title: Stromal processing peptidase binds transit peptides and initiates their ATP-dependent turnover in chloroplasts.
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.
authors.affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: S
authors.initials: S
authors.lastname: Richter
authors.forename: G K
authors.initials: GK
authors.lastname: Lamppa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002614:Chelating Agents / Q000494:pharmacology; D002736:Chloroplasts / Q000201:enzymology* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D059828:Plant Cells; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature
chemical_list: D002614:Chelating Agents; D010618:Phenanthrolines; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D008666:Metalloendopeptidases; C071986:stromal processing peptidase; D005033:Ethylmaleimide; C025205:1,10-phenanthroline