Analysis of the Interactions of Preproteins with the Import Machinery over the Course of Protein Import into Chloroplasts

Abstract

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.

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

“…The available evidence indicates that the two proteins act in concert to recognize the chloroplast targeting peptide (Kouranov and Schnell, 1997;Sveshnikova et al, 2000;Chen et al, 2000a). Toc159 is thought to act as the primary receptor of transit sequences (Hirsch et al, 1994;Perry and Keegstra, 1994;Kouranov and Schnell, 1997). An alternative model in which Toc34 functions as the primary receptor and Toc159 as a GTP-dependent translocation motor has also been proposed (Schleiff et al, 2003).…”

“…A C-terminal Mdomain (membrane anchor domain) lacking predicted hydrophobic transmembrane helices anchors the proteins in the outer membrane (Hirsch et al, 1994;Kessler et al, 1994). In addition, precursor proteins were shown to directly interact with the M-domain (Kouranov and Schnell, 1997).…”