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 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 Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

1996 ◽  
Vol 134 (2) ◽  
pp. 315-327 ◽  
Author(s):  
Y Ma ◽  
A Kouranov ◽  
S E LaSala ◽  
D J Schnell
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Small Subunit ◽  
Chloroplast Envelope ◽  
Stable Complex ◽  
Nucleoside Triphosphate ◽  
Cross Linking ◽  
Outer Envelope ◽  
Bisphosphate Carboxylase ◽  
Precursor Proteins

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

scholarly journals Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons

2017 ◽  
Author(s):  
Iniyan Ganesan ◽  
Lan-Xin Shi ◽  
Mathias Labs ◽  
Steven M. Theg
Keyword(s):  
Pore Size ◽  
Protein Unfolding ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Transit Peptide ◽  
Small Subunit ◽  
Chloroplast Envelope ◽  
Residual Structure ◽  
Folded Proteins

ABSTRACTThe degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, which has historically been the thought for the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that medium-sized tightly folded proteins such as the 22 kDa dihydrofolate reductase (DHFR) can be tolerated by TOC and TIC. Chimeric DHFR fused with RuBisCO small subunit transit peptide (tp22DHFR) was found to be imported into chloroplasts in complex with its stabilizing ligand, methotrexate (MTX), in a folded conformation. Following import, both mature tp22DHFR and MTX were found in the chloroplast stroma. A subsaturating concentration of MTX was used to exclude the possibility that MTX was stripped off tp22DHFR, independently imported into the chloroplasts, and reassociated with imported tp22DHFR. Independent MTX import was further excluded by use of fluorescein conjugated MTX (FMTX), which has very slow membrane transport rates relative to unconjugated MTX. The TOC/TIC pore size was determined by probing the translocons with particles of fixed diameter and found to be greater than 25.6 Å, large enough to support folded DHFR import. The pore size is also larger than those of the mitochondrial protein translocons that have a requirement for protein unfolding.SIGNIFICANCEThe chloroplast TOC and TIC translocons are responsible for the import of up to 95% of all chloroplast proteins and are therefore essential for plastid biogenesis and photosynthesis. However, the mechanisms of protein import into chloroplasts are not well understood. The TOC/TIC translocons have long been suggested to have a strong unfoldase activity relative to other comparable protein translocons. Here, we present data suggesting that this is not true, and that instead, they possess a relatively large pore size. This identifies TOC and TIC as rather unique protein translocons capable of transporting folded proteins across a double membrane barrier, which has important implications in the mechanisms of TOC/TIC function and biogenesis of photosynthetic proteins.Classification - Biochemistry

scholarly journals Synthesis of the small subunit of ribulose-bisphosphate carboxylase from genes cloned into plasmids containing the SP6 promoter

1986 ◽  
Vol 240 (3) ◽  
pp. 709-715 ◽  
Author(s):  
S Anderson ◽  
S M Smith
Keyword(s):  
Dna Sequences ◽  
Protein Import ◽  
Transit Peptide ◽  
Small Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Free System ◽  
Pisum Sativum L

DNA sequences encoding ribulose 1,5-bisphosphate carboxylase small subunit precursor from Pisum sativum L. have been transcribed from plasmids containing the SP6 promoter, and translated in a wheat germ cell-free system. The small subunit precursor polypeptide, its N-terminal leader sequence (transit peptide) and the mature small subunit have each been synthesized independently from three different plasmid constructs. The precursor polypeptide is imported into isolated pea chloroplasts and processed to the mature small subunit by a stromal proteinase. The mature polypeptide is neither imported, nor subject to proteolysis by stromal extracts. The transit peptide alone is very rapidly degraded by a stromal proteinase activity which can be inhibited by EDTA or 1,10-phenanthroline. The use of these gene constructs helps to establish the crucial role of the transit peptide in protein import into the chloroplast.

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 The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs Involved in Chloroplast Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Mingjiu Li ◽  
Hannes Ruwe ◽  
Michael Melzer ◽  
Astrid Junker ◽  
Goetz Hensel ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Ribosomal Proteins ◽  
Protein Import ◽  
Chloroplast Development ◽  
Transit Peptide ◽  
Growth Conditions ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Chloroplast Transit Peptide ◽  
Pale Green

The Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7, or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.

scholarly journals The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-functionalized Paralogs involved in Chloroplast Development

2021 ◽  
Author(s):  
Mingjiu Li ◽  
Hannes Ruwe ◽  
Michael Melzer ◽  
Astrid Junker ◽  
Götz Hensel ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Ribosomal Proteins ◽  
Protein Import ◽  
Chloroplast Development ◽  
Transit Peptide ◽  
Growth Conditions ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Chloroplast Transit Peptide ◽  
Pale Green

ABSTRACTThe Arabidopsis gene Chloroplast Import Apparatus 2 (CIA2) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries. CIA2-like (CIL) is the paralogous gene of CIA2. We generated a cil mutant by site-directed mutagenesis and compared it with cia2 and cia2cil double mutant. Phenotype of the cil mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to cia2, the cia2cil mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs. In silico analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of cia2 with HvCMF3, HvCMF7 or with a truncated CIA2 lacking the predicted cTP could partially rescue the pale-green phenotype of cia2. These data are discussed with respect to potentially overlapping functions between CIA2, CIL and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.HIGHLIGHTThe nucleus-localized CCT domain proteins CIA2 and CIL in Arabidopsis and the homologous chloroplast-localized HvCMF3 and HvCMF7 in barley retained partially overlapping functions in chloroplast development.

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