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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 contains a binding site for the intact transit peptide. a, Schematic representation of the binding assay (see Materials and Methods). Recombinant SPP was immobilized onto streptavidin-coated magnetic beads that bound to its biotin-containing peptide tag. [35S]methionine-labeled preparations of FD transit peptide (T) or its subfragment (SF) were added. Using a magnet, the supernatant containing the unbound substrate was separated from the immobilized SPP fraction with the bound substrate. b, Binding of FD transit peptide to immobilized SPP. Total amount of substrate used in one reaction, control lane 1; supernatant containing unbound substrate, lane 2; bound substrate independently eluted at NaCl concentrations between 0 and 1,000 mM, lanes 3–8; substrate still bound to immobilized SPP after washing at 1,000 mM NaCl, lane 9. c, Binding of FD transit peptide to immobilized SPP in the presence of 5 mM 1,10-phenanthroline. Substrate, control lane 1; supernatant, lane 2; bound substrate independently eluted at NaCl concentrations of 200, 500, and 1,000 mM, lanes 3–5; substrate bound to immobilized SPP after washing at 1,000 mM NaCl, lane 6. d, Binding of FD transit peptide to streptavidin-coated magnetic beads incubated with extract of E. coli cells. Substrate, control lane 1; supernatant, lane 2; bound substrate eluted from the magnetic beads fraction at 500 mM NaCl, lane 3. Binding of subfragment of FD transit peptide to immobilized SPP. Substrate, control lane 4; supernatant, lane 5; bound substrate eluted from immobilized SPP at 500 mM NaCl, lane 6.
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Figure 3: SPP contains a binding site for the intact transit peptide. a, Schematic representation of the binding assay (see Materials and Methods). Recombinant SPP was immobilized onto streptavidin-coated magnetic beads that bound to its biotin-containing peptide tag. [35S]methionine-labeled preparations of FD transit peptide (T) or its subfragment (SF) were added. Using a magnet, the supernatant containing the unbound substrate was separated from the immobilized SPP fraction with the bound substrate. b, Binding of FD transit peptide to immobilized SPP. Total amount of substrate used in one reaction, control lane 1; supernatant containing unbound substrate, lane 2; bound substrate independently eluted at NaCl concentrations between 0 and 1,000 mM, lanes 3–8; substrate still bound to immobilized SPP after washing at 1,000 mM NaCl, lane 9. c, Binding of FD transit peptide to immobilized SPP in the presence of 5 mM 1,10-phenanthroline. Substrate, control lane 1; supernatant, lane 2; bound substrate independently eluted at NaCl concentrations of 200, 500, and 1,000 mM, lanes 3–5; substrate bound to immobilized SPP after washing at 1,000 mM NaCl, lane 6. d, Binding of FD transit peptide to streptavidin-coated magnetic beads incubated with extract of E. coli cells. Substrate, control lane 1; supernatant, lane 2; bound substrate eluted from the magnetic beads fraction at 500 mM NaCl, lane 3. Binding of subfragment of FD transit peptide to immobilized SPP. Substrate, control lane 4; supernatant, lane 5; bound substrate eluted from immobilized SPP at 500 mM NaCl, lane 6.

Mentions: We investigated if SPP specifically binds the intact transit peptide and its subfragment using an in vitro binding assay (Fig. 3 a). A protocol was established for preparation of [35S]methionine-labeled FD transit peptide and its subfragment (see Materials and Methods). Aliquots of a FD transit peptide preparation were incubated with immobilized SPP for five minutes. Each immobilized SPP fraction was separated from the supernatant and individually washed with a solution of NaCl at different concentrations for elution of bound transit peptide. To determine the amount of transit peptide that remained bound to immobilized SPP at 1,000 mM NaCl, one SPP fraction was washed in 1,000 mM NaCl and the bound material was liberated for analysis by boiling in the presence of gel-loading buffer. A representative supernatant, all eluates, and the material released by boiling were subjected to tricine SDS-PAGE to analyze by both autoradiography (Fig. 3 b) and PhosphorImager scanning for quantification. The supernatant was separated from the immobilized SPP fraction and contained unbound transit peptide (Fig. 3 b, lane 2). Washing of the immobilized SPP fraction without NaCl did not elute detectable amounts of transit peptide (Fig. 2 b, lane 3). Washing with low NaCl (50 mM) released 3% of the substrate originally added to one reaction (Fig. 2 b, lane 4). Using a higher NaCl concentration (100 mM) for washing of another immobilized fraction, a substantially greater amount of transit peptide, 13%, was released (Fig. 3 b, lane 5). However, if parallel fractions were washed with even higher concentrations of NaCl, the amount of eluted transit peptide did not increase, not even at 1,000 mM NaCl (Fig. 3 b, lanes 6–8). Surprisingly, however, boiling of the SPP fraction after washing with 1,000 mM NaCl released an additional 7% of the original substrate added to the reaction (Fig. 3 b, lane 9). Together, the eluate at 1,000 mM NaCl and its corresponding SPP fraction contained in total 20%, i.e. 13% + 7%, of the original substrate, and thus was equivalent to the portion of transit peptide initially bound by immobilized SPP in one binding reaction. Two-thirds of the bound transit peptide was released from SPP at high NaCl concentration, but, importantly, one third remained bound. This observation suggested that SPP may have two states of transit peptide binding, and one of these has an especially high affinity for the intact transit peptide.


