Import and assembly of the beta-subunit of chloroplast coupling factor 1 (CF1) into isolated intact chloroplasts

The transit peptide gene of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana plumbaginifolia was fused to the tentoxin-resistant beta-subunit gene of chloroplast coupling factor 1 (CF1) from Nicotiana tabacum via a linker sequence. The consequent fusion gene encodes the entire gene sequences of both the transit peptide and the beta-subunit of CF1 without a single change of amino acid residues. The fusion gene was in vitro expressed in a coupled transcription-translation system as a 62-kDa precursor and was imported into isolated intact chloroplasts of Nicotiana longiflora. The imported precursor was found to be processed to the expected mature beta-subunit size. Evidence is presented that radioactively labeled beta-subunit was incorporated into mature CF1 and not just nonspecifically associated with the thylakoid membranes. Since the fusion protein containing only the transit peptide sequence was imported and apparently correctly processed, it may not be necessary to include N-terminal amino acids of the mature small subunit for correct proteolytic cleavage in the chloroplast stroma.