CHARACTERIZATION OF THE IMPORT PROCESS OF A TRANSIT PEPTIDE INTO CHLOROPLASTS

In order to get insight into the functioning of transit sequences in c hloroplast protein transport, the import of the full-length transit pe ptide of ferredoxin (trfd) was investigated. trfd rapidly associated w ith chloroplasts under import conditions and becomes protected against externally added proteases. Import of radiolabeled trfd is inhibited equally efficiently by nonlabeled trfd as well as by the intact precur sor of ferredoxin. This strongly suggests that trfd enters the general import pathway of proteins into chloroplasts. trfd import was stimula ted by ATP, which is the first demonstration that ATP is involved in m embrane translocation of a targeting signal. Imported trfd was membran e-associated but was also partially degraded by internal proteases, mo st likely present in the stroma, indicating that the membrane-associat ed fraction of trfd is en route to its functional localization. The de gradation products are exported out of the organelle. In contrast to t he import of the precursor of ferredoxin, the import of trfd was indep endent of protease-sensitive components on the chloroplast surface, in dicating that the initial binding of precursor proteins may be facilit ated by transit sequence-lipid interactions.