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Stromal processing peptidase binds transit peptides and initiates their ATP-dependent turnover in chloroplasts.

Richter S, Lamppa GK - J. Cell Biol. (1999)

Bottom Line: We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release.A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent.Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

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Transit peptide turnover by a chloroplast extract yields a discrete subfragment before degradation. Using preFD and preHSP21 as substrates, time courses of processing reactions with chloroplast extract were carried out and analyzed by standard SDS-PAGE (a) or tricine SDS-PAGE (b and c). If inhibition by 1,10-phenanthroline or EDTA was tested, the reactions were interrupted by adding the inhibitor and then continued for up to 30 min. a, [35S]cysteine-labeled preFD used to monitor generation of mature FD. b, [35S]methionine-labeled preFD used to monitor generation of FD transit peptide and its subfragment, lanes 1–6; inhibitors were added after 2 min, lanes 7–9. c, [35S]methionine-labeled preHSP21, lanes 1–6; inhibitors were added after 5 min, lanes 7–9.
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Figure 5: Transit peptide turnover by a chloroplast extract yields a discrete subfragment before degradation. Using preFD and preHSP21 as substrates, time courses of processing reactions with chloroplast extract were carried out and analyzed by standard SDS-PAGE (a) or tricine SDS-PAGE (b and c). If inhibition by 1,10-phenanthroline or EDTA was tested, the reactions were interrupted by adding the inhibitor and then continued for up to 30 min. a, [35S]cysteine-labeled preFD used to monitor generation of mature FD. b, [35S]methionine-labeled preFD used to monitor generation of FD transit peptide and its subfragment, lanes 1–6; inhibitors were added after 2 min, lanes 7–9. c, [35S]methionine-labeled preHSP21, lanes 1–6; inhibitors were added after 5 min, lanes 7–9.

Mentions: The intact transit peptide was identified as an initial product of precursor processing by SPP. However, the fate of the transit peptides in vivo is unknown. They are not stable upon in vitro import into the chloroplast using preFD as a substrate (van't Hof and de Kruijff 1995; also, Richter, S., and G. Lamppa, unpublished results). SPP has the capability to trim transit peptides, but to complete their turnover in the chloroplast, one predicts that other degradative activities are needed. Precursor processing by a chloroplast extract was analyzed to explore this prediction. Using preFD and preHSP21 as substrates, within five minutes both the mature protein and the transit peptide appeared at the same time (Fig. 5, a–c, lanes 2 and 3). After ten minutes, conversion of the transit peptide to a subfragment was observed (Fig. 5b and Fig. c, lanes 4 and 5). The initial appearance and trimming of the transit peptide within the first 20 min of the reaction using the chloroplast extract resembled the pattern found for processing using immobilized SPP (Fig. 2, a and c). In sharp contrast to the results using immobilized SPP in a processing reaction, however, the subfragment was then quickly degraded by the chloroplast extract (Fig. 5b and Fig. c, lane 6). These results indicate that transit peptides are most likely trimmed in the chloroplast by SPP in a discrete step before their final degradation.


Stromal processing peptidase binds transit peptides and initiates their ATP-dependent turnover in chloroplasts.

Richter S, Lamppa GK - J. Cell Biol. (1999)

Transit peptide turnover by a chloroplast extract yields a discrete subfragment before degradation. Using preFD and preHSP21 as substrates, time courses of processing reactions with chloroplast extract were carried out and analyzed by standard SDS-PAGE (a) or tricine SDS-PAGE (b and c). If inhibition by 1,10-phenanthroline or EDTA was tested, the reactions were interrupted by adding the inhibitor and then continued for up to 30 min. a, [35S]cysteine-labeled preFD used to monitor generation of mature FD. b, [35S]methionine-labeled preFD used to monitor generation of FD transit peptide and its subfragment, lanes 1–6; inhibitors were added after 2 min, lanes 7–9. c, [35S]methionine-labeled preHSP21, lanes 1–6; inhibitors were added after 5 min, lanes 7–9.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2164977&req=5

Figure 5: Transit peptide turnover by a chloroplast extract yields a discrete subfragment before degradation. Using preFD and preHSP21 as substrates, time courses of processing reactions with chloroplast extract were carried out and analyzed by standard SDS-PAGE (a) or tricine SDS-PAGE (b and c). If inhibition by 1,10-phenanthroline or EDTA was tested, the reactions were interrupted by adding the inhibitor and then continued for up to 30 min. a, [35S]cysteine-labeled preFD used to monitor generation of mature FD. b, [35S]methionine-labeled preFD used to monitor generation of FD transit peptide and its subfragment, lanes 1–6; inhibitors were added after 2 min, lanes 7–9. c, [35S]methionine-labeled preHSP21, lanes 1–6; inhibitors were added after 5 min, lanes 7–9.
Mentions: The intact transit peptide was identified as an initial product of precursor processing by SPP. However, the fate of the transit peptides in vivo is unknown. They are not stable upon in vitro import into the chloroplast using preFD as a substrate (van't Hof and de Kruijff 1995; also, Richter, S., and G. Lamppa, unpublished results). SPP has the capability to trim transit peptides, but to complete their turnover in the chloroplast, one predicts that other degradative activities are needed. Precursor processing by a chloroplast extract was analyzed to explore this prediction. Using preFD and preHSP21 as substrates, within five minutes both the mature protein and the transit peptide appeared at the same time (Fig. 5, a–c, lanes 2 and 3). After ten minutes, conversion of the transit peptide to a subfragment was observed (Fig. 5b and Fig. c, lanes 4 and 5). The initial appearance and trimming of the transit peptide within the first 20 min of the reaction using the chloroplast extract resembled the pattern found for processing using immobilized SPP (Fig. 2, a and c). In sharp contrast to the results using immobilized SPP in a processing reaction, however, the subfragment was then quickly degraded by the chloroplast extract (Fig. 5b and Fig. c, lane 6). These results indicate that transit peptides are most likely trimmed in the chloroplast by SPP in a discrete step before their final degradation.

Bottom Line: We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release.A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent.Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
A stromal processing peptidase (SPP) cleaves a broad range of precursors targeted to the chloroplast, yielding proteins for numerous biosynthetic pathways in different compartments. SPP contains a signature zinc-binding motif, His-X-X-Glu-His, that places it in a metallopeptidase family which includes the mitochondrial processing peptidase. Here, we have investigated the mechanism of cleavage by SPP, a late, yet key event in the import pathway. Recombinant SPP removed the transit peptide from a variety of precursors in a single endoproteolytic step. Whereas the mature protein was immediately released, the transit peptide remained bound to SPP. SPP converted the transit peptide to a subfragment form that it no longer recognized. We conclude that SPP contains a specific binding site for the transit peptide and additional proteolysis by SPP triggers its release. A stable interaction between SPP and an intact transit peptide was directly demonstrated using a newly developed binding assay. Unlike recombinant SPP, a chloroplast extract rapidly degraded both the transit peptide and subfragment. A new degradative activity, distinguishable from SPP, was identified that is ATP- and metal-dependent. Our results indicate a regulated sequence of events as SPP functions during precursor import, and demonstrate a previously unrecognized ATP-requirement for transit peptide turnover.

Show MeSH