The precursor of POR (pPOR), synthesized from a corresponding full-length barley cDNA clone by coupling in vitro transcription and translation, is enzymatically active and converts PChlide to chlorophyllide (Chlide) in a light- and NADPH-dependent manner. Chlorophyllide formed catalytically remains tightly but noncovalently bound to the precursor protein and stabilizes a transport-incompetent conformation of pPOR. As shown by in vitro processing experiments, the chloroplast transit peptide in the Chlide-pPOR complex appears to be masked and thus is unable to physically interact with the outer plastid envelope membrane. In contrast, the chloroplast transit peptide in the naked pPOR (without its substrates and its product attached to it) and in the pPOR-substrate complexes, such as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independently of the mature region of the polypeptide, and thus is able to bind to the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexes were exposed to light during a short preincubation, the enzymatically produced Chlide slowed down the actual translocation step, giving rise to the sequential appearance of two partially processed translocation intermediates. However, ongoing translocation induced by feeding the chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was able to override these two early blocks in translocation, suggesting that the plastid import machinery has a substantial capacity to denature a tightly folded, envelope-bound precursor protein. Together, our results show that pPOR with Chlide attached to it is impaired both in the ATP-dependent step of binding to a receptor protein component of the outer chloroplast envelope membrane, as well as in the PChlide-dependent step of precursor translocation.

scholarly journals A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts

2000 ◽  
Vol 351 (2) ◽  
pp. 377-384 ◽  
Author(s):  
Yan-Yun LIU ◽  
Naheed KADERBHAI ◽  
Mustak A. KADERBHAI
Keyword(s):  
Protein Import ◽  
Transit Peptide ◽  
Small Subunit ◽  
Precursor Protein ◽  
Chloroplast Envelope ◽  
Globular Domain ◽  
Dependent Manner ◽  
Chloroplast Transit Peptide ◽  
Bisphosphate Carboxylase ◽  
Isolated Chloroplasts

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b5 (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence–processed Tp–Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.

scholarly journals Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX)

2016 ◽  
Vol 113 (12) ◽  
pp. 3383-3388 ◽  
Author(s):  
Armin Springer ◽  
ChulHee Kang ◽  
Sachin Rustgi ◽  
Diter von Wettstein ◽  
Christiane Reinbothe ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Critical Role ◽  
Transit Peptide ◽  
Physiological Changes ◽  
Pathogen Defense ◽  
Chloroplast Transit Peptide ◽  
Selective Destruction ◽  
Plastid Envelope ◽  
Volatile Aldehydes

Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall.

scholarly journals Maize defective kernel5 is a bacterial TamB homologue required for chloroplast envelope biogenesis

2019 ◽  
Vol 218 (8) ◽  
pp. 2638-2658 ◽  
Author(s):  
Junya Zhang ◽  
Shan Wu ◽  
Susan K. Boehlein ◽  
Donald R. McCarty ◽  
Gaoyuan Song ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Phosphate Uptake ◽  
Soluble Sugars ◽  
Chloroplast Envelope ◽  
Envelope Membrane ◽  
Double Membrane ◽  
Plastid Envelope ◽  
Membrane Envelope ◽  
Bacterial Outer Membrane ◽  
Outer Membrane Biogenesis

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins.

scholarly journals Synthesis and transfer of galactolipids in the chloroplast envelope membranes of Arabidopsis thaliana

2016 ◽  
Vol 113 (38) ◽  
pp. 10714-10719 ◽  
Author(s):  
Amélie A. Kelly ◽  
Barbara Kalisch ◽  
Georg Hölzl ◽  
Sandra Schulze ◽  
Juliane Thiele ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Chloroplast Envelope ◽  
Envelope Membrane ◽  
Lipid Transfer ◽  
Terminal Sequence ◽  
Outer Envelope ◽  
Outer Envelope Membrane ◽  
Envelope Reconstruction ◽  
Envelope Membranes

Galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] are the hallmark lipids of photosynthetic membranes. The galactolipid synthases MGD1 and DGD1 catalyze consecutive galactosyltransfer reactions but localize to the inner and outer chloroplast envelopes, respectively, necessitating intermembrane lipid transfer. Here we show that the N-terminal sequence of DGD1 (NDGD1) is required for galactolipid transfer between the envelopes. Different diglycosyllipid synthases (DGD1, DGD2, and Chloroflexus glucosyltransferase) were introduced into the dgd1-1 mutant of Arabidopsis in fusion with N-terminal extensions (NDGD1 and NDGD2) targeting to the outer envelope. Reconstruction of DGDG synthesis in the outer envelope membrane was observed only with diglycosyllipid synthase fusion proteins carrying NDGD1, indicating that NDGD1 enables galactolipid translocation between envelopes. NDGD1 binds to phosphatidic acid (PA) in membranes and mediates PA-dependent membrane fusion in vitro. These findings provide a mechanism for the sorting and selective channeling of lipid precursors between the galactolipid pools of the two envelope membranes.

scholarly journals Analytical ultracentrifugation and preliminary X-ray studies of the chloroplast envelope quinone oxidoreductase homologue fromArabidopsis thaliana

2015 ◽  
Vol 71 (4) ◽  
pp. 455-458 ◽  
Author(s):  
Sarah Mas y mas ◽  
Cécile Giustini ◽  
Jean-Luc Ferrer ◽  
Norbert Rolland ◽  
Gilles Curien ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
Alternative Pathway ◽  
Transit Peptide ◽  
Chloroplast Envelope ◽  
Quinone Oxidoreductase ◽  
Chloroplast Targeting ◽  
Data Set ◽  
Chloroplast Transit Peptide ◽  
Chloroplast Envelope Membrane ◽  
Living Organisms

Quinone oxidoreductases reduce a broad range of quinones and are widely distributed among living organisms. The chloroplast envelope quinone oxidoreductase homologue (ceQORH) fromArabidopsis thalianabinds NADPH, lacks a classical N-terminal and cleavable chloroplast transit peptide, and is transported through the chloroplast envelope membrane by an unknown alternative pathway without cleavage of its internal chloroplast targeting sequence. To unravel the fold of this targeting sequence and its substrate specificity, ceQORH fromA. thalianawas overexpressed inEscherichia coli, purified and crystallized. Crystals of apo ceQORH were obtained and a complete data set was collected at 2.34 Å resolution. The crystals belonged to space groupC2221, with two molecules in the asymmetric unit.

scholarly journals A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide.

1995 ◽  
Vol 15 (11) ◽  
pp. 6206-6212 ◽  
Author(s):  
C Reinbothe ◽  
K Apel ◽  
S Reinbothe
Keyword(s):  
Product Formation ◽  
In Vitro Transcription ◽  
Precursor Protein ◽  
Dependent Manner ◽  
Hordeum Vulgare L ◽  
Nadph:Protochlorophyllide Oxidoreductase ◽  
Catalytically Active ◽  
Protease Treatment ◽  
Energy Dependent

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light- and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.

scholarly journals Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants

2007 ◽  
Vol 282 (49) ◽  
pp. 35536-35545 ◽  
Author(s):  
Yuzuru Tozawa ◽  
Akira Nozawa ◽  
Takuya Kanno ◽  
Takakuni Narisawa ◽  
Shinji Masuda ◽  
...  
Keyword(s):  
Stringent Response ◽  
Transit Peptide ◽  
Genetic System ◽  
Land Plants ◽  
Synthetase Activity ◽  
Chloroplast Transit Peptide ◽  
Ef Hand ◽  
Genes Encoding

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5′-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.

scholarly journals Plastocyanin Transit Peptide Interacts with Potato virus X Coat Protein, While Silencing of Plastocyanin Reduces Coat Protein Accumulation in Chloroplasts and Symptom Severity in Host Plants

