A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Clark, SK; Theg, Steven M.

doi:10.1091/mbc.8.5.923

Cited by 161 publications

(113 citation statements)

References 45 publications

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“…Export of folded proteins had only been demonstrated experimentally in vitro for the plant equivalent of Tat, the ⌬pH-dependent import system of thylakoids. Clark and Theg (15) showed that prefolded and chemically crosslinked BPTI could be imported into thylakoids when linked to the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17). However, BPTI is a very small protein (56 residues, 6.5 kDa) and therefore not typical of native Tat and ⌬pH substrates.…”

Section: Discussionmentioning

confidence: 99%

“…Processes such as cofactor incorporation or the assembly of protein subunits hinge on the formation of secondary or tertiary structure. In vitro experiments in the thylakoid system have shown that: (i) bovine pancreatic trypsin inhibitor (BPTI) fused to a ⌬pH-dependent pathway precursor is transported into thylakoids, even when BPTI was permanently folded by using irreversible disulfide cross-linkers that prevented any unfolding during the translocation process (15); and (ii) dihydrofolate reductase (DHFR) bound to methotrexate, a ligand that greatly stabilizes the folded state of the protein, is specifically targeted to the thylakoid lumen by the ⌬pH-dependent pathway (16). However, BPTI and DHFR are significantly smaller (56 and 187 aa, respectively) than typical ⌬pH-dependent polypeptide substrates.…”

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confidence: 99%

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Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

DeLisa

Tullman

Georgiou

2003

Proc. Natl. Acad. Sci. U.S.A.

279

385

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To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F AB antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F AB protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

DeLisa

Tullman

Georgiou

2003

Proc. Natl. Acad. Sci. U.S.A.

279

385

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“…1 and 2). Working in parallel with the well characterized Sec pathway, the Tat system uses an entirely different mechanism and has the unique ability to transport fully folded proteins across coupled membranes (3,4). In general, it appears to be used primarily for the transport of proteins that are either obliged to fold prior to translocation, or which fold too tightly or rapidly for the Sec system to handle.…”

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confidence: 99%

Essential Cytoplasmic Domains in the Escherichia coli TatC Protein

Allen

Barrett

Ray

et al. 2002

Journal of Biological Chemistry

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The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.

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“…We also observed that the import reaction continued in these experiments for at least 30 min without slowing, and sometimes even longer. This is longer than that usually reported for in vitro import reactions (38,41), which may be due to our keeping the chloroplasts in complete darkness during the reaction, thereby avoiding the formation of potentially harmful reactive oxygen species. This phenomenon was not investigated further.…”

Section: Effect Of Inhibitors On Intrinsic Background Atpase Activitymentioning

confidence: 77%

Energetic cost of protein import across the envelope membranes of chloroplasts

Shi

Theg

2012

Proc. Natl. Acad. Sci. U.S.A.

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Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ∼3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranesonly the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ∼650 ATP molecules. This translates to a ΔG protein transport of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ∼0.6% of the total light-saturated energy output of the organelle. T he majority of proteins in cells are synthesized in the cytoplasm on free or membrane-bound ribosomes. The fraction of proteins that are translocated across one or more cellular membranes has been estimated to be almost 50% in eukaryotic cells (1) and as high as 30% in bacteria (2, 3). Thus, protein movement out of the cytoplasm across membranes represents a considerable cellular activity involving numerous and varied protein transport systems. Protein translocation across membranes is also generally an energy-requiring process, making it a potentially costly activity for cells (4).The nature of the energy inputs to the different protein translocation systems has been extensively studied. We know, for example, that the posttranslational transport of proteins into the endoplasmic reticulum requires ATP hydrolysis alone (4-6), and that the transport of proteins into the thylakoid lumen or across the bacterial plasma membrane on the Tat pathway requires only the transmembrane protonmotive force with no contribution of ATP hydrolysis (7,8). A more complex energy input is required for many other protein transport systems. For instance, the import of proteins into the mitochondrial matrix requires both ATP hydrolysis and the Δψ across the inner mitochondrial membrane (9-11), and bacterial secretion and protein transport to the thylakoid lumen on their respective Sec pathways use ATP hydrolysis and the protonmotive force (3,7,12,13).In contrast to the abundance of work defining the nature of the energy driving protein transporters, few attempts have been made to quantitate the amount of energy required for these reactions. Without this information, it is not possible to assess the amount of metabolic energy that is spent on the cell's considerable protein trafficking activity. One such measurement of the energy required for protein transport is the so-called translo...

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A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Cited by 161 publications

References 45 publications

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway

Essential Cytoplasmic Domains in the Escherichia coli TatC Protein

Energetic cost of protein import across the envelope membranes of chloroplasts

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