Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Abstract

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.

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

“…These results are supported by the crystal structure of the pea Toc34 homodimer identifying sequence motifs conserved in both atToc33 and atToc159 which are required for dimerization and chloroplast targeting of soluble atToc159 (Sun et al, 2002;Weibel et al, 2003). These findings suggest that atToc159 and its homologues may function as soluble factors in chloroplast protein import (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002). The Toc GTPase-system is strikingly analogous to the SRP system (which also employs a targeting mechanism based on two homotypic GTP-binding proteins) (Keenan et al, 2001) as well as to the Sec-system: SecA is present as soluble protein in the cytosol but also as a membrane-attached protein in the outer bacterial membrane and may drive protein translocation across the periplasmic membrane (Economou and Wickner, 1994) in an ATP-dependent, sewing machine-type fashion.…”

“…After 15 min centrifugation at 100 000 · g at 4°C the supernatant was removed and used for immunoprecipitation. Hundred microlitres solubilized membranes were incubated on a rolling shaker for 60 min at 4°C with 3 lg affinity-purified atToc75 antibody (Hiltbrunner et al, 2001b); as a control an equal amount of unspecific rabbit IgG was used. Twenty microlitres packed Protein A Sepharose beads (Amersham Biosciences) were added and the samples were incubated for another 60 min.…”

“…Samples were then concentrated as described elsewhere (Wessel and Flu¨gge, 1984) and analyzed by SDS-PAGE and autoradiography according to standard protocols. Immunodetection of atToc75 was done as described (Hiltbrunner et al, 2001b).…”

“…Indeed, a cytosolic member of the 14-3-3 family of proteins has been identified that binds to phosphorylated transit sequences (Waegemann and Soll, 1996) and thus forms a guidance complex to deliver precursor proteins to the Toc-complex (May and Soll, 2000). Recently, we have discovered that atToc159 also exists in an abundant soluble form, partitioning between the outer chloroplast membrane and the cytosol (Hiltbrunner et al, 2001b). Cytosolic atToc159 is directly targeted to the Toc-complex in a homotypic interaction with atToc33 mediated by the G-domains of the two proteins (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002;Lee et al, 2003;Weibel et al, 2003).…”

“…Recently, we have discovered that atToc159 also exists in an abundant soluble form, partitioning between the outer chloroplast membrane and the cytosol (Hiltbrunner et al, 2001b). Cytosolic atToc159 is directly targeted to the Toc-complex in a homotypic interaction with atToc33 mediated by the G-domains of the two proteins (Hiltbrunner et al, 2001b;Bauer et al, 2002;Smith et al, 2002;Lee et al, 2003;Weibel et al, 2003). These results are supported by the crystal structure of the pea Toc34 homodimer identifying sequence motifs conserved in both atToc33 and atToc159 which are required for dimerization and chloroplast targeting of soluble atToc159 (Sun et al, 2002;Weibel et al, 2003).…”