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© The Rockefeller University Press,
0021-9525/1999//33 $5.00
The Journal of Cell Biology, Volume 147, Number 1,
, 1999 33-44
Original Article |
Stromal Processing Peptidase Binds Transit Peptides and Initiates Their Atp-Dependent Turnover in Chloroplasts
gklamppa{at}midway.uchicago.edu
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.
Key Words: chloroplast • metallopeptidase • protein degradation • stromal processing peptidase • transit peptide
© 1999 The Rockefeller University Press
1.used in this paper: FD, ferredoxin; HSP21, heat shock protein 21; LHCP, light-harvesting chlorophyll a/b binding protein; MPP, mitochondrial processing peptidase; NEM, N-ethylmaleimide; preFD, precursor ferredoxin; preHSP21, precursor heat shock protein 21; preLHCP, precursor light-harvesting chlorophyll a/b binding protein; preRBCA, precursor ribulose-1,5-bisphosphate carboxylase/oxygenase activase; preRBCS, precursor ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit; RBCA, ribulose-1,5-bisphosphate carboxylase/oxygenase activase; RBCS, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit; SPP, stromal processing peptidase
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