Limits...
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.

Show MeSH

Related in: MedlinePlus

SPP generates intact transit peptides. a, Amino acid sequences of transit peptides: preFD (Smeekens et al. 1985), preHSP21 (Vierling et al. 1988), preLHCP (Lamppa et al. 1985) and mutant of preLHCP with a modification indicated by open arrow heads (Clark et al. 1989), preRBCA (Werneke et al. 1988), and preRBCS (Coruzzi et al. 1984). Radiolabeled methionines incorporated into transit peptides during in vitro translation are indicated in bold. SPP processing sites are shown by filled arrows, and lengths of transit peptides are noted. b, Processing of [35S]methionine-labeled precursors by recombinant SPP. The long exposure times needed to detect the transit peptides (T) on the autoradiograms usually resulted in a loss of resolution of the precursor (P) and the mature protein (M). Closed circles mark an unidentified [35S]-labeled product detected in most of the substrate preparations. Name of each mature protein is given above each panel.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2164977&req=5

Figure 1: SPP generates intact transit peptides. a, Amino acid sequences of transit peptides: preFD (Smeekens et al. 1985), preHSP21 (Vierling et al. 1988), preLHCP (Lamppa et al. 1985) and mutant of preLHCP with a modification indicated by open arrow heads (Clark et al. 1989), preRBCA (Werneke et al. 1988), and preRBCS (Coruzzi et al. 1984). Radiolabeled methionines incorporated into transit peptides during in vitro translation are indicated in bold. SPP processing sites are shown by filled arrows, and lengths of transit peptides are noted. b, Processing of [35S]methionine-labeled precursors by recombinant SPP. The long exposure times needed to detect the transit peptides (T) on the autoradiograms usually resulted in a loss of resolution of the precursor (P) and the mature protein (M). Closed circles mark an unidentified [35S]-labeled product detected in most of the substrate preparations. Name of each mature protein is given above each panel.

Mentions: To determine if an intact transit peptide is usually an initial cleavage product, other chloroplast protein precursors were tested in processing reactions with recombinant SPP. In addition to preFD, the precursors of heat shock protein 21 (HSP21), LHCP, ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RBCA), and RBCS, which contain two or more methionines in the transit peptide (Fig. 1 a), were synthesized by in vitro translation as [35S]methionine-labeled polypeptides. Each substrate was incubated with recombinant SPP for five minutes. A fragment that was similar in mobility to the FD transit peptide was observed for each reaction upon tricine SDS-PAGE (Fig. 1 b). Thus, in all cases, SPP initially generated what appeared to be an intact transit peptide by a single endoproteolytic step.


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

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

SPP generates intact transit peptides. a, Amino acid sequences of transit peptides: preFD (Smeekens et al. 1985), preHSP21 (Vierling et al. 1988), preLHCP (Lamppa et al. 1985) and mutant of preLHCP with a modification indicated by open arrow heads (Clark et al. 1989), preRBCA (Werneke et al. 1988), and preRBCS (Coruzzi et al. 1984). Radiolabeled methionines incorporated into transit peptides during in vitro translation are indicated in bold. SPP processing sites are shown by filled arrows, and lengths of transit peptides are noted. b, Processing of [35S]methionine-labeled precursors by recombinant SPP. The long exposure times needed to detect the transit peptides (T) on the autoradiograms usually resulted in a loss of resolution of the precursor (P) and the mature protein (M). Closed circles mark an unidentified [35S]-labeled product detected in most of the substrate preparations. Name of each mature protein is given above each panel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: SPP generates intact transit peptides. a, Amino acid sequences of transit peptides: preFD (Smeekens et al. 1985), preHSP21 (Vierling et al. 1988), preLHCP (Lamppa et al. 1985) and mutant of preLHCP with a modification indicated by open arrow heads (Clark et al. 1989), preRBCA (Werneke et al. 1988), and preRBCS (Coruzzi et al. 1984). Radiolabeled methionines incorporated into transit peptides during in vitro translation are indicated in bold. SPP processing sites are shown by filled arrows, and lengths of transit peptides are noted. b, Processing of [35S]methionine-labeled precursors by recombinant SPP. The long exposure times needed to detect the transit peptides (T) on the autoradiograms usually resulted in a loss of resolution of the precursor (P) and the mature protein (M). Closed circles mark an unidentified [35S]-labeled product detected in most of the substrate preparations. Name of each mature protein is given above each panel.
Mentions: To determine if an intact transit peptide is usually an initial cleavage product, other chloroplast protein precursors were tested in processing reactions with recombinant SPP. In addition to preFD, the precursors of heat shock protein 21 (HSP21), LHCP, ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RBCA), and RBCS, which contain two or more methionines in the transit peptide (Fig. 1 a), were synthesized by in vitro translation as [35S]methionine-labeled polypeptides. Each substrate was incubated with recombinant SPP for five minutes. A fragment that was similar in mobility to the FD transit peptide was observed for each reaction upon tricine SDS-PAGE (Fig. 1 b). Thus, in all cases, SPP initially generated what appeared to be an intact transit peptide by a single endoproteolytic step.

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