2009 ◽  
Vol 22 (12) ◽  
pp. 1523-1534 ◽  
Author(s):  
Y. Qiao ◽  
H. F. Li ◽  
S. M. Wong ◽  
Z. F. Fan
Keyword(s):  
Coat Protein ◽  
Potato Virus ◽  
Symptom Severity ◽  
Host Plants ◽  
Transit Peptide ◽  
Potato Virus X ◽  
Precursor Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Accumulation

Potato virus X coat protein (PVXCP) is, through communication with host proteins, involved in processes such as virus movement and symptom development. Here, we report that PVXCP also interacts with the precursor of plastocyanin, a protein involved in photosynthesis, both in vitro and in vivo. Yeast two-hybrid analysis indicated that PVXCP interacted with only the plastocyanin transit peptide. In subsequent bimolecular fluorescence complementation assays, both proteins were collocated within chloroplasts. Western blot analyses of chloroplast fractions showed that PVXCP could be detected in the envelope, stroma, and lumen fractions. Transmission electron microscopy demonstrated that grana were dilated in PVX-infected Nicotiana benthamiana. Furthermore, virus-induced gene silencing of plastocyanin by prior infection of N. benthamiana using a Tobacco rattle virus vector reduced the severity of symptoms that developed following subsequent PVX infection as well as the accumulation of PVXCP in isolated chloroplasts. However, PVXCP could not be detected in pea chloroplasts in an in vitro re-uptake assay using the plastocyanin precursor protein. Taken together, these data suggest that PVXCP interacts with the plastocyanin precursor protein and that silencing the expression of this protein leads to reduced PVXCP accumulation in chloroplasts and ameliorates symptom severity in host plants.

scholarly journals Polynucleotide Phosphorylase Functions as Both an Exonuclease and a Poly(A) Polymerase in Spinach Chloroplasts

2001 ◽  
Vol 21 (16) ◽  
pp. 5408-5416 ◽  
Author(s):  
Shlomit Yehudai-Resheff ◽  
Merav Hirsh ◽  
Gadi Schuster
Keyword(s):  
Transit Peptide ◽  
Rna Degradation ◽  
Polynucleotide Phosphorylase ◽  
Chloroplast Transit Peptide ◽  
Stem Loop ◽  
Rna Molecules ◽  
Spinach Chloroplasts ◽  
Endonucleolytic Cleavage ◽  
Exonucleolytic Degradation

ABSTRACT The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli,polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of Piand ADP. As expected of a phosphorylase, Pi enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the completeArabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.

scholarly journals Maize defective kernel5 is a bacterial tamB homolog required for chloroplast envelope biogenesis

2018 ◽  
Author(s):  
Junya Zhang ◽  
Shan Wu ◽  
Susan K. Boehlein ◽  
Donald R. McCarty ◽  
Gaoyuan Song ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Phosphate Uptake ◽  
Soluble Sugars ◽  
Chloroplast Envelope ◽  
Membrane Biogenesis ◽  
Envelope Membrane ◽  
Plastid Envelope ◽  
Membrane Envelope ◽  
Bacterial Outer Membrane ◽  
Outer Membrane Biogenesis

ABSTRACTChloroplasts are of prokaryotic origin with a double membrane envelope that separates plastid metabolism from the cytosol. Envelope membrane proteins integrate the chloroplast with the cell, but the biogenesis of the envelope membrane remains elusive. We show that the maize defective kernel5 (dek5) locus is critical for plastid membrane biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. We show that dek5 encodes a protein homologous to rice SUBSTANDARD STARCH GRAIN4 (SSG4) and E.coli tamB. TamB functions in bacterial outer membrane biogenesis. The DEK5 protein is localized to the chloroplast envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Both proteomics and antibody-based analyses show that dek5 chloroplasts have reduced levels of chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable metabolite transport.

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