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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 peptides are converted to subfragments by SPP. Time courses of processing by immobilized SPP were carried out using [35S]methionine-labeled precursors as substrates. Separation of precursor (P) and products (mature protein, M; transit peptide, T; subfragment of transit peptide, SF) was done by tricine SDS-PAGE, if not otherwise noted. PreFD alone (a) and with 5 mM 1,10-phenanthroline added after 2 min (b). PreHSP21 without (c) and with 5 mM 1,10-phenanthroline added after 5 min (d). PreLHCP wild-type (e) and mutant (f). The 90-min incubation was done in a separate experiment (e and f, lane 8). PreRBCA (g), the top shows a standard SDS-PAGE used to monitor generation of mature RBCA, the middle shows generation of T and SF1, and the bottom shows an enlargement of lanes 2–5 of the middle panel. PreRBCA (h), to detect SF2, the time for tricine SDS-PAGE was reduced. Autoradiograms with different exposure times were used for presentation of the top and the bottom part of a gel, as indicated by brackets at right.
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Figure 2: Transit peptides are converted to subfragments by SPP. Time courses of processing by immobilized SPP were carried out using [35S]methionine-labeled precursors as substrates. Separation of precursor (P) and products (mature protein, M; transit peptide, T; subfragment of transit peptide, SF) was done by tricine SDS-PAGE, if not otherwise noted. PreFD alone (a) and with 5 mM 1,10-phenanthroline added after 2 min (b). PreHSP21 without (c) and with 5 mM 1,10-phenanthroline added after 5 min (d). PreLHCP wild-type (e) and mutant (f). The 90-min incubation was done in a separate experiment (e and f, lane 8). PreRBCA (g), the top shows a standard SDS-PAGE used to monitor generation of mature RBCA, the middle shows generation of T and SF1, and the bottom shows an enlargement of lanes 2–5 of the middle panel. PreRBCA (h), to detect SF2, the time for tricine SDS-PAGE was reduced. Autoradiograms with different exposure times were used for presentation of the top and the bottom part of a gel, as indicated by brackets at right.

Mentions: Despite the large mass of diverse transit peptides that enter the organelle, nothing is known about their turnover. To investigate if SPP is involved, time courses of processing reactions were carried out using the precursors shown in Fig. 1 a and recombinant SPP, which was immobilized onto magnetic beads (see Materials and Methods). After 2 min, the mature protein and the transit peptide appeared simultaneously in each reaction, although the processing of each precursor progressed at a different rate (Fig. 2, a, c, e, and g; not shown for RBCS). Interestingly, using the precursors for FD, HSP21 and LHCP, we found that the initial generation of intact transit peptide was followed by its conversion to one detectable subfragment. The transit peptide of RBCA gave rise to two subfragments (RBCA: Fig. 2g and Fig. h). Conversion of the RBCS transit peptide could not be directly monitored. Instead, it nearly disappeared after one hour, probably because the conversion products containing the labeled methionines were too small to be seen upon tricine SDS-PAGE. Progression of this trimming reaction differed considerably between the transit peptides examined. At one extreme, the conversion of the RBCA transit peptide to subfragment 1 was completed in <10 min (Fig. 2 g), whereas an incubation time of 90 min was needed for full conversion of the LHCP transit peptide to a smaller form (Fig. 2 e). Nevertheless, based on our results with four substrates, trimming of transit peptides as another function of SPP is apparently a common step upon precursor processing.


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

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

Transit peptides are converted to subfragments by SPP. Time courses of processing by immobilized SPP were carried out using [35S]methionine-labeled precursors as substrates. Separation of precursor (P) and products (mature protein, M; transit peptide, T; subfragment of transit peptide, SF) was done by tricine SDS-PAGE, if not otherwise noted. PreFD alone (a) and with 5 mM 1,10-phenanthroline added after 2 min (b). PreHSP21 without (c) and with 5 mM 1,10-phenanthroline added after 5 min (d). PreLHCP wild-type (e) and mutant (f). The 90-min incubation was done in a separate experiment (e and f, lane 8). PreRBCA (g), the top shows a standard SDS-PAGE used to monitor generation of mature RBCA, the middle shows generation of T and SF1, and the bottom shows an enlargement of lanes 2–5 of the middle panel. PreRBCA (h), to detect SF2, the time for tricine SDS-PAGE was reduced. Autoradiograms with different exposure times were used for presentation of the top and the bottom part of a gel, as indicated by brackets at right.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Transit peptides are converted to subfragments by SPP. Time courses of processing by immobilized SPP were carried out using [35S]methionine-labeled precursors as substrates. Separation of precursor (P) and products (mature protein, M; transit peptide, T; subfragment of transit peptide, SF) was done by tricine SDS-PAGE, if not otherwise noted. PreFD alone (a) and with 5 mM 1,10-phenanthroline added after 2 min (b). PreHSP21 without (c) and with 5 mM 1,10-phenanthroline added after 5 min (d). PreLHCP wild-type (e) and mutant (f). The 90-min incubation was done in a separate experiment (e and f, lane 8). PreRBCA (g), the top shows a standard SDS-PAGE used to monitor generation of mature RBCA, the middle shows generation of T and SF1, and the bottom shows an enlargement of lanes 2–5 of the middle panel. PreRBCA (h), to detect SF2, the time for tricine SDS-PAGE was reduced. Autoradiograms with different exposure times were used for presentation of the top and the bottom part of a gel, as indicated by brackets at right.
Mentions: Despite the large mass of diverse transit peptides that enter the organelle, nothing is known about their turnover. To investigate if SPP is involved, time courses of processing reactions were carried out using the precursors shown in Fig. 1 a and recombinant SPP, which was immobilized onto magnetic beads (see Materials and Methods). After 2 min, the mature protein and the transit peptide appeared simultaneously in each reaction, although the processing of each precursor progressed at a different rate (Fig. 2, a, c, e, and g; not shown for RBCS). Interestingly, using the precursors for FD, HSP21 and LHCP, we found that the initial generation of intact transit peptide was followed by its conversion to one detectable subfragment. The transit peptide of RBCA gave rise to two subfragments (RBCA: Fig. 2g and Fig. h). Conversion of the RBCS transit peptide could not be directly monitored. Instead, it nearly disappeared after one hour, probably because the conversion products containing the labeled methionines were too small to be seen upon tricine SDS-PAGE. Progression of this trimming reaction differed considerably between the transit peptides examined. At one extreme, the conversion of the RBCA transit peptide to subfragment 1 was completed in <10 min (Fig. 2 g), whereas an incubation time of 90 min was needed for full conversion of the LHCP transit peptide to a smaller form (Fig. 2 e). Nevertheless, based on our results with four substrates, trimming of transit peptides as another function of SPP is apparently a common step upon precursor processing.

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
Related in: MedlinePlus