The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts

Abstract

The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a ␤-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon.

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

“…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. Similarly, Toc159 may provide a GTP-dependent push to aid precursor translocation across the outer chloroplast envelope (Schleiff et al, 2003;Wallas et al, 2003).…”

Chloroplast protein import: solve the GTPase riddle for entry

“…The crystal structure of Toc34 [14], in conjunction with solution-and solid-phase binding assays [7,15,16], demonstrates that dimerization of the GTPase proteins occurs in the GDP-bound state and is precluded by bound GTP. How can these different observations be explained?…”

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

“…The major components of the TOC machinery include the preprotein receptors Toc159 and Toc33 (Toc34 in pea), which are membrane-bound GTPases, and Toc75, a β-barrel protein that forms a cation-selective channel through which preproteins cross the outer membrane (6)(7)(8). Toc75 also is implicated in the insertion of outer membrane proteins that lack an N-terminal transit peptide (9,10). An additional TOC complex component, Toc64, has a tetratricopeptide repeat domain and serves as a receptor site for cytosolic Hsp90/ 70 and their chloroplast-bound substrates (11,12).…”