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.

Abstract

Abstract. The interactions of precursor proteins with components of the chloroplast envelope were investigated during the 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-l,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 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 triphosphateinduced insertion of the transit sequence across the outer membrane.

The Arabidopsisppi1Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins[W]

“…In support of this model, preproteins can be cross-linked to pea Toc34 (psToc34) and/or psToc159 very early during import into pea chloroplasts (Perry and Keegstra, 1994;Ma et al, 1996;Kouranov and Schnell, 1997), and a direct and specific interaction between preproteins and psToc34 has been observed in vitro (Sveshnikova et al, 2000;Schleiff et al, 2002). However, not all cross-linking studies resulted in the identification of psToc34 (Perry and Keegstra, 1994;Ma et al, 1996), and in those that did, psToc34 appeared to interact with the mature region of the preprotein rather than with the transit peptide, as would be expected of a receptor (Kouranov and Schnell, 1997).…”

“…Protein import into chloroplasts can be divided into three distinct stages in vitro based on energetic requirements (Olsen et al, 1989;Olsen and Keegstra, 1992;Ma et al, 1996;Kouranov and Schnell, 1997). These stages are assumed to correspond to sequential steps in the import process that occurs in vivo.…”

“…Both proteins are anchored in the outer envelope by COOH-terminal domains and project their GTP binding domains into the cytosol. Although their precise mode of action remains unclear (Sveshnikova et al, 2000;Hiltbrunner et al, 2001b), Toc159 and Toc34 interact with incident preproteins (Ma et al, 1996;Kouranov and Schnell, 1997) and mediate their transfer to the translocation channel, of which Toc75 is a major component. Toc75 has a ␤ -barrel structure comprising 16 amphiphilic ␤ -strands and forms a channel with a pore size of ‫ف‬ 14 to 26 Å (Hinnah et al, 2002).…”

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

“…These are termed the Toc-(translocon at the outer chloroplast membrane) and Tic-complexes (translocon at the inner chloroplast membrane). In pea, the Toc-complex consists of three major components forming a stable complex Ma et al, 1996). Toc159 (the number indicates the molecular mass in kD) and Toc34 are surface exposed, GTP-binding integral membrane proteins (Hirsch et al, 1994;Kessler et al, 1994;Seedorf et al, 1995;Chen et al, 2000a).…”

Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif-Containing Protein, CIA2

“…Several translocon components have been identified from pea chloroplasts by cross-linking or coimmunoprecipitating with importing precursor proteins (reviewed by Schleiff and Soll, 2000). They are collectively named Toc (for translocon at the outer envelope membrane of chloroplasts) and Tic (for translocon at the inner envelope membrane of chloroplasts) proteins (Schnell et al, 1997).Among the Toc components identified, Toc159 is proposed to function as the transit peptide receptor (Perry and Keegstra, 1994;Ma et al, 1996). A considerable amount of evidence indicates that Toc75 is the major component of the protein-translocating channel in the outer membrane (Hinnah et al, 1997;Reumann et al, 1999).…”