CytochromefEncoded by the Chloroplast Genome Is Imported into Thylakoids via the SecA-Dependent Pathway

Nohara, Tetsuya; Asai, Tomomi; Nakai, Masaaki; Sugiura, Masahiro; Endo, Toshiya

doi:10.1006/bbrc.1996.1051

Cited by 33 publications

(17 citation statements)

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“…This is also in agreement with the observation that the N-terminal SP of pCytf can target ␤-galactosidase to the E. coli inner membrane in a SecAdependent manner (9). Furthermore, (non-physiological) posttranslational in vitro import studies into isolated thylakoids (35) or intact chloroplasts (36) indicate that pCytf membrane insertion is cpSecA-dependent.…”

Section: Figsupporting

confidence: 88%

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Röhl¹,

Wijk

2001

Journal of Biological Chemistry

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Using a homologous chloroplast translation system, we have reconstituted insertion and processing of the chloroplast-encoded thylakoid protein cytochrome f (pCytf). Cross-linking demonstrated that pCytf nascent chains when attached to the 70 S ribosome tightly interact with cpSecA, but this is strictly dependent on thylakoid membranes and a functional signal peptide. This indicates that cpSecA is only operative in pCytf biogenesis when it is bound to the membrane, most likely as part of the Sec translocon. No evidence for interaction between the 54-kDa subunit of the chloroplast signal recognition particle (cpSRP) and the pCytf nascent chain could be detected, suggesting that pCytf, in contrast to the polytopic D1 protein, does not require cpSRP for targeting. Insertion of pCytf occurred only co-translationally, resulting in processing and accumulation of both the processed signal peptide and the mature protein in the thylakoid. This co-translational membrane insertion and processing required a functional signal peptide and was inhibited by azide, demonstrating that cpSecA is essential for translocation of the soluble luminal domain. pCytf also associated posttranslationally with thylakoids, but the soluble N-terminal domain could not be translocated into the lumen. This is the first study in which synthesis, targeting, and insertion of a chloroplast-encoded thylakoid membrane protein is reconstituted from exogenous transcripts and using the chloroplast translational machinery.

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Section: Figsupporting

confidence: 88%

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Röhl¹,

Wijk

2001

Journal of Biological Chemistry

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“…The synthesized mRNAs were purified and subjected to in vitro translation either with a wheat germ or with a reticulocyte cell‐free system in the presence of [4,5‐ 3 H]leucine (Amersham). In vitro import of the radiolabeled precursor proteins into isolated pea chloroplasts and thylakoids and suborganellar fractionation after import were performed as described previously [35–37]. For the experimental details, see the figure captions, also.…”

Section: Methodsmentioning

confidence: 99%

“…Published procedures were used for growing plants, recombinant DNA technique, subcellular and suborganellar fractionations, SDS‐PAGE, fluorography, and immunoblotting analyses [35–37].…”

Section: Methodsmentioning

confidence: 99%

Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids

et al. 1999

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We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein. The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide. The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids. Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein. The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll a/b-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.z 1999 Federation of European Biochemical Societies.

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“…In bacterial cells, secretory proteins are translocated across the cytoplasmic membrane through a channel consisting of SecY/E/G proteins with the aid of a translocation ATPase, SecA. In chloroplasts, homologues of SecA (cpSecA), SecY (cpSecY) and SecE (cpSecE) have been identified and shown to mediate transport of a class of thylakoid lumenal proteins in vitro and in vivo (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Another class of thylakoid lumenal proteins is translocated across the thylakoid membranes via a transport pathway that requires ⌬pH across the thylakoid membranes as a sole energy source and Hcf106 protein in the thylakoid membranes (13)(14)(15).…”

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confidence: 99%

Sec-dependent Pathway and ΔpH-dependent Pathway Do Not Share a Common Translocation Pore in Thylakoidal Protein Transport

Asai¹,

Shinoda²,

Nohara³

et al. 1999

Journal of Biological Chemistry

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Thylakoidal proteins of plant chloroplasts are transported to thylakoids via several different pathways, including the ⌬pH-dependent and the Sec-dependent pathways. In this study, we asked if these two pathways utilize a common translocation pore. A fusion protein consisting of a 23-kDa subunit of the oxygen evolving complex and Escherichia coli biotin carboxyl carrier protein was biotinylated in E. coli cells and purified. When incubated with isolated pea thylakoids in the absence of avidin, the purified fusion protein was imported into the thylakoids via the ⌬pH-dependent pathway. However in the presence of avidin, the fusion protein became lodged in the thylakoid membranes, with its N terminus reaching the thylakoidal lumen, while its C-terminal segment complexed with avidin exposed on the thylakoidal surface. The translocation intermediate of the fusion protein inhibited the import of authentic 23-kDa subunit, suggesting that it occupies a putative translocation pore for the ⌬pH-dependent pathway. However the intermediate did not block import of the 33-kDa subunit of the oxygen evolving complex, which is a substrate for the Sec-dependent pathway. These results provide evidence against the possibility of a common translocation pore shared by the Sec-dependent pathway and the ⌬pH-dependent pathway.Nuclear-encoded thylakoidal proteins are synthesized in the cytosol, are imported into the chloroplast stroma, and are subsequently inserted into or translocated across the thylakoid membranes. Recent evidence has shown that thylakoidal proteins are transported to the thylakoids via several different pathways, including the Sec-dependent and the ⌬pH-dependent pathways (1, 2). In bacterial cells, secretory proteins are translocated across the cytoplasmic membrane through a channel consisting of SecY/E/G proteins with the aid of a translocation ATPase, SecA. In chloroplasts, homologues of SecA (cpSecA), SecY (cpSecY) and SecE (cpSecE) have been identified and shown to mediate transport of a class of thylakoid lumenal proteins in vitro and in vivo (3-12). Another class of thylakoid lumenal proteins is translocated across the thylakoid membranes via a transport pathway that requires ⌬pH across the thylakoid membranes as a sole energy source and Hcf106 protein in the thylakoid membranes (13-15). This ⌬pH pathway appears to have ability to translocate folded proteins that are not accepted by the Sec-dependent pathway (16 -18). Recent studies have revealed that a protein transport pathway similar to the thylakoidal ⌬pH-dependent pathway is present in bacterial cells (19 -21).Although the Sec-dependent and the ⌬pH-dependent pathways appear to utilize some pathway-specific components, they could share common translocation pore in the thylakoid membranes. In vitro competition experiments have shown that the import of a certain substrate protein into isolated thylakoids is effectively competed with saturating amounts of another competitor protein which utilizes the same thylakoidal transport pathway as the substrate protein but...

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CytochromefEncoded by the Chloroplast Genome Is Imported into Thylakoids via the SecA-Dependent Pathway

Cited by 33 publications

References 0 publications

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System

Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids

Sec-dependent Pathway and ΔpH-dependent Pathway Do Not Share a Common Translocation Pore in Thylakoidal Protein Transport

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