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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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Selective degradation of the FD transit peptide and its subfragment in the chloroplast. [35S]methionine-labeled FD transit peptide (open square) and its subfragment (open triangle), as well as [35S]cysteine-labeled mature FD (open circle), were used as substrates in degradation assays with chloroplast extract. Reactions were analyzed by standard SDS-PAGE (mature FD) or tricine SDS-PAGE (transit peptide, subfragment) and quantified. a, Percent of substrate remaining upon incubation in chloroplast extract. b, Percent of substrate remaining upon 30 min incubation in chloroplast extract in the presence of 1,10-phenanthroline.
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Figure 6: Selective degradation of the FD transit peptide and its subfragment in the chloroplast. [35S]methionine-labeled FD transit peptide (open square) and its subfragment (open triangle), as well as [35S]cysteine-labeled mature FD (open circle), were used as substrates in degradation assays with chloroplast extract. Reactions were analyzed by standard SDS-PAGE (mature FD) or tricine SDS-PAGE (transit peptide, subfragment) and quantified. a, Percent of substrate remaining upon incubation in chloroplast extract. b, Percent of substrate remaining upon 30 min incubation in chloroplast extract in the presence of 1,10-phenanthroline.

Mentions: We analyzed the fate of the FD transit peptide and its subfragment separately. [35S]methionine-labeled FD transit peptide and its subfragment were prepared (see Materials and Methods) and used as substrates in separate reactions with chloroplast extract. We observed rapid conversion of the transit peptide and complete degradation of the subfragment within 30 min (Fig. 6 a). Mature FD was prepared by incubation (30 min) of [35S]cysteine-labeled preFD with immobilized SPP. The supernatant of this processing reaction was added directly to a chloroplast extract. Mature FD was relatively stable over 30 min (Fig. 6 a); we observed only a 15% loss of fragment within the first 2 min. The distinct stability of mature FD compared with its transit peptide in the chloroplast extract demonstrates that selective degradation of the latter occurred.


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

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

Selective degradation of the FD transit peptide and its subfragment in the chloroplast. [35S]methionine-labeled FD transit peptide (open square) and its subfragment (open triangle), as well as [35S]cysteine-labeled mature FD (open circle), were used as substrates in degradation assays with chloroplast extract. Reactions were analyzed by standard SDS-PAGE (mature FD) or tricine SDS-PAGE (transit peptide, subfragment) and quantified. a, Percent of substrate remaining upon incubation in chloroplast extract. b, Percent of substrate remaining upon 30 min incubation in chloroplast extract in the presence of 1,10-phenanthroline.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164977&req=5

Figure 6: Selective degradation of the FD transit peptide and its subfragment in the chloroplast. [35S]methionine-labeled FD transit peptide (open square) and its subfragment (open triangle), as well as [35S]cysteine-labeled mature FD (open circle), were used as substrates in degradation assays with chloroplast extract. Reactions were analyzed by standard SDS-PAGE (mature FD) or tricine SDS-PAGE (transit peptide, subfragment) and quantified. a, Percent of substrate remaining upon incubation in chloroplast extract. b, Percent of substrate remaining upon 30 min incubation in chloroplast extract in the presence of 1,10-phenanthroline.
Mentions: We analyzed the fate of the FD transit peptide and its subfragment separately. [35S]methionine-labeled FD transit peptide and its subfragment were prepared (see Materials and Methods) and used as substrates in separate reactions with chloroplast extract. We observed rapid conversion of the transit peptide and complete degradation of the subfragment within 30 min (Fig. 6 a). Mature FD was prepared by incubation (30 min) of [35S]cysteine-labeled preFD with immobilized SPP. The supernatant of this processing reaction was added directly to a chloroplast extract. Mature FD was relatively stable over 30 min (Fig. 6 a); we observed only a 15% loss of fragment within the first 2 min. The distinct stability of mature FD compared with its transit peptide in the chloroplast extract demonstrates that selective degradation of the latter occurred.

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