The C terminus of a chloroplast precursor modulates its interaction with the translocation apparatus and PIRAC.
The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Delta52, Delta67, and Delta74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of (35)S-prSSU. Both IC(50) and K(i) values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Delta52 were properly processed and accumulated within the chloroplast, whereas Delta67 and Delta74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.
Dabney-Smith
C
C
Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996, USA.
van Den Wijngaard
P W
PW
Treece
Y
Y
Vredenberg
W J
WJ
Bruce
B D
BD
D001667:Binding, Competitive; D002736:Chloroplasts / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013329:Structure-Activity Relationship
D007473:Ion Channels; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D012273:Ribulose-Bisphosphate Carboxylase