2 COMPONENTS OF THE CHLOROPLAST PROTEIN IMPORT APPARATUS, IAP86 AND IAP75, INTERACT WITH THE TRANSIT SEQUENCE DURING THE RECOGNITION AND TRANSLOCATION OF PRECURSOR PROTEINS AT THE OUTER ENVELOPE

The interactions of precursor proteins with components of the chloropl ast envelope were investigated during the early stages of protein impo rt using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75 , cross-linked to the transit sequence of the precursor to the small s ubunit of ribulose-1,5-bisphosphate carboxylase (pS) in a precursor bi nding assay. In the presence of concentrations of ATP or GTP that supp ort maximal precursor binding to the envelope, cross-linking to the tr ansit sequence occurred predominantly with IAP75 and a previously unid entified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brou ght into the vicinity of the outer membrane under these conditions. IA P75 and a third import component, IAP34, were coimmunoprecipitated wit h IAP86 antibodies from solubilized envelope membranes, indicating tha t these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosp hate-induced insertion of the transit sequence across the outer membra ne.