Structural properties of the chloroplast stromal processing peptidase required for its function in transit peptide removal.
The stromal processing peptidase (SPP) catalyzes removal of transit peptides from a diversity of precursor proteins imported into chloroplasts. SPP contains an HXXEH zinc-binding motif characteristic of members of the metallopeptidase family M16. We previously found that the three steps of precursor processing by SPP (i.e. transit peptide binding, removal, and conversion to a degradable subfragment) are mediated by features that reside in the C-terminal 10-15 residues of the transit peptide. In this study, we performed a mutational analysis of SPP to identify structural elements that determine its function. SPP loses the ability to proteolytically remove the transit peptide when residues of the HXXEH motif, found in an N-terminal region, are mutated. Deletion of 240 amino acids from its C terminus also abolishes activity. Interestingly, however, SPP can still carry out the initial binding step, recognizing the C-terminal residues of the transit peptide. Hence, transit peptide binding and removal are two separable steps of the overall processing reaction. Transit peptide conversion to a subfragment also depends on the HXXEH motif. The precursor of SPP, containing an unusually long transit peptide itself, is not proteolytically active. Thus, the SPP precursor is synthesized as a latent form of the metallopeptidase.
Richter
Stefan
S
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
Lamppa
Gayle K
GK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000378:metabolism*; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D060365:Chloroplast Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; C078737:chloroplast transit peptides; D008666:Metalloendopeptidases; C071986:stromal processing peptidase; D015032:Zinc