Article
Mutations at the stromal processing peptidase cleavage site of a thylakoid lumen protein precursor affect the rate of processing but not the fidelity
The Journal of Biological Chemistry
(1994)
Abstract
Nuclear-encoded stromal proteins are imported into the chloroplast by means of presequences, or transit peptides, which are removed after import by a stromal processing peptidase (SPP); the presequences of thylakoid lumen proteins are processed by SPP at intermediate sites prior to transport of these proteins across the thylakoid membrane. SPP has been previously shown to be a highly specific enzyme, but the basis for the reaction specificity is unclear, because the cleavage sites of different substrates display virtually no primary structure similarity. We have examined the influence of the cleavage site residues on the SPP reaction mechanism by introducing mutations at these positions (denoted -1 and +1, relative to the SPP cleavage site) within the presequence of the lumenal 33-kDa photosystem II protein. Substitution of the -1 Arg by Ala or Met leads to a 5-7-fold reduction in the rate of processing, whereas substitution by Glu almost completely blocks cleavage. The replacement of the +1 Ala by Lys likewise almost completely blocks cleavage. None of the introduced -1 mutations affect cleavage fidelity; we show that all three mutants are cleaved only at the correct site. All of the mutant precursors are efficiently imported into the thylakoid lumen of intact chloroplasts, indicating that this cleavage event is not an important element of the overall import pathway. The results indicate that the identity of the -1 residue, within the context of a given presequence, is important in terms of influencing processing efficiency, but that the site of cleavage is specified by other determinants. At least a proportion of the other determinants are likely to be in close proximity to the cleavage site, since the deletion of a 7-residue section spanning this site completely blocks processing.
Disciplines
Publication Date
1994
Publisher Statement
This research was originally published in The Journal of Biological Chemistry. Bassham DC, Creighton AM, Karnauchov I, Herrmann RG, Klösgen RB, Robinson C. Mutations at the stromal processing peptidase cleavage site of a thylakoid lumen protein precursor affect the rate of processing but not the fidelity. The Journal of Biological Chemistry. 1994; 269:16062-16066. © the American Society for Biochemistry and Molecular Biology
Citation Information
Diane C. Bassham, Alison M. Creighton, Ivan Karnauchov, Reinhold G. Herrmann, et al.. "Mutations at the stromal processing peptidase cleavage site of a thylakoid lumen protein precursor affect the rate of processing but not the fidelity" The Journal of Biological Chemistry Vol. 269 Iss. 23 (1994) p. 16062 - 16066 Available at: http://works.bepress.com/diane-bassham/10/