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

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

SPP contains a binding site for the intact transit peptide. a, Schematic representation of the binding assay (see Materials and Methods). Recombinant SPP was immobilized onto streptavidin-coated magnetic beads that bound to its biotin-containing peptide tag. [35S]methionine-labeled preparations of FD transit peptide (T) or its subfragment (SF) were added. Using a magnet, the supernatant containing the unbound substrate was separated from the immobilized SPP fraction with the bound substrate. b, Binding of FD transit peptide to immobilized SPP. Total amount of substrate used in one reaction, control lane 1; supernatant containing unbound substrate, lane 2; bound substrate independently eluted at NaCl concentrations between 0 and 1,000 mM, lanes 3–8; substrate still bound to immobilized SPP after washing at 1,000 mM NaCl, lane 9. c, Binding of FD transit peptide to immobilized SPP in the presence of 5 mM 1,10-phenanthroline. Substrate, control lane 1; supernatant, lane 2; bound substrate independently eluted at NaCl concentrations of 200, 500, and 1,000 mM, lanes 3–5; substrate bound to immobilized SPP after washing at 1,000 mM NaCl, lane 6. d, Binding of FD transit peptide to streptavidin-coated magnetic beads incubated with extract of E. coli cells. Substrate, control lane 1; supernatant, lane 2; bound substrate eluted from the magnetic beads fraction at 500 mM NaCl, lane 3. Binding of subfragment of FD transit peptide to immobilized SPP. Substrate, control lane 4; supernatant, lane 5; bound substrate eluted from immobilized SPP at 500 mM NaCl, lane 6.
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Figure 3: SPP contains a binding site for the intact transit peptide. a, Schematic representation of the binding assay (see Materials and Methods). Recombinant SPP was immobilized onto streptavidin-coated magnetic beads that bound to its biotin-containing peptide tag. [35S]methionine-labeled preparations of FD transit peptide (T) or its subfragment (SF) were added. Using a magnet, the supernatant containing the unbound substrate was separated from the immobilized SPP fraction with the bound substrate. b, Binding of FD transit peptide to immobilized SPP. Total amount of substrate used in one reaction, control lane 1; supernatant containing unbound substrate, lane 2; bound substrate independently eluted at NaCl concentrations between 0 and 1,000 mM, lanes 3–8; substrate still bound to immobilized SPP after washing at 1,000 mM NaCl, lane 9. c, Binding of FD transit peptide to immobilized SPP in the presence of 5 mM 1,10-phenanthroline. Substrate, control lane 1; supernatant, lane 2; bound substrate independently eluted at NaCl concentrations of 200, 500, and 1,000 mM, lanes 3–5; substrate bound to immobilized SPP after washing at 1,000 mM NaCl, lane 6. d, Binding of FD transit peptide to streptavidin-coated magnetic beads incubated with extract of E. coli cells. Substrate, control lane 1; supernatant, lane 2; bound substrate eluted from the magnetic beads fraction at 500 mM NaCl, lane 3. Binding of subfragment of FD transit peptide to immobilized SPP. Substrate, control lane 4; supernatant, lane 5; bound substrate eluted from immobilized SPP at 500 mM NaCl, lane 6.
Mentions: We investigated if SPP specifically binds the intact transit peptide and its subfragment using an in vitro binding assay (Fig. 3 a). A protocol was established for preparation of [35S]methionine-labeled FD transit peptide and its subfragment (see Materials and Methods). Aliquots of a FD transit peptide preparation were incubated with immobilized SPP for five minutes. Each immobilized SPP fraction was separated from the supernatant and individually washed with a solution of NaCl at different concentrations for elution of bound transit peptide. To determine the amount of transit peptide that remained bound to immobilized SPP at 1,000 mM NaCl, one SPP fraction was washed in 1,000 mM NaCl and the bound material was liberated for analysis by boiling in the presence of gel-loading buffer. A representative supernatant, all eluates, and the material released by boiling were subjected to tricine SDS-PAGE to analyze by both autoradiography (Fig. 3 b) and PhosphorImager scanning for quantification. The supernatant was separated from the immobilized SPP fraction and contained unbound transit peptide (Fig. 3 b, lane 2). Washing of the immobilized SPP fraction without NaCl did not elute detectable amounts of transit peptide (Fig. 2 b, lane 3). Washing with low NaCl (50 mM) released 3% of the substrate originally added to one reaction (Fig. 2 b, lane 4). Using a higher NaCl concentration (100 mM) for washing of another immobilized fraction, a substantially greater amount of transit peptide, 13%, was released (Fig. 3 b, lane 5). However, if parallel fractions were washed with even higher concentrations of NaCl, the amount of eluted transit peptide did not increase, not even at 1,000 mM NaCl (Fig. 3 b, lanes 6–8). Surprisingly, however, boiling of the SPP fraction after washing with 1,000 mM NaCl released an additional 7% of the original substrate added to the reaction (Fig. 3 b, lane 9). Together, the eluate at 1,000 mM NaCl and its corresponding SPP fraction contained in total 20%, i.e. 13% + 7%, of the original substrate, and thus was equivalent to the portion of transit peptide initially bound by immobilized SPP in one binding reaction. Two-thirds of the bound transit peptide was released from SPP at high NaCl concentration, but, importantly, one third remained bound. This observation suggested that SPP may have two states of transit peptide binding, and one of these has an especially high affinity for the intact transit peptide.

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