Structural and Guanosine Triphosphate/Diphosphate Requirements for Transit Peptide Recognition by the Cytosolic Domain of the Chloroplast Outer Envelope Receptor, Toc34

Abstract

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.

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

“…In the first model, Toc34 and Toc159 remain stably associated with the outer envelope membrane and interact with incident preproteins directly, much like the mitochondrial protein import receptors Tom20 and Tom22 (Pfanner and Geissler, 2001). 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).…”

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

“…This could provide a part of the molecular switch that triggers the insertion of Toc159 and the initiation of preprotein translocation. Others have proposed that Toc33/34 acts as the primary transit peptide receptor and that Toc159 functions only as a component of the translocation motor (7,12,25). However, all current data can be reconciled by the model in which the two Toc GTPases act coordinately to regulate transit peptide recognition and initiate membrane translocation by interacting directly with each other in a manner regulated by their intrinsic GTPase activities.…”

Chloroplast protein import: solve the GTPase riddle for entry

“…(i) Initial studies demonstrated that Toc34 binds to a variety of chloroplast preproteins, suggesting that it functions as a receptor for protein import [9]. Phosphorylation of the transit peptide, although not essential for preprotein recognition by Toc34, promotes high-affinity binding to Toc34 [10].…”