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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.

Show MeSH
Schematic representation of transit peptide removal and turnover in the chloroplast. Step 1, A precursor entering the stroma with its NH2-terminal transit peptide first is recognized by SPP. A single endoproteolytic cleavage by SPP releases the mature protein. The transit peptide remains bound by SPP. Step 2, SPP releases the transit peptide by its conversion to a subfragment. The regenerated SPP is free for a new enzyme–substrate interaction. Step 3, The liberated subfragment is degraded by an ATP-dependent, soluble metallopeptidase.
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Figure 8: Schematic representation of transit peptide removal and turnover in the chloroplast. Step 1, A precursor entering the stroma with its NH2-terminal transit peptide first is recognized by SPP. A single endoproteolytic cleavage by SPP releases the mature protein. The transit peptide remains bound by SPP. Step 2, SPP releases the transit peptide by its conversion to a subfragment. The regenerated SPP is free for a new enzyme–substrate interaction. Step 3, The liberated subfragment is degraded by an ATP-dependent, soluble metallopeptidase.

Mentions: The experiments presented in this study provide evidence that transit peptide removal and turnover are regulated processes, and significantly, degradation of the transit peptide is an ATP-dependent step not previously recognized in the general import pathway. Our observations have been incorporated into a model (Fig. 8) that makes a number of important predictions about how SPP carries out its function during precursor import.


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

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

Schematic representation of transit peptide removal and turnover in the chloroplast. Step 1, A precursor entering the stroma with its NH2-terminal transit peptide first is recognized by SPP. A single endoproteolytic cleavage by SPP releases the mature protein. The transit peptide remains bound by SPP. Step 2, SPP releases the transit peptide by its conversion to a subfragment. The regenerated SPP is free for a new enzyme–substrate interaction. Step 3, The liberated subfragment is degraded by an ATP-dependent, soluble metallopeptidase.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Schematic representation of transit peptide removal and turnover in the chloroplast. Step 1, A precursor entering the stroma with its NH2-terminal transit peptide first is recognized by SPP. A single endoproteolytic cleavage by SPP releases the mature protein. The transit peptide remains bound by SPP. Step 2, SPP releases the transit peptide by its conversion to a subfragment. The regenerated SPP is free for a new enzyme–substrate interaction. Step 3, The liberated subfragment is degraded by an ATP-dependent, soluble metallopeptidase.
Mentions: The experiments presented in this study provide evidence that transit peptide removal and turnover are regulated processes, and significantly, degradation of the transit peptide is an ATP-dependent step not previously recognized in the general import pathway. Our observations have been incorporated into a model (Fig. 8) that makes a number of important predictions about how SPP carries out its function during precursor import.

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