Minisymposium 18: Protein Targeting

Abs # 32003: Transit peptide binding, removal, and degradation during processing of chloroplast precursor proteins.

Presenter:	Richter, Stefan , srichter@uchicago.edu
Authors	Richter, Stefan  (A)   Lamppa, Gayle K (A)
Affiliations:	(A): Department of Molecular Genetics and Cell Biology, University of Chicago

Most chloroplast proteins are nuclear-encoded and synthesized in the cytosol as precursors with an N-terminal transit peptide which directs import across the choloroplast envelope into the stroma. We demonstrated that a stromal processing peptidase (SPP) removes transit peptides from an array of precursors involved in different biosynthetic pathways and destined for different chloroplast compartments. Experiments to study the mechanism of transit peptide removal revealed a succession of events catalyzed by SPP. SPP initially recognizes a precursor by binding to its transit peptide. It then cleaves the precursor to liberate the mature protein, while the interaction between SPP and the transit peptide is maintained. We discovered that SPP carries out an additional cleavage by converting the transit peptide to a subfragment which triggers its release from the enzyme. In a reaction independent of SPP, the subfragment is subsequently degraded in the stroma. Our analysis revealed a new proteolytic activity that depends on ATP and metal ions. Surprisingly, the degradative activity may hydrolyze other unrelated peptides as well. Therefore, SPP may not only be critical for precursor maturation in the import pathway, but it may provide an important link to a newly recognized intraorganellar proteolytic system. To identify structural determinants of the processing reaction we searched for elements involved in the SPP-precursor interaction. We initiated a mutant analysis of SPP which is a metallopeptidase containing the zinc-binding motif HXXEH. When the N-terminal histidine of the motif was mutated to leucine, processing was abolished. The glutamic acid of the motif is believed to polarize a water molecule for nucleophilic attack on the scissile bond of a precursor. Mutation of the residue to glutamine inactivated SPP. Significantly, both SPP mutants still bind the transit peptide. Thus, transit peptide binding by SPP does not depend on an active catalytic site. Competition experiments using synthetic oligopeptides of a transit peptide were performed to examine which regions of the transit peptide interact with SPP. Strikingly, the C-terminal 12-mer of the transit peptide blocked binding and removal of the intact transit peptide as well as its conversion to the subfragment. Hence this region alone is necessary to mediate each of the three steps catalyzed by SPP in a processing reaction that finally leads to the release of a degradable transit peptide subfragment.