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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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SPP has a different affinity for the products generated during processing. Aliquots of processing reactions with immobilized SPP using preFD and preHSP21 as substrates were collected at intervals over a 60-min period and immediately separated into the supernatant and the immobilized SPP fraction before analysis by standard SDS-PAGE (a) or tricine SDS-PAGE (b–e). [35S]cysteine-labeled preFD, used to monitor generation of mature FD, supernatant (a). [35S]methionine-labeled preFD, used to monitor generation of FD transit peptide and its subfragment, supernatant (b), and immobilized SPP fraction (c). [35S]methionine-labeled preHSP21 supernatant (d), and immobilized SPP fraction (e). Substrate control (C), lane 1.
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Figure 4: SPP has a different affinity for the products generated during processing. Aliquots of processing reactions with immobilized SPP using preFD and preHSP21 as substrates were collected at intervals over a 60-min period and immediately separated into the supernatant and the immobilized SPP fraction before analysis by standard SDS-PAGE (a) or tricine SDS-PAGE (b–e). [35S]cysteine-labeled preFD, used to monitor generation of mature FD, supernatant (a). [35S]methionine-labeled preFD, used to monitor generation of FD transit peptide and its subfragment, supernatant (b), and immobilized SPP fraction (c). [35S]methionine-labeled preHSP21 supernatant (d), and immobilized SPP fraction (e). Substrate control (C), lane 1.

Mentions: The finding that SPP does not bind the FD transit peptide subfragment showed that trimming of the transit peptide alters SPP's affinity for the transit peptide, and suggested this may serve as a specific step to trigger its release from SPP. To understand the temporal relationship between transit peptide conversion to the subfragment form and release from SPP, time courses of precursor processing by immobilized SPP were carried out. Binding of the processing products to SPP was monitored by separate analysis of the supernatant and the immobilized SPP fraction. Using preFD and preHSP21 as substrates, during the first 10 min of processing, both mature proteins, FD and HSP21, and their respective transit peptides were produced simultaneously, but they were found in different fractions (Fig. 4). The mature proteins were immediately released into the supernatant (Fig. 4, a and d, lanes 2–4). In contrast, the intact transit peptides remained bound to immobilized SPP (Fig. 4c and Fig. e, lanes 2–4). Beginning at 10 min, proteolytic conversion of the transit peptide coincided with the release of its subfragment into the supernatant (Fig. 4b and Fig. d, lanes 5–7). This demonstrates that generation and release of the mature protein is accompanied by an initial accumulation of the intact transit peptide bound to SPP. Subsequently, limited proteolysis by SPP releases the subfragment, perhaps, to clear a binding site for new substrate.


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

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

SPP has a different affinity for the products generated during processing. Aliquots of processing reactions with immobilized SPP using preFD and preHSP21 as substrates were collected at intervals over a 60-min period and immediately separated into the supernatant and the immobilized SPP fraction before analysis by standard SDS-PAGE (a) or tricine SDS-PAGE (b–e). [35S]cysteine-labeled preFD, used to monitor generation of mature FD, supernatant (a). [35S]methionine-labeled preFD, used to monitor generation of FD transit peptide and its subfragment, supernatant (b), and immobilized SPP fraction (c). [35S]methionine-labeled preHSP21 supernatant (d), and immobilized SPP fraction (e). Substrate control (C), lane 1.
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Related In: Results  -  Collection

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Figure 4: SPP has a different affinity for the products generated during processing. Aliquots of processing reactions with immobilized SPP using preFD and preHSP21 as substrates were collected at intervals over a 60-min period and immediately separated into the supernatant and the immobilized SPP fraction before analysis by standard SDS-PAGE (a) or tricine SDS-PAGE (b–e). [35S]cysteine-labeled preFD, used to monitor generation of mature FD, supernatant (a). [35S]methionine-labeled preFD, used to monitor generation of FD transit peptide and its subfragment, supernatant (b), and immobilized SPP fraction (c). [35S]methionine-labeled preHSP21 supernatant (d), and immobilized SPP fraction (e). Substrate control (C), lane 1.
Mentions: The finding that SPP does not bind the FD transit peptide subfragment showed that trimming of the transit peptide alters SPP's affinity for the transit peptide, and suggested this may serve as a specific step to trigger its release from SPP. To understand the temporal relationship between transit peptide conversion to the subfragment form and release from SPP, time courses of precursor processing by immobilized SPP were carried out. Binding of the processing products to SPP was monitored by separate analysis of the supernatant and the immobilized SPP fraction. Using preFD and preHSP21 as substrates, during the first 10 min of processing, both mature proteins, FD and HSP21, and their respective transit peptides were produced simultaneously, but they were found in different fractions (Fig. 4). The mature proteins were immediately released into the supernatant (Fig. 4, a and d, lanes 2–4). In contrast, the intact transit peptides remained bound to immobilized SPP (Fig. 4c and Fig. e, lanes 2–4). Beginning at 10 min, proteolytic conversion of the transit peptide coincided with the release of its subfragment into the supernatant (Fig. 4b and Fig. d, lanes 5–7). This demonstrates that generation and release of the mature protein is accompanied by an initial accumulation of the intact transit peptide bound to SPP. Subsequently, limited proteolysis by SPP releases the subfragment, perhaps, to clear a binding site for new substrate.

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