Two 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

• Published in: The Journal of cell biology 1996-07, Vol.134 (2), p.315-327
• Main Authors: Ma, Y. (The State University of New Jersey, Newark, NJ.), Kouranov, A, LaSala, S.E, Schnell, D.J
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Biological Transport; CHLOROPLASTE; Chloroplasts; Chloroplasts - metabolism; CLOROPLASTO; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Gels; GTP-Binding Proteins - metabolism; Hydrolysis; IMMUNOLOGIE; Immunoprecipitation; Imports; INMUNOLOGIA; MEMBRANAS CELULARES; MEMBRANE CELLULAIRE; Membrane proteins; Membrane Proteins - metabolism; METABOLISME DES PROTEINES; METABOLISMO PROTEICO; P branes; Peptides - metabolism; PISUM SATIVUM; Pisum sativum - metabolism; Plant Proteins; PRECURSEUR D'ENZYME; PRECURSORES DE ENZIMAS; Protein precursors; Protein Precursors - metabolism; Protein Sorting Signals - metabolism; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; Rabbits; Ribulose-Bisphosphate Carboxylase - metabolism; RUBISCO; String theory
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.134.2.315

The interactions of precursor proteins with components of the chloroplast envelope were investigated during toe early stages of protein import 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 subunit of ribulose-1,5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 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 brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex to 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 triphosphate-induced insertion of the transit sequence across the outer membrane