Abstract
C-terminally truncated precursors of wheat light-harvesting chlorophyll a/b binding protein (LHCP) were synthesized to investigate the origin of the two forms (about 25 kD and 26 kD) of the mature protein observed upon in vitro import into the chloroplast. Precursors p delta 13 and p delta 27, lacking 13 and 27 amino acids, respectively, were successfully imported, and both gave rise to two smaller forms proportional to the size of their deletions. These results demonstrate that there are two N-terminal sites that are cleaved during import of the LHCP precursor, undoubtedly contributing to the heterogeneity of LHCP found in vivo. Significantly, p delta 27 yielded only 50% of mature LHCP when compared with wild type. Although the products of p delta 27 import were localized to the thylakoids, in contrast to p delta 13 they were not correctly inserted into the membranes, indicating that residues essential for this step are missing. p delta 27 is distinguished from p delta 13 by lacking the carboxy end of a domain highly conserved between LHCP of photosystems II and I. Other specific precursor mutants with larger C-terminal deletions were not efficiently transported into the organelle in time course experiments, nor did they insert directly into the thylakoids using chloroplast lysates, in an assay independent of translocation across the envelope. In addition, the mutant p delta 18n, lacking the first 18 amino acids of mature LHCP, was only found bound to the chloroplast envelope. However, both p delta 18n and the mature protein, i.e., LHCP, synthesized in vitro without its 34-amino acid transit peptide inserted into the thylakoids in chloroplast lysates. The overall conformation of the mutant precursor polypeptides was probed using the chloroplast soluble processing enzyme in an organelle-free reaction optimized for the LHCP precursor and the more general protease trypsin. A tightly folded, protease-resistant conformation was not apparent to explain the loss of efficient import.
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