Effect of mutations on the binding and translocation functions of a chloroplast transit peptide.
We studied transport and binding to intact chloroplasts of 10 mutants in three regions of the transit peptide of a precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (transphosphorylating), E.C.4.1.1.39]. Transport was assayed in a reconstituted system using isolated pea chloroplasts and radioactively labeled precursor. Binding to the chloroplast envelope was assayed in a similar manner using chloroplasts pretreated with nigericin. Most mutants showed a dramatically decreased capacity of binding, although some of them transported relatively well. The accumulation of the mutant proteins inside the chloroplast as a function of time was examined. Although the authentic small subunit precursor was imported rapidly, uptake of most mutant precursors was considerably slower and continued until the last time point examined. In terms of assigning functions to individual regions, we found that at least the middle region and parts of the amino and the carboxyl termini of the transit peptide are more important for receptor binding than for translocation. A two-step processing mechanism has been postulated for the maturation of the small subunit precursor. This model predicts the occurrence of processing intermediates. When precursors carrying carboxyl-terminal deletions were presented to the chloroplast, no defined intermediates could be detected. Instead, a number of proteins, probably resulting from aberrant processing, accumulated simultaneously inside the chloroplasts.
Reiss
B
B
Max-Planck-Institut für Züchtungsforschung, Köln, Federal Republic of Germany.
Wasmann
C C
CC
Schell
J
J
Bohnert
H J
HJ
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010946:Plants, Medicinal; D011499:Protein Processing, Post-Translational; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
D012273:Ribulose-Bisphosphate Carboxylase