Cloning and Characterization of Sec and Ffh Genes from the Cyanobacterium Synechococcus PCC7942

Nakai, Masaaki; Nohara, Tetsuya; Sugita, Daigo; Endo, Toshiya

doi:10.1007/978-94-009-0173-5_576

Cited by 9 publications

(10 citation statements)

References 9 publications

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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%

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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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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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“…A subset of lumenal proteins, including plastocyanin (PC), PSI‐F and the 33 kDa photosystem II protein (33K) are synthesized with bipartite presequences that contain two signals in tandem. The first ‘envelope transit’ signal specifies translocation across the envelope and is usually removed by the stromal processing peptidase (SPP), after which the second signal directs translocation across the thylakoid membrane by a prokaryotic‐type, Sec‐dependent mechanism that relies on ATP and the presence of a stromal SecA molecule (Hulford et al ., 1994; Karnauchov et al ., 1994; Mant et al ., 1994; Nakai et al ., 1994; Yuan et al ., 1994). The second signal is then removed by a lumen‐facing thylakoidal processing peptidase (TPP) that is mechanistically similar to bacterial signal peptidases (Halpin et al ., 1989).…”

Section: Introductionmentioning

confidence: 99%

Pathway specificity for a ΔpH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein

Bogsch

Brink

Robinson

1997

EMBO J

140

118

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Cleavable N-terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec-type or delta pH-driven protein translocase. The targeting signals specify choice of translocation pathway, yet all resemble typical bacterial 'signal' peptides in possessing a charged N-terminus (N-domain), hydrophobic core region (H-domain) and more polar C-terminal region (C-domain). We have previously shown that a twin-arginine motif in the N-domain is essential for targeting by the delta pH-dependent pathway, but it has remained unclear why targeting signals for this system (transfer peptides) are not recognized by the Sec apparatus. We show here that the conserved charge distribution around the H-domain in the 23K transfer peptide (twin-Arg in the N-domain, Lys in the C-domain) constitutes a 'Sec-avoidance' signal. The C-domain Lys, while not important for delta pH-dependent targeting, is the only barrier to Sec-dependent translocation; its removal generates an apparently perfect signal peptide. Conversely, insertion of twin-Arg into the N-domain of a Sec substrate has little effect, as has insertion of a C-domain Lys, but the combined substitutions almost totally block transport. We also show that the 23K mature protein is incapable of being targeted by the Sec pathway, and it is proposed that the role of the Sec-avoidance motif in the transfer peptide is to prevent futile interactions with the Sec apparatus.

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“…In fact, the situation is considerably more complex and there is now good evidence that lumenal proteins are transported by two completely different mechanisms (reviewed in Robinson and K1osgen, 1994). SecA is involved in this mechanism, strongly suggesting that the translocation mechanism was indeed inherited from a prokaryotic ancestor (Nakai et al, 1994;Yuan et al, 1994). SecA is involved in this mechanism, strongly suggesting that the translocation mechanism was indeed inherited from a prokaryotic ancestor (Nakai et al, 1994;Yuan et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase.

et al. 1995

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The delta pH‐driven and Sec‐related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH‐dependent system contain a common twin‐arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH‐driven translocation process; substitution of the arg‐arg by gln‐gln or even arg‐lys totally blocks translocation across the thylakoid membrane, and replacement by lys‐arg reduces the rate of translocation by > 100‐fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin‐arg motif into a Sec‐dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec‐type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

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Cloning and Characterization of Sec and Ffh Genes from the Cyanobacterium Synechococcus PCC7942

Cited by 9 publications

References 9 publications

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

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

Pathway specificity for a ΔpH-dependent precursor thylakoid lumen protein is governed by a 'Sec-avoidance' motif in the transfer peptide and a 'Sec-incompatible' mature protein

A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase.

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