Poster: Protein Targeting & Vesicular Trafficking

Abs # 1192: 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

The majority of 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 the transit peptide from an array of precursors involved in different biosynthetic pathways and destined for different chloroplast compartments. The transit peptide determines precursor binding and cleavage and is then converted to a degradable substrate by SPP. SPP contains a zinc-binding motif, HXXEH, and its activity depends on metal ions. When the N-terminal zinc-ligand of the HXXEH-motif was mutated to leucine, processing was abolished. The glutamic acid of the HXXEH-motif is believed to polarize a water molecule for nucleophilic attack on the scissile bond of the substrate. Mutation of this residue to glutamine inactivated SPP. Significantly, both SPP mutants still bind the transit peptide. Thus, transit peptide binding does not depend on an active catalytic site. To study the mechanism of the processing reaction, we performed competition experiments using oligopeptides of the transit peptide of the ferredoxin precursor. We found that precursor binding and processing depend on the interaction of SPP with the C-terminus of the transit peptide. SPP terminates the interaction by a second cleavage, converting the transit peptide to a subfragment form which is subsequently degraded by an ATP-dependent metallopeptidase. Analysis of the degradative activity suggests that it may recognize 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.