MUTATIONS AT THE STROMAL PROCESSING PEPTIDASE CLEAVAGE SITE OF A THYLAKOID LUMEN PROTEIN-PRECURSOR AFFECT THE RATE OF PROCESSING BUT NOT THE FIDELITY

Nuclear-encoded stromal proteins are imported into the chloroplast by means of presequences, or transit peptides, which are removed after im port by a stromal processing peptidase (SPP); the presequences of thyl akoid 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 fo r the reaction specificity is unclear, because the cleavage sites of d ifferent substrates display virtually no primary structure similarity. We have examined the influence of the cleavage site residues on the S PP reaction mechanism by introducing mutations at these positions (den oted -1 and +1, relative to the SPP cleavage site) within the preseque nce 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 proc essing, whereas substitution by Glu almost completely blocks cleavage. The replacement of the +1 Ala by Lys likewise almost completely block s cleavage. None of the introduced -1 mutations affect cleavage fideli ty; we show that all three mutants are cleaved only at the correct sit e. All of the mutant precursors are efficiently imported into the thyl akoid lumen of intact chloroplasts, indicating that this cleavage even t is not an important element of the overall import pathway. The resul ts 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 determ inants. 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.