Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein

We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.