Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco

Chaumont, François; Silva-Filho, Márcio C.; Thomas, Dominique; Leterme, S.; Boutry, Marc

doi:10.1007/bf00023559

Cited by 49 publications

(56 citation statements)

References 43 publications

(58 reference statements)

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“…For instance, we found similar rates of in vitro mitochondrial import for TPT5-CAT or TPT23-CAT as for a construct linking CAT to a truly mitochondrial presequence, that of the mitochondrial ATPase ␤ subunit, and yet in vivo the latter construct was imported efficiently into mitochondria (18).…”

Section: Fig 2 Import Of Tpt5-cat and Tpt23-cat Into Isolated Chlormentioning

confidence: 62%

“…The CAT encoding sequence was isolated by HindIII and BamHI digestion of plasmid pBin35ScatE9Ј (18) and inserted into the corresponding sites of SK(ϩ) Bluescript, resulting in the CAT plasmid. The HindIII site previously used for inserting targeting sequences (8,18) is localized 75 nucleotides upstream of the CAT translation initiation codon.…”

Section: Methodsmentioning

confidence: 99%

“…The HindIII site previously used for inserting targeting sequences (8,18) is localized 75 nucleotides upstream of the CAT translation initiation codon. When translated, this region encodes a cryptic mitochondrial cleavage site (18). Using polymerase chain reaction, therefore, we engineered a new CAT gene provided with a HindIII site 10 nucleotides upstream of the CAT translation initiation codon.…”

Section: Methodsmentioning

confidence: 99%

“…The TPT5-CAT and TPT23-CAT plasmids were digested with BamHI and partial HindIII, releasing the fragments TPT5-CAT and TPT23-CAT. These fragments were inserted into the corresponding sites of Bin35ScatE9Ј (18), digested previously with HindIII and BamHI, producing the plant transformation vectors Bin35S-TPT5-CAT and Bin35S-TPT23-CAT, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Fractionation of Tobacco Cells and Protein Quantitation-Subcellular fractions were obtained from 10 g of leaves as described previously (18), except that homogenization was performed in 50 ml of homogenization buffer and that 0.2% (w/v) polyvinylpyrrolidone was added to the buffer.…”

Section: Methodsmentioning

confidence: 99%

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Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

Silva-Filho¹,

Wieers²,

Flügge³

et al. 1997

Journal of Biological Chemistry

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The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH 2 -terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

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Section: Fig 2 Import Of Tpt5-cat and Tpt23-cat Into Isolated Chlormentioning

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

Silva-Filho¹,

Wieers²,

Flügge³

et al. 1997

Journal of Biological Chemistry

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When green carbon plants meet synthetic biology

Wang,

Zhang,

Dai

et al. 2023

Modern Agriculture

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Recycling carbon dioxide (CO2) into chemicals or fuels presents a promising avenue for mitigating carbon emissions and addressing the energy crisis. Plants serve as the ideal platform for the production of materials and chemicals, thanks to their innate capacity to directly use CO2 in the synthesis of various organic compounds. While conventional methods for enhancing plant CO2 fixation may reach their limits, novel technological solutions are imperative. Synthetic biology has illuminated the potential for biosynthesising multiple carbon sources through artificial CO2 fixation pathways in vitro. Recent breakthroughs in photorespiratory bypasses and artificial carboxylation modules offer significant promise for engineering plants to improve carbon fixation, guiding the design and development of plants with more efficient CO2 utilisation. In this context, we begin by summarising recent progress in designing or engineering in vitro CO2 fixation pathways, as well as those solely established in microbes. Subsequently, we delineate strategies employed to enhance CO2 fixation in plants. Finally, we explore potential methods for introducing artificial CO2 fixation pathways into plants. These advancements are critical in advancing synthetic biology's efforts to tackle future challenges related to food and energy scarcity.

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Imaging and Analysis of Mitochondrial Dynamics in Living Cells

Ekanayake

Zawily

Paszkiewicz

et al. 2015

Methods in Molecular Biology

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One of the most striking features of plant mitochondria when visualized in living tissue is their dynamism. The beauty of cytoplasmic streaming, driving, and being driven by the motility of mitochondria and other small organelles belies the complexity of the process. Equally, capturing that dynamism and investigating the genes, proteins, and mechanisms underpinning the processes using molecular cell biology and bioimaging is a complex process. It requires the generation of fluorescent protein constructs, stable transgenic plants sometimes expressing multiple fusions, and generation of mutants, even before one is ready for analytical experimentation. Here, we describe some of the key tools and methods necessary to investigate plant mitochondrial dynamics.

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Truncated presequences of mitochondrial F1-ATPase β subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco

Cited by 49 publications

References 43 publications

Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

When green carbon plants meet synthetic biology

Imaging and Analysis of Mitochondrial Dynamics in Living Cells

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