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Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

Chen KY, Li HM - Plant J. (2006)

Bottom Line: It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40.Two different precursor proteins were shown to associate with the same complexes.Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

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Identifying a suitable time point during active import for identification of the translocon complex(a) [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C. At each time point, a portion of the import mixture was removed and the import reaction terminated by diluting with cold import buffer. An equal amount of protein was loaded in each lane.(b) Chase after 20-min import. [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C for 20 min. Chloroplasts were isolated and resuspended in import buffer containing 3 mm ATP. A portion of the chloroplasts were immediately re-isolated (chase time 0 min), or the reaction was incubated further at 20°C for 10 or 20 min (chase time 10 and 20 min). An equal amount of protein was loaded in each lane.
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fig01: Identifying a suitable time point during active import for identification of the translocon complex(a) [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C. At each time point, a portion of the import mixture was removed and the import reaction terminated by diluting with cold import buffer. An equal amount of protein was loaded in each lane.(b) Chase after 20-min import. [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C for 20 min. Chloroplasts were isolated and resuspended in import buffer containing 3 mm ATP. A portion of the chloroplasts were immediately re-isolated (chase time 0 min), or the reaction was incubated further at 20°C for 10 or 20 min (chase time 10 and 20 min). An equal amount of protein was loaded in each lane.

Mentions: To find a suitable time point during active import for identification of the translocon complex, import time-course experiments were performed with isolated pea chloroplasts and in vitro-translated [35S]-labeled precursors to the small subunit of Rubisco (prRBCS). To maximize the signal intensity for further analyses, one volume of chloroplasts (at a concentration of 1 mg chlorophyll ml−1) was mixed with one volume of [35S]prRBCS. To prolong the time span of active import in order to increase the chance of observing intermediates, import was performed at 20°C instead of 25°C. Under these conditions, the amount of mature RBCS imported continued to increase for about 60 min (Figure 1a). The 20-min time point was chosen for further analyses because chloroplasts at this time point contained a large amount of prRBCS and the amount of RBCS imported was increasing linearly. When chloroplasts from a 20-min import reaction were isolated and further incubated in a buffer containing 3 mm ATP but without additional prRBCS, the prRBCS molecules associated with chloroplasts were gradually chased into mature RBCS (Figure 1b), indicating that the prRBCS molecules were on an active course of import.


Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

Chen KY, Li HM - Plant J. (2006)

Identifying a suitable time point during active import for identification of the translocon complex(a) [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C. At each time point, a portion of the import mixture was removed and the import reaction terminated by diluting with cold import buffer. An equal amount of protein was loaded in each lane.(b) Chase after 20-min import. [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C for 20 min. Chloroplasts were isolated and resuspended in import buffer containing 3 mm ATP. A portion of the chloroplasts were immediately re-isolated (chase time 0 min), or the reaction was incubated further at 20°C for 10 or 20 min (chase time 10 and 20 min). An equal amount of protein was loaded in each lane.
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Related In: Results  -  Collection

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fig01: Identifying a suitable time point during active import for identification of the translocon complex(a) [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C. At each time point, a portion of the import mixture was removed and the import reaction terminated by diluting with cold import buffer. An equal amount of protein was loaded in each lane.(b) Chase after 20-min import. [35S]prRBCS was incubated with isolated chloroplasts under import conditions at 20°C for 20 min. Chloroplasts were isolated and resuspended in import buffer containing 3 mm ATP. A portion of the chloroplasts were immediately re-isolated (chase time 0 min), or the reaction was incubated further at 20°C for 10 or 20 min (chase time 10 and 20 min). An equal amount of protein was loaded in each lane.
Mentions: To find a suitable time point during active import for identification of the translocon complex, import time-course experiments were performed with isolated pea chloroplasts and in vitro-translated [35S]-labeled precursors to the small subunit of Rubisco (prRBCS). To maximize the signal intensity for further analyses, one volume of chloroplasts (at a concentration of 1 mg chlorophyll ml−1) was mixed with one volume of [35S]prRBCS. To prolong the time span of active import in order to increase the chance of observing intermediates, import was performed at 20°C instead of 25°C. Under these conditions, the amount of mature RBCS imported continued to increase for about 60 min (Figure 1a). The 20-min time point was chosen for further analyses because chloroplasts at this time point contained a large amount of prRBCS and the amount of RBCS imported was increasing linearly. When chloroplasts from a 20-min import reaction were isolated and further incubated in a buffer containing 3 mm ATP but without additional prRBCS, the prRBCS molecules associated with chloroplasts were gradually chased into mature RBCS (Figure 1b), indicating that the prRBCS molecules were on an active course of import.

Bottom Line: It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40.Two different precursor proteins were shown to associate with the same complexes.Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.

ABSTRACT
The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

Show MeSH