THE PLANT CELL, Vol 2, Issue 2 173-184, Copyright © 1990 by American Society of Plant Biologists
Loss of Efficient Import and Thylakoid Insertion Due to N- and C-Terminal Deletions in the Light-Harvesting Chlorophyll a/b Binding Protein
S. E. Clark, J. E. Oblong and G. K. Lamppa
Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 E. 58th Street, Chicago, Illinois 60637
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.
