Insertion of OEP14 into the Outer Envelope Membrane Is Mediated by Proteinaceous Components of Chloroplasts

Abstract

Most chloroplastic outer envelope membrane proteins are synthesized in the cytosol at their mature size without a cleavable targeting signal. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. The insertion has been assumed to be mediated by a spontaneous mechanism or by interaction solely with the lipid components of the outer membrane. However, we show here that insertion of an outer membrane protein requires some trypsin-sensitive and some N -ethylmaleimide-sensitive components of chloroplasts. Association and insertion of the outer membrane protein are saturable and compete with the import of another outer membrane protein. These data suggest that import of chloroplastic outer membrane proteins occurs at specific proteinaceous sites on chloroplasts. INTRODUCTIONMost chloroplastic proteins are encoded by the nuclear genome and synthesized in the cytosol. Nuclear-encoded chloroplastic proteins can be divided into roughly two groups on the basis of the presence or absence of cleavable targeting signals. Proteins in the first group are synthesized as higher molecular weight precursors with N-terminal extensions called transit peptides. Import of these precursor proteins into chloroplasts requires ATP and some thermolysin-sensitive receptor proteins on the chloroplastic surface (Cline et al., 1985;Olsen et al., 1989;Theg et al., 1989). This group of proteins includes all proteins destined for the interior of chloroplasts and at least one protein destined for the outer envelope membrane Tranel et al., 1995). The second group of proteins consists of most of the outer membrane proteins identified so far. These outer membrane proteins are synthesized at their mature size in the cytosol without a cleavable transit peptide (Salomon et al., 1990;Li et al., 1991;Ko et al., 1992;Fischer et al., 1994;Kessler et al., 1994;Seedorf et al., 1995;Bolter et al., 1999). The import mechanism for the first group of proteins has been studied extensively, and many protein components of the import machinery have been identified (Chen and Schnell, 1999;Keegstra and Cline, 1999;May and Soll, 1999). In contrast, very little is known about how the outer membrane proteins in the second group are targeted and inserted into the outer membrane.Two unique characteristics mark the import of these outer membrane proteins. For almost all of these proteins, thermolysin pretreatment of chloroplasts and ATP removal have no effect on insertion of the proteins into the outer membrane (Salomon et al., 1990;Li et al., 1991;Ko et al., 1992;Fischer et al., 1994). Because these results suggest that insertion of these proteins does not require any surfaceexposed chloroplastic proteins or energy, their insertion has generally been assumed to be accomplished by a spontaneous mechanism or through interaction with the lipid components of the outer membrane (Bruce, 1998;Keegstra and Cline, 1999).However, the hypotheses of spontaneous insertion or interaction with lipids both have their p...

Chloroplast envelope membranes: a dynamic interface between plastids and the cytosol

“…Insertion of the overwhelming part of the outer envelope membrane proteins was first though not to require either surface-exposed receptors or energy and had generally been assumed to be accomplished by a spontaneous mechanism or through interaction with the lipid components of the outer membrane Cline 1999, Schleiff andKlösgen 2001). Although early work suggested otherwise, the beststudied outer membrane proteins are now known to use both proteins within the chloroplast and NTPs for insertion (Tsai et al 1999;Tu and Li 2000;Tu et al 2004;Hofmann and Theg 2005).…”

Genomic reduction and evolution of novel genetic membranes and protein-targeting machinery in eukaryote-eukaryote chimaeras (meta-algae)

“…Unfortunately, it is not known how Toc34 and most other OM proteins are inserted. They have hydrophobic insertion sequences at the N-terminus as in OEP14 (Tu & Li 2001) or at the C-terminus as in OEP7 and Toc34. Although OEP7 may simply use its highly discriminating insertion sequence (Lee et al 2001) to recognize the special galactolipids and phospholipid composition of the OM ) specific proteins are probably needed to mediate insertion of others, including Toc34 and OEP14 (Tsai et al 1999).…”

Tic21 Is an Essential Translocon Component for Protein Translocation across the Chloroplast Inner Envelope Membrane

“…Samples were analyzed by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and detected by alkaline phosphatase-conjugated secondary antibodies. All electrophoresis was performed as described (Tu and Li, 2000). Insertion mutant information was obtained from the SIGnAL website at http://signal.salk.edu and further confirmed by DNA sequencing.…”