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

• Published in: The Journal of cell biology 1999-10, Vol.147 (1), p.33-43
• Main Authors: Richter, S, Lamppa, G.K
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino acids; binding; Binding Sites; Biological Transport; Cell biology; Cellular biology; Chelating Agents - pharmacology; Chlorophylls; Chloroplasts; Chloroplasts - enzymology; Chloroplasts - metabolism; enzyme activity; Enzymes; Ethylmaleimide - pharmacology; Hydrogen-Ion Concentration; Imports; Metal ions; Metalloendopeptidases - antagonists & inhibitors; Metalloendopeptidases - metabolism; Molecular Sequence Data; Original; Peptides; Phenanthrolines - pharmacology; Plant Cells; Plant Proteins - chemistry; Plant Proteins - metabolism; Plants - enzymology; Plants - metabolism; Protein Binding - drug effects; Protein precursors; Protein Precursors - chemistry; Protein Precursors - metabolism; Protein Processing, Post-Translational - drug effects; Protein Sorting Signals - chemistry; Protein Sorting Signals - metabolism; proteinases; Proteins; proteolysis; Recombinant Proteins - metabolism; signal peptide; Sodium Chloride - pharmacology; synthetic peptides; Temperature
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.147.1.33

